Effects of Auricularia auricula residue on soil physicochemical properties, microbial community composition and diversity, and rice yield

Effects of Auricularia auricula residue on soil physicochemical properties, microbial community composition and diversity, and rice yield | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Effects of Auricularia auricula residue on soil physicochemical properties, microbial community composition and diversity, and rice yield Weidong Yuan, Tingxuan Zong, Bin Yu, Ya Xin, Jia Lu, Qin Qiu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6661042/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The return of mushroom residue to the field is an effective measure to improve soil fertility and maintain agroecosystem productivity. We investigated the effects of returning Auricularia auricula residue to the field on the soil nutrients, enzyme activities, and microbial communities in a rice– A. auricula rotation farmland. The trial was designed with three treatments: K0 (no mushroom residue), F1 (3600 kg/667 m² residue), and F2 (4500 kg/667 m² residue). The return of A. auricula residue to the field significantly increased the soil nutrient content. The contents of ammonium nitrogen, total nitrogen, total potassium, total phosphorus, available potassium (QK), available phosphorus, and organic matter in the F2 treatment increased by 180.49%, 70.41%, 16.3%, 54.35%, 137.33%, 38.84%, and 59.29%, respectively, compared to those of the K0 treatment. The activities of the soil enzymes urease, sucrase, β-glucosidase (β-GC), and acetyl-β- d -glucosidase significantly increased by 32.98%, 407.78%, 206.85%, and 186.26%, respectively, in the F2 treatment compared to those of K0; catalase and leucine aminopeptidase activities were increased by 244.42% and 130.90% in the F1 treatment compared to those of K0. Moreover, A. auricula residue return to the field increased the Chao1 and Shannon indices of the bacterial community, but decreased the diversity of the fungal community. Redundancy analysis showed that QK, β-GC, and urease were the main factors driving the changes in microbial communities. In conclusion, returning A. auricula mushroom residue to the field can enhance soil ecological functions by improving soil nutrients, enzyme activities, and microbial community structure. Earth and environmental sciences/Environmental sciences Earth and environmental sciences/Environmental sciences/Environmental impact Mushroom residue Rice–Auricularia auricula rotation Soil fertility Enzyme activity Microbial diversity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction China has a high species diversity and abundant fungal resources, making it one of the world’s largest producers of edible mushrooms. Specifically, China’s annual production of edible mushrooms accounts for more than 70% of the total global production 1 . However, with rapid development of the edible mushroom industry, the production of mushroom residue by-products has also shown a sharp increase. According to Hu et al. 2 , the annual output of mushroom residue in China has exceeded 8.0 × 10 7 tons. As an accompanying by-product of the production process of edible fungi, mushroom residue is rich in organic matter (45.3–67.8%), total nitrogen (TN; 1.2–2.5%), total phosphorus (TK; 0.4–0.8%), polysaccharides, amino acids, and other nutrients 3 . From the perspective of resource recycling and reuse, mushroom residue has significant potential value. Multiple studies have shown that properly processed mushroom residue can be converted into high-quality organic fertilizer 4 . The use of these fertilizers has proven to significantly enhance the soil structure, improve water retention and aeration, and increase microbial activity in the soil. In addition, polysaccharides in mushroom residue promote plant growth and enhance resistance 5 . Therefore, the use of mushroom residue not only recycles agricultural waste but also provides a new solution for enhancing soil and promoting sustainable agricultural development. The return of mushroom residue to the field has been widely used in global agricultural production practices as an effective measure to improve the soil environment and enhance fertility 6 . It is now well-established that the return of mushroom residue to the field not only provides the necessary nutrients for crop growth 7 , but also has the effect of regulating the physical shelf 8 and optimizing the biological function 9 of the soil. Tang et al. 10 showed that the return of mushroom residue to the field can significantly increase the TN, TK, and humus contents in the soil of single-season planted crops, thereby effectively improving soil fertility. Zhang et al. 11 found that the nitrogen and organic matter contents of soil were significantly increased after the implementation of shiitake and Agaricus bisporu s mushroom residue in a rice–wheat rotation farmland. Soil enzymes and microorganisms are essential indicators of soil biological properties, significantly influencing processes such as soil nutrient cycling and organic matter decomposition 12 . The decomposition and transformation processes of mushroom residue returned to the field are largely driven by soil microorganisms. The incorporation of mushroom residue into the soil provides a rich and diverse carbon source, promoting the proliferation and growth of soil microbial communities 13 . Wang et al. 14 demonstrated that returning mushroom residue to the field alters the community structure of Proteobacteria and increases the abundance of bacteria associated with complex organic matter degradation. Peng et al. 15 revealed that combining mushroom residue with chemical fertilizer significantly enhanced soil fertility, improved soil enzyme activity and bacterial abundance, and reshaped the bacterial community structure compared to the impacts of chemical fertilizer alone. Therefore, studying soil microorganisms is highly beneficial for maintaining and optimizing soil ecosystem functions. Rice– Auricularia auricula rotation is a new eco-efficient cultivation mode that can fully utilize the land, light, and temperature resources in the autumn and winter after rice production. This rotation system can also reduce the occurrence of diseases and pests in the cultivation process of A. auricula while increasing the yield of rice, thereby achieving significant ecological and economic benefits 16 . Given this background, the aim of this study was to investigate the effects of A. auricula residue returned to the field on soil nutrients, enzyme activities, bacterial and fungal diversity, and microbial community structure under a rice– A. auricula rotation system. Toward this end, we analyzed the patterns of soil nutrient and microbial community changes and their intrinsic relationships with soil enzyme activity. This research seeks to provide a scientific foundation for the rational utilization of A. auricula residue and establishment of a practice for the precise regulation of soil nutrients to ultimately realize an enhanced crop yield and improved soil biological functions. Results Effects of mushroom residue application on soil nutrients, physicochemical properties, extracellular enzyme activities, and rice yield The application of mushroom residue significantly influenced the soil nutrient content. As shown in Fig. 1 a-b and Table S1 , with increasing amounts of mushroom residue, the levels of AN, TN, TK, TP, QK, QP, and organic matter exhibited a gradual increase, reaching their highest values in the F2 treatment. In contrast, the NN content was the highest in the K0 treatment at 1.90 mg/kg. Therefore, mushroom residue application effectively promoted the accumulation of organic matter and nutrients in the soil. Additionally, the pH, WAT, and EC showed gradual increases with higher mushroom residue application levels (Fig. 1 c and Table S1 ). These results demonstrate that using A. auricula residue enhances soil nutrient levels and influences soil physico-chemical properties. Soil enzymes play a crucial role in nearly all chemical reactions within the soil and are highly sensitive to environmental changes. As shown in Fig. 1 d-e and Table S2, the activities of UE, SC, β-GC, and NAG exhibited gradual increases with higher mushroom residue application levels, reaching their peak values in the F2 treatment. In contrast, the activities of CAT and LAP showed an initial increase followed by a decrease, peaking in the F1 treatment. This suggests that an appropriate amount of mushroom residue is more conducive to the expression of oxidative stress–related enzymes. Conversely, NR activity displayed an opposite trend, initially decreasing and then increasing with a higher level of mushroom residue application, reaching its highest value in the K0 treatment at 5.06 U/g. These results indicate that the addition of A. auricula residue significantly influences the activities of various extracellular enzymes in the soil. However, different enzymes exhibited distinct response patterns to mushroom residue application, which is attributed to their unique characteristics and functional roles in soil ecological processes. The application of A. auricula residue also significantly influenced rice yield. As shown in Fig. 1 E and Supplementary Table S3, rice yield exhibited a gradual increase with higher mushroom residue application levels, reaching its peak value in the F2 treatment at 646 kg/667 m², representing a significant increase of 21.57% compared to that of the K0 treatment. The F1 treatment followed, with a yield of 623.36 kg/667 m², which was 17.14% higher than that of the K0 treatment. These results demonstrate that the rice– A. auricula rotation system significantly enhances rice yield. Effects of mushroom residue application on soil microbial diversity Significant differences in bacterial and fungal diversity were observed among the treatments (Fig. 2 ). Alpha-diversity analysis of the bacterial communities (Fig. 2 a) revealed that the ACE, Chao1, Shannon, and Simpson indices were the highest in the F2 treatment, with no significant differences between the F2 and F1 treatments; compared to those of the K0 group, these indices increased by 9.26%, 9.2%, 1.65%, and 0.03%, respectively. In contrast, fungal communities (Fig. 2 b) exhibited the lowest ACE, Chao1, and Shannon indices in the F2 treatment, which decreased by 52.68%, 22.61%, and 51.81%, respectively, relative to those of K0. The Simpson index was the lowest in the F1 treatment (no significant difference from F2), showing a 3.05% reduction compared to that of K0. These results indicate that mushroom residue application effectively enhances bacterial richness (Chao1), evenness (ACE), and diversity (Shannon and Simpson), but reduces fungal richness, evenness, and diversity to some extent. The composition of the soil bacteria communities at the phylum and genus levels differed significantly among treatments after the return of the mushroom residue to the field. At the phylum level (Fig. 3 a), dominant taxa (mean relative abundance ≥ 2.4%) included Acidobacteriota (29.13–38.13%), Proteobacteria (26.41–30.7%), Chloroflexi (5.99–6.47%), Myxococcota (4.13–9.09%), Gemmatimonadota (3.28–5.13%), Methylomirabilota (1.13–4.25%), Desulfobacterota (1.61–3.96%), Nitrospirota (2.07–3.96%), and Bacteroidota (1.85–2.68%). The relative abundances of Myxococcota and Desulfobacterota increased with higher mushroom residue application levels, while the relative abundances of Gemmatimonadota, Methylomirabilota, Nitrospirota, and Bacteroidota peaked in the F1 treatment (with moderate residue application). To analyze the relevant data more accurately, unclassified data were removed at the genus level (Fig. 3 b). The top 10 genera were Candidatus_Solibacter (2.64–4.23%), Anaeromyxobacter (1.7–4.25%), Bryobacter (1.61–2.92%), Candidatus_Koribacter (1.04–2.38%), Rhodanobacter (0.62–3.15%), Haliangium (0.62–2.25%), and Acidibacter (0.62–1.58%). The relative abundances of Anaeromyxobacter and Haliangium significantly increased with higher levels of residue application, whereas those of Candidatus_Koribacter and Rhodanobacter decreased. Significant differences in fungal community composition at the phylum and genus levels were also observed among the treatments. At the phylum level (Fig. 3 c), the dominant taxa (mean relative abundance ≥ 1%) included Ascomycota (51.47–63.73%), Basidiomycota (4.26–31.69%), Mortierellomycota (1.69–11.63%), Rozellomycota (2.03–5.35%), Chytridiomycota (0.62–4.61%), and Olpidiomycota (0.08–5.13%). Compared to those of the K0 treatment, the relative abundances of Ascomycota and Basidiomycota significantly increased with mushroom residue application. For a more precise analysis, unclassified taxa were excluded at the genus level (Fig. 3 d). The top 10 fungal genera were Agrocybe (0.04–26.0%), Mortierella (1.66–11.27%), Schizothecium (1.49–7.19%), Pyrenochaetopsis (1.14–5.69%), Fusarium (0.13–7.53%), Trichoderma (0.15–6.7%), Podospora (0.2–5.93%), Echria (0.47–1.57%), Sarocladium (0.09–2.53%), and Fusicolla (0.11–1.96%). Compared to those of the K0 treatment, the relative abundances of Agrocybe, Schizothecium , Pyrenochaetopsis, Fusarium, Podospora , Echria , and Sarocladium significantly increased. Notably, the relative abundance of Sarocladium increased with higher levels of mushroom residue returned to the field, while Trichoderma, Mortierella , and Fusicolla had their highest relative abundances in the treatment without the application of mushroom residue. RDA of soil nutrients, enzyme activities, and microbial diversity RDA revealed significant correlations between soil nutrients, enzyme activities, and bacterial and fungal communities following the return of A. auricula residue to the field. As shown in Fig. 4 a, RDA1 explained 98.94% of the total variance between soil nutrients, enzyme activities, and bacterial communities, with QK, β-GC, and urease identified as the primary factors influencing the bacterial community composition. Similarly, Fig. 4 b demonstrates that RDA1 accounted for 92.21% of the variance between soil nutrients, enzyme activities, and fungal communities, with QK, β-GC, and urease also being the dominant drivers of fungal community changes. These results indicate that under the rice– Auricularia rotation system, the return of mushroom residue to the field significantly alters soil nutrients and enzyme activities, which are closely associated with shifts in microbial community structure. Correlation analysis of soil microbial diversity with soil nutrients, enzyme activities, and rice yield As shown in Fig. 5 a, significant correlations were observed between soil bacteria and soil fertility as well as rice yield. Specifically, the relative abundances of the genera Anaeromyxobacter, Geothrix, Haliangium , and Sideroxydans exhibited highly significant positive correlations with AN and yield, while the relative abundances of the genera Dokdonella, Holophaga, Nitrosospira, Pseudolabrys, Rhodanobacter , and Candidatus_Koribacter showed highly significant negative correlations with AN and yield. Additionally, Geothrix and Novosphingobium were positively correlated with TP; Sideroxydans was positively correlated with TK, TP, and yield; Anaeromyxobacter was positively correlated with TK, QK, and yield; and Haliangium was positively correlated with TK, TP, QK, and yield. In contrast, Candidatus_Koribacter, Dokdonella, Holophaga , Nitrosospira, Rhodanobacter , and Rhodoferax were negatively correlated with QK, while the relative abundances of Dokdonella, Holophaga, Nitrosospira , and Rhodanobacter showed significant negative correlations with TP. Significant correlations were also found between soil bacteria and enzyme activities. Anaeromyxobacter, Haliangium , and Sideroxydans were positively correlated with β-GC and sucrase, Geothrix was positively correlated with NAG and urease, and Novosphingobium was positively correlated with β-GC and urease. Conversely, Dokdonella, Holophaga , Nitrosospira , and Rhodanobacter were negatively correlated with β-GC, NAG, and sucrase; Candidatus_Koribacter was negatively correlated with β-GC and sucrase; Pseudolabrys was negatively correlated with β-GC; and Rhodoferax was negatively correlated with NAG and NR. As illustrated in Fig. 5 b, significant correlations were observed between soil fungi, soil fertility, and rice yield. Specifically, Hypholoma, Nigrospora , and Tetraplosphaeria exhibited highly significant positive correlations with QK, AN, and yield; Lecythophora and Tetraploa were positively correlated with QK, QP, and yield; Phaeosphaeria was positively correlated with AN, TK, TP, and yield; and Sarocladium was positively correlated with AN and yield. In contrast, Articulospora, Cystofilobasidium, Oidiodendron , and Penicillium showed highly significant negative correlations with AN and TK, while Fusicolla, Mortierella, Talaromyces , and Paraphaeosphaeria were negatively correlated with AN, TP, and yield. Significant correlations were also found between soil fungi and enzyme activities. Funiliomyces, Hypholoma, Tetraplosphaeria , and Nigrospora were positively correlated with SC and β-GC, while Apiosordaria, Articulospora, Fusicolla, Mortierella, Paraphaeosphaeria , Penicillium , and Talaromyces were negatively correlated with sucrase and β-GC activities. Additionally, positive correlations were found for Hypholoma with NAG; Lecythophora with sucrase; Ochroconis with urease, NAG, and β-GC; and Tetraploa with sucrase. Conversely, negative correlations were found for Cystofilobasidium with NAG and β-GC; Mortierella with NAG; Oidiodendron with NAG and TN; and Paraphaeosphaeria , Penicillium , and Talaromyces with NAG. Discussion Changes in soil physico-chemical properties and nutrient content are closely associated with the return of mushroom residue to the field. Following mushroom residue application, significant increases were observed in soil organic matter, TN, TP, TK, QK, QP, and AN. Hao et al. 17 demonstrated that the return of Stropharia rugosoannulata residue effectively enhanced soil organic carbon, nitrogen, and potassium contents, aligning with our findings. Additionally, Chen et al. 18 noted that mushroom residue, which is rich in lignin and polysaccharides, can slowly release nutrients through microbial degradation, consistent with the 59.29% increase in organic matter observed in the F2 treatment in this study. Mushroom residue contains substantial organic matter and nutrients, and its appropriate application not only significantly improves the soil nutrient content and structure but also enhances the water retention capacity and mitigates soil acidity or alkalinity 19 . The soil pH in the F1 and F2 treatments was significantly higher than that in the K0 treatment, consistent with findings reported by Frąc et al. 19 This indicates that mushroom residue application effectively alleviates soil acidification and its adverse effects, thereby promoting nutrient uptake and improving crop yield and nutritional status. Overall, the results of this study highlight that the rational application of A. auricula residue in rice-cultivated soils significantly enhances the soil nutrient content, offering substantial benefits for improving soil quality and increasing rice yield. Enzyme activity has been proposed as a biological indicator of soil quality and is closely linked to soil functions. Specifically, soil urease catalyzes the hydrolysis of urea, generating ammonia, a vital nitrogen source for crop growth 20 . β-GC converts cellobiose into glucose, a nutrient essential for plants 21 . Sucrase facilitates the hydrolysis of organic matter into glucose and fructose, playing a critical role in increasing soluble nutrients in the soil 22 . NAG is crucial for promoting soil carbon and nitrogen cycling and is closely associated with soil microorganisms, indirectly reflecting the intensity and direction of biochemical processes in the soil 23 . The significant increases in β-GC, urease, and NAG activities found in this study indicate that mushroom residue input accelerates carbon and nitrogen cycling 20 . During the decomposition of mushroom residue returned to the field, soil aggregate and pore structures are altered, enhancing the buffering capacity and water retention, thereby providing carriers for various enzymes. Additionally, the mushroom residue supplies abundant energy substrates for soil microorganisms, optimizing their habitat and stimulating metabolic activities. This in turn promotes the secretion of enzymes involved in carbon, nitrogen, and phosphorus cycling, which enter the soil through vigorous root exudation and microbial activity, leading to increased enzyme activity and faster carbon and nitrogen turnover 24 . Nannipieri et al. 20 emphasized that soil enzyme activity is directly related to substrate availability, and suggested that the cellulose and chitin in mushroom residue may provide specific substrates for β-GC. Furthermore, in paddy field experiments, Zhang et al. 25 found that sucrase activity was significantly positively correlated with the QP content, supporting the synergistic effect observed in this study, where the sucrase activity increased by 407.78% alongside a 38.84% rise in QP. In this study, the activities of sucrase, NAG, catalase, urease, LAP, and β-GC all increased after A. auricula residue application, with urease, β-GC, sucrase, and NAG activities rising with higher mushroom residue application levels. These findings further demonstrate that the return of A. auricula residue to the field effectively promotes the transformation of enzyme activities related to carbon and nitrogen cycling, facilitating the decomposition of slow-release nutrients into readily available forms for plant uptake. This process provides additional nutrients for plant growth, playing a significant role in soil nutrient cycling and ecosystem stability. Soil microorganisms play a pivotal role in soil ecosystems, driving nutrient cycling and organic matter degradation; therefore, soil microbes are recognized as key indicators of soil health 26 . The results of this study demonstrate that the return of mushroom residue to the field significantly influences the diversity and community structure of soil bacteria and fungi. Specifically, mushroom residue application notably enhanced bacterial diversity and richness while reducing fungal diversity and richness, consistent with the findings of Su et al. 27 In addition, Six et al. 28 suggested that bacteria, compared to fungi, preferentially utilize readily decomposable resources. The addition of mushroom residue provides heterotrophic bacteria with exogenous organic materials and nutrients, promoting their proliferation, reducing intercommunity competition, and thereby enhancing bacterial diversity in paddy soils 29 . Although the return of A. auricula residue to the field impacted fungal diversity and richness, it significantly increased the relative abundance of Sarocladium while reducing the relative abundances of Trichoderma , Mortierella , and Fusicolla , thereby modulating the fungal community structure and composition. Furthermore, the composition of bacterial and fungal communities varied among treatments 30 . In this study, the return of A. auricula residue to the field increased the relative abundances of beneficial bacterial phyla such as Acidobacteriota, Proteobacteria, Chloroflexi, Myxococcota, and Desulfobacterota. Proteobacteria play a crucial role in soil carbon, nitrogen, and sulfur cycling and exhibit strong resistance to heavily polluted soils 31 , particularly under heavy metal stress 32 . Acidobacteriota can degrade cellulose 33 and participate in iron cycling and other ecosystem functions 34 . Chloroflexi generates energy through photosynthesis and facilitates carbon transformation and utilization under low nutrient availability 35 . With mushroom residue addition, the relative abundances of Myxococcota and Desulfobacterota significantly increased, accelerating organic matter decomposition 36 , providing more nutrients for plant growth, and preventing sulfide accumulation that could harm plants. Chen et al. 18 also found that organic waste application significantly increased the relative abundances of Proteobacteria and Acidobacteriota, which was directly linked to the input of carbon and nitrogen sources from the residue. These microorganisms play key roles in carbon and nitrogen cycling, further validating the promotion of functional microbes by mushroom residue return to the field. Compared to those found in the K0 treatment, the F1 and F2 treatments exhibited higher relative abundances of the genera Anaeromyxobacter , Haliangium , Candidatus_Koribacter , and Rhodanobacter . Anaeromyxobacter , an anaerobic bacterium, participates in the redox processes of multivalent heavy metals, stabilizes radioactive elements, and metabolizes organic halides 37 . Haliangium enhances soil carbon cycling, accelerates organic matter decomposition, and provides nutrients for plants 38 . Candidatus_Koribacter is essential for degrading various organic compounds, including cellulose, hemicellulose, starch, and chitin 39 . Rhodanobacter promotes nitrification pathways and utilizes Fe²⁺ for denitrification 40 . Li et al. 41 reported that return of organic material to the field can suppress the proliferation of pathogenic fungi (e.g., Fusarium ) while promoting the colonization of beneficial fungi (e.g., Trichoderma ) by regulating the soil pH and carbon-to-nitrogen ratio, consistent with the observed trends in fungal community structure in this study. The increase in beneficial microorganisms in the soil can promote plant growth and enhance yield 42 . Therefore, the diverse microbial community formed by the return of A. auricula residue to the field contributes to soil improvement and the maintenance of high soil functionality. Influence of the soil environment on microbial communities Soil nutrients, enzyme activities, and microbial communities are key factors maintaining the rhizosphere microenvironment, interacting with and constraining one another. RDA revealed that QK, β-GC, and urease were the primary drivers of the shifts observed in bacterial and fungal community structures after the return of mushroom residue to the field. In a life-cycle assessment, Dorr et al. 43 highlighted that the return of mushroom residue to the field reduces the risk of nitrogen leaching by regulating microbial functions, further validating the positive correlation between AN and bacterial abundance observed in this study. Spearman correlation heatmaps indicated that after the return of A. auricula residue to the field, bacterial genera such as Geothrix, Novosphingobium , Promicromonospora, Anaeromyxobacter, Haliangium , and Sideroxydans significantly and positively influenced soil properties, including QP, TP, pH, QK, AN, TK, and organic matter, as well as the activities of soil enzymes such as urease, sucrase, β-GC, and NAG. A systematic review 20 emphasized the central role of soil enzymes in carbon, nitrogen, and phosphorus cycling, noting that β-GC and urease activities are significantly positively correlated with organic matter decomposition rates, providing a theoretical basis for the observed increase in enzyme activities observed in this study. Soil organic matter supplies ample substrates for enzymes such as urease and sucrase, thereby enhancing their activities. Urease catalyzes urea hydrolysis to produce ammonia and carbon dioxide, providing nitrogen and carbon sources for Geothrix . During metabolism, Geothrix generates organic acids, whose carboxyl and hydroxyl groups bind with calcium ions in calcium phosphate, forming soluble complexes that promote phosphate dissolution and increase soil-available phosphorus. Sucrase catalyzes the hydrolysis of sucrose into glucose and fructose; therefore, the enhanced activity of sucrase increases available carbon sources in the soil, providing more energy for bacterial growth. In a paddy field experiment, Huang et al. 44 found that increased sucrase activity is closely linked to higher soil-available phosphorus, indicating that enzyme activities improve phosphorus availability by promoting organic phosphorus mineralization, corroborating the findings of the present study. Bacteria such as Geothrix and Novosphingobium utilize nutrients released by the activities of these enzymes for their growth and metabolism. Additionally, the return of mushroom residue to the field effectively reduced the abundance of pathogenic fungi such as Ceratocystis and Fusarium . In summary, the return of A. auricula residue to the field can foster sustainable and healthy soil ecosystems through interactions between microorganisms and soil physicochemical properties. Conclusions Under the rice– A. auricula rotation mode, the return of mushroom residue to the field can effectively increase the soil nutrient content, soil enzyme activity, and the diversity and abundance of soil microorganisms. High-throughput sequencing technology showed that the return of mushroom residue to the field could significantly increase the Chao1 and Shannon diversity indices of soil bacteria and decrease those of fungi. The dominant bacterial phyla were Acidobacteria and Proteobacteria, while Ascomycota and Basidiomycota were identified as the major fungal taxa, with soil organic matter, QK, β-GC, and urease activity being the main factors affecting the composition of soil bacterial and fungal communities in the farmland under rice– A. auricula rotation. In conclusion, the return of A. auricula residue to the field has significant potential for achieving soil improvement under the rice– A. auricula rotation pattern, which is important for improving soil quality and agricultural production. Methods Experimental site The experiment was carried out at the Hecheng Edible Fungi Cooperative, located in Tonglu County, Hangzhou, Zhejiang Province, China. In mid-October 2022, after the rice harvest, A. auricula cultivation was initiated using sawdust and bran as the primary substrates, with each bag weighing approximately 1.6–1.7 kg. Approximately 8,000 or 10,000 bags were deployed per mu (667 m²). Harvesting was completed by mid-April 2023, yielding an average dry mushroom residue of 450 g per bag. The mushroom residue was then directly returned to the field after bag removal. Rice (variety Yongyou 1540) was transplanted in early June 2023, with a fertilization rate of 30 kg/667 m² compound fertilizer and 10 kg/667 m² urea. Field management followed standard cultivation practices. Experimental sampling and design The experiment included three treatments: treatment 1 (K0) involved no application of mushroom residue, treatment 2 (F1) involved the application of mushroom residue at 3,600 kg/667 m² (8,000 bags), and treatment 3 (F2) involved the application of mushroom residue at 4,500 kg/667 m² (10,000 bags). Each treatment was set up with three replicates and each replicate covered 1 mu (667 m²). Rice yields were measured in all paddies for each replicate. Rice was threshed and sun-dried (adjusted to a 14% moisture content) to obtain the yield. After the rice harvest, soil samples were collected from the plough layer (0–20 cm) using an “S”-shaped five-point sampling method. Five random soil samples were mixed, sieved through a 1-mm mesh, and divided into two portions: one portion was air-dried for soil fertility and enzyme activity analysis and the other was stored at − 20°C for soil microbial sequencing. Soil fertility analysis Nitrate nitrogen (NN) was determined by the phenol disulfonic acid colorimetric method 45 and ammonium nitrogen (AN) was determined by the potassium chloride leaching–indophenol blue colorimetric method 46 . Available potassium (QK) and available phosphorus (QP) were measured using the flame photometry and Olsen-P method, respectively 47 . The total nitrogen (TN) was determined by the Kjeldahl method 48 , total potassium (TK) was determined by flame photometry 49 , total phosphorus (TP) was determined by the molybdenum-antimony anti-colorimetric method 48 , and organic matter (OMT) was analyzed using the potassium dichromate oxidation method 50 . Soil water content (WAT) was determined by the drying method 51 . Soil pH and electrical conductivity (EC) were measured using a pH meter (Sartorius PB-10, Germany) and a conductivity meter (DDSJ-308F, Leici, China), respectively, after extraction with deionized water (soil:water = 1:5) 6,51 . Soil enzyme activity analysis Leucine aminopeptidase (LAP) activity was quantified using microplate fluorometric analysis 23 and sucrase (SC) activity was assessed using the 3,5-dinitrosalicylic acid colorimetric method 52 . Nitrate reductase (NR) activity was determined by the sulfanilamide colorimetric method 53 . Catalase (CAT) activity was measured by potassium permanganate titration 54 . Soil urease (UE) activity was determined using the sodium phenolate–sodium hypochlorite colorimetric method 52 . β-Glucosidase (β-GC) and acetyl-β-D-glucosaminidase (NAG) activities were analyzed via the nitrophenol colorimetric method 21 . DNA extraction, polymerase chain reaction (PCR) amplification, and sequencing of soil samples For each treatment, a 0.5 g soil sample was accurately weighed and genomic DNA was extracted using the TGuide Magnetic Bead Soil DNA Kit (Tiangen Biotech, DP812). Using the extracted DNA as a template, bacterial 16S rRNA genes were amplified at the V4 region with primers 515F (5′-GTGYCAGCMGCCGCGGTAA-3′) and 806R2 (5′-GGACTACNVGGGTWTCTTAAT-3′), while fungal ITS genes were amplified in the ITS1 region with primers ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS2 (5′-GCTGCGTTCTTCATCGATGC-3′). The PCR system consisted of 50 ng DNA, 0.3 µL each forward and reverse primer (10 µM), 5 µL KOD FX Neo Buffer, 2 µL dNTPs (2 mM each), 0.2 µL KOD FX Neo, and ddH 2 O to a final volume of 10 µL. PCR conditions were as follows: initial denaturation at 95°C for 3 min; 25 cycles of 95°C for 30 s, 50°C for 30 s, and 72°C for 40 s; final extension at 72°C for 5 min; and hold at 4°C. PCR products were purified using VAHTSTM DNA Clean Beads and recovered by 1.8% agarose gel electrophoresis. High-throughput sequencing was performed on the Illumina NovaSeq 6000 platform. Data processing and statistical analyses Statistical analyses were conducted using SPSS 27.0. Differences among treatments in soil fertility indicators and enzyme activities were compared with one-way analysis of variance and Duncan’s test (significance level P < 0.05). Primer sequences were identified and trimmed using cutadapt v2.7.8, low-quality sequences were filtered using Trimmomatic v0.33, and paired-end reads were assembled using FLASH v1.2.11. The spliced data were denoised and chimeric sequences were removed using QIIME2 v2020.6 to obtain final valid data (non-chimeric reads). Alpha diversity indices, including the Chao1, Simpson, Shannon, and ACE indices, were determined by QIIME2 v2020.6. Taxonomic classification of bacterial and fungal communities was achieved by aligning feature sequences to the respective Silva 138 and UNITE reference databases, with dominant species identified at the genus and phylum levels based on relative abundance. Redundancy analysis (RDA) using origin 21.0 and heatmap correlation analysis by the Spearman Rank Correlation Coefficient Algorithm were employed to elucidate the interactions between environmental factors and microbial community structures. Abbreviations TN total nitrogen TK total phosphorus NN nitrate nitrogen AN ammonium nitrogen QK available potassium QP available phosphorus TP total phosphorus OMT organic matter WAT water content EC electrical conductivity LAP leucine aminopeptidase SC sucrase NR nitrate reductase CAT catalase UE urease β-GC β-Glucosidase NAG acetyl-β-D-glucosaminidase RDA redundancy analysis Declarations Author contributions W.Y., J.S., and L.M. conceived and designed the experiments; T.Z., B.Y., and Y.X. collected the samples; W.Y., J.L. and Q.Q. analyzed the data and wrote the manuscript; and J.S. and L.M. revised and approved the final version of the paper. W.Y., T.Z., and B.Y. contributed to the work equally and should be regarded as co-first authors. Funding Project of Collaborative Extension of Major Agricultural Technologies of Zhejiang (No. 2021XTTGSYJ02-1) and the China Agriculture Research System of MOF and MARA (No. CARS-20). Competing interests The authors declare no competing interests. Availability of data and material The data of this study are included in the article or the Supplementary Materials. The raw sequence data of microorganisms reported in this paper have been deposited in the Genome Sequence Archive (GSA) database under accession numbers CRA******. All the data have been submitted. The application is currently undergoing review and the number allocation is pending. The Project number is PRJCA040766, the biological Biosample number is subSAM142064, and the GSA application number is subCRA042610. The details will be added to the manuscript promptly after the number is assigned. Correspondence and requests for materials should be addressed to L.M. or J.S. References Qiao, Y. et al. Comprehensive evaluation on effect of planting and breeding waste composts on the yield, nutrient utilization, and soil environment of baby cabbage. J. Environ. Manage. 341 , 117941 (2023). Hu, L. et al. Fungi residue returning: effects on soil physicochemical characters, microorganisms and enzyme activity in vegetable field. Chin. Agric. Sci. Bull. 36 (1), 98–104 (2020). Zhang, L. & Sun, X. Changes in physical, chemical, and microbiological properties during the two-stage co-composting of green waste with spent mushroom compost and biochar. Bioresource Technol. 171 , 274–284 (2014). Majchrowska-Safaryan, A., Pakuła, K. & Becher, M. The influence of spent mushroom substrate fertilization on the selected properties of arable soil. Environ. Prot. Nat. Resour. 31 , 28–34 (2020). Xiao, W. et al. Effect of polysaccharides of nine edible mushroom seed bodies on the growth of rice seedlings. Acta Edulis Fungi . 27 (1), 69–74 (2020). Chen, L. et al. Short-term responses of soil nutrients, heavy metals and microbial community to partial substitution of chemical fertilizer with spent mushroom substrates (SMS). Sci. Total Environ. 844 , 157064 (2022). Xiao, J. et al. Meta-analysis of biochar application effects on soil fertility and yieldsof fruit and vegetables in greenhouse. J. Plant. Nutr. Fertilizers . 24 (1), 228–236 (2018). Li, X. et al. Effects of fertilizer with gypsum application on growing development and nutrient uptake of potting corn in coastal saline soil. J. Anhui Sci. Technol. Univ. 25 (6), 23–28 (2011). Zhang, N. et al. Response of soil nutrients and microbial communities to increased application of mushroom dreg in albic soil. Chin. Agric. Sci. Bull. 41 (2), 49–55 (2025). Tang, Q., Liu, W., Huang, H., Peng, Z. & Deng, L. Responses of crop yield, soil fertility, and heavy metals to spent mushroom residues application. Plants 13 (5), 663 (2024). Zhang, H. et al. Effects of combined application of chemical fertilizers with different mushroom residues on soil fertility and enzymes activities. J. Soil. Water Conserv. 36 (6), 364–370 (2022). Li, Y., Zhang, L., Fang, S., Tian, Y. & Guo, J. Variation of soil enzyme activity and microbial biomass in poplar plantations of different genotypes and stem spacings. J. Forestry Res. 29 (4), 963–972 (2018). Li, F. et al. Effect of mushroom residue on soil property and crop and research progress in its recycling. J. Agr Sci. Tech. 17 (3), 100–106 (2015). Wang, Q. et al. Long-term fertilization changes bacterial diversity and bacterial communities in the maize rhizosphere of Chinese Mollisols. Appl. Soil. Ecol. 125 , 88–96 (2018). Peng, X. et al. Effects of returning Stropharia rugosoannulata cultivation residue to paddy fields on rice growth and soil environment. Acta Agriculturae Universitatis Jiangxiensis . 45 (2), 349–360 (2023). Hu, Y., Ma, J., Ye, Z., Di, C. & Wang, X. Effects of continous application of edibe fungus residue on soil fertility preperties under rice-edible fungus rotation system. J. Soil. Water Conserv. 27 (6), 172–176 (2013). Hao, H. et al. Effects of the rice-mushroom rotation pattern on soil properties and microbial community succession in paddy fields. Front. Microbiol. 15 , 1449922 (2024). Chen, Y. et al. Organic amendments shift the phosphorus-correlated microbial co-occurrence pattern in the peanut rhizosphere network during long-term fertilization regimes. Appl. Soil. Ecol. 124 , 229–239 (2018). Frąc, M. et al. Mycobiome composition and diversity under the long-term application of spent mushroom substrate and chicken manure. Adv. Agron. 11 (3), 410 (2021). Nannipieri, P., Trasar-Cepeda, C. & Dick, R. P. Soil enzyme activity: a brief history and biochemistry as a basis for appropriate interpretations and meta-analysis. Biol. Fert Soils . 54 (1), 11–19 (2018). Eivazi, F. & Tabatabai, M. A. Factors affecting glucosidase and galactosidase activities in soils. Soil Biology Biochemistry . 22 (7), 891–897 (1990). Zhu, J. G., Chu, H., Xie, Z. B. & Yagi, K. Effects of lanthanum on nitrification and ammonification in three Chinese soils. Nutr. Cycl. Agroecosys . 63 , 309–314 (2002). Burns, R. G. et al. Soil enzymes in a changing environment: current knowledge and future directions. Soil Biology Biochemistry . 58 , 216–234 (2013). Borase, D. N. et al. Long-term impact of diversified crop rotations and nutrient management practices on soil microbial functions and soil enzymes activity. Ecol. Indic. 114 , 106322 (2020). Zhang, W. et al. Effects of adding vermicompost on soil fertility and enzyme activity of acidic paddy soil. J. Nuclear Agricultural Sci. 39 (3), 615–623 (2025). Zhao, J. et al. Manipulation of the rhizosphere microbial community through application of a new bio-organic fertilizer improves watermelon quality and health. PLoS One . 13 (2), e0192967 (2018). Su, Y. et al. Linking soil microbial community dynamics to straw-carbon distribution in soil organic carbon. Sci. Rep. 10 (1), 5526 (2020). Six, J., Frey, S. D., Thiet, R. K. & Batten, K. M. Bacterial and fungal contributions to carbon sequestration in agroecosystems. Soil Sci. Soc. Am. J. 70 , 555–569 (2006). Subhashini, D. Growth promotion and increased potassium uptake of tobacco by potassium-mobilizing bacterium Frateuria aurantia grown at different potassium levels in vertisols. Commun. Soil. Sci. Plan. 46 , 210–220 (2014). Brenzinger, K. et al. Steering microbiomes by organic amendments towards climate-smart agricultural soils. Bio Fert Soils . 57 (8), 1053–1074 (2021). Rampelotto, P. H., de Siqueira Ferreira, A., Barboza, A. D. & Roesch, L. F. Changes in diversity, abundance, and structure of soil bacterial communities in Brazilian Savanna under different land use systems. Microb. Ecol. 66 (3), 593–607 (2013). Li, X. et al. Changes in the composition and diversity of microbial communities during anaerobic nitrate reduction and Fe(II) oxidation at circumneutral pH in paddy soil. Soil Biology Biochemistry . 94 , 70–79 (2016). Lee, S. H., Ka, J. O. & Cho, J. C. Members of the phylum Acidobacteria are dominant and metabolically active in rhizosphere soil. FEMS Microbiol. Lett. 285 (2), 263–269 (2008). Wang, Y. et al. Comparison of the levels of bacterial diversity in freshwater, intertidal wetland, and marine sediments by using millions of illumina tags. Appl. Environ. Microb. 78 (23), 8264–8271 (2012). Fullerton, H. & Moyer, C. L. Comparative single-cell genomics of Chloroflexi from the Okinawa trough deep-subsurface biosphere. Appl. Environ. Microb. 82 (10), 3000–3008 (2016). Guan, Y. et al. Effects of continuous straw returning on soil functional microorganisms and microbial communities. J. Microbiol. 61 (1), 49–62 (2023). Prakash, O. et al. Geobacter daltonii sp. nov., an Fe(III)- and uranium(VI)-reducing bacterium isolated from a shallow subsurface exposed to mixed heavy metal and hydrocarbon contamination. Internatonal J. Syst. Evolutionary Microbiol. 60 (3), 546–553 (2010). Dai, W. et al. Phylogenetic diversity of stochasticity-dominated predatory myxobacterial community drives multi-nutrient cycling in typical farmland soils. Sci. Total Environ. 871 , 161680 (2023). Zhang, X. et al. Responses of soil nutrients and microbial communities to intercropping medicinal plants in moso bamboo plantations in subtropical China. Environ. Sci. Pollut R . 27 (2), 2301–2310 (2020). Cao, X. et al. Effects of iron-based substrate on coupling of nitrification, aerobic denitrification and Fe(II) autotrophic denitrification in tidal flow constructed wetlands. Bioresource Technol. 361 , 127657 (2022). Li, J., Yang, Y., Wen, J., Mo, F. & Liu, Y. Continuous manure application strengthens the associations between soil microbial function and crop production: Evidence from a 7-year multisite field experiment on the Guanzhong Plain. Agriculture, Ecosystems & Environment 338, 108082 (2022). Garbeva, P., van Veen, J. A. & van Elsas, J. D. Microbial diversity in soil: selection microbial populations by plant and soil type and implications for disease suppressiveness. Annu. Rev. Phytopathol. 42 , 243–270 (2004). Dorr, E., Koegler, M., Gabrielle, B. & Aubry, C. Life cycle assessment of a circular, urban mushroom farm. J. Clean. Prod. 288 , 125668 (2020). Huang, M. et al. Effects of different tillage measures during summer fallow seasonontents and enzyme activities in dryland wheat field. Soil. Fertilizer Sci. China . 8 , 30–42 (2024). Norman, R. J. & Stucki, J. W. The determination of nitrate and nitrite in soil extracts by ultraviolet spectrophotometry. Soil Sci. Soc. Am. J. 45 (2), 347–353 (1981). Rhine, E. D., Mulvaney, R. L., Pratt, E. J. & Sims, G. K. Improving the Berthelot reaction for determining ammonium in soil extracts and water. Soil Sci. Soc. Am. J. 62 , 473–480 (1998). Ying, B., Xiao, S., Xiong, K., Cheng, Q. & Luo, J. Comparative studies of the distribution characteristics of rocky desertification and land use/land cover classes in typical areas of Guizhou province, China. Environ. Earth Sci. 71 (2), 631–645 (2014). Vance, E. D., Brookes, P. C. & Jenkinson, D. S. An extraction method for measuring soil microbial biomass C. Soil Biology Biochemistry . 19 (6), 703–707 (1987). Cambardella, C. A. & Elliott, E. T. Particulate soil organic-matter changes across a grassland cultivation sequence. Soil Sci. Soc. Am. J. 56 , 777–783 (1992). Dai, L. et al. Organic matter regulates ammonia-oxidizing bacterial and archaeal communities in the surface sediments of Ctenopharyngodon idellus aquaculture ponds. Front. Microbiol. 9 , 2290 (2018). Zhang, Q. et al. Effects of different irrigation levels on the yield and quality of the Cyperus esculentus L. at tuber stage. Acta Agrestia Sinica . 32 (11), 3636–3645 (2024). Du, Y., Bao, Y., Liu, X. & Zhang, X. Effects of tartary buckwheat rotation on enzyme activities and microorganisms in rhizosphere soil of cultivated potato in Yunnan Province. J. Agr Sci. Tech. 26 (5), 192–200 (2024). Wang, L. et al. Effects of bio-organic fertilizer with microbial inoculants on the growth, physiological characteristics, yield and quality of garlic seedlings. J. Gansu Agricultural Univ. 58 (5), 63–70 (2022). Li, D. et al. Effects of crop rotation on the growth, quality and soil environment of Euryale ferox . J. Chin. Med. Mater. 47 (1), 12–21 (2024). Additional Declarations No competing interests reported. Supplementary Files Supplementarydata.doc Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6661042","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":464588715,"identity":"9de5cfa8-12a8-4725-9cbe-87570cdeb7e1","order_by":0,"name":"Weidong Yuan","email":"","orcid":"","institution":"Hangzhou Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Weidong","middleName":"","lastName":"Yuan","suffix":""},{"id":464588716,"identity":"185c7ede-41e4-427d-a36c-36c388a4ebc9","order_by":1,"name":"Tingxuan Zong","email":"","orcid":"","institution":"Zhejiang Agricultural Technical Extension Center","correspondingAuthor":false,"prefix":"","firstName":"Tingxuan","middleName":"","lastName":"Zong","suffix":""},{"id":464588717,"identity":"b0812f9b-da64-42dc-b927-456033fca3b1","order_by":2,"name":"Bin Yu","email":"","orcid":"","institution":"Hangzhou Agricultural Technical Extension Center","correspondingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"Yu","suffix":""},{"id":464588718,"identity":"68364cc3-3384-4383-b368-6632695b481c","order_by":3,"name":"Ya Xin","email":"","orcid":"","institution":"Hangzhou Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ya","middleName":"","lastName":"Xin","suffix":""},{"id":464588719,"identity":"0e570dfd-ed51-41b9-b48e-dc56668969f5","order_by":4,"name":"Jia Lu","email":"","orcid":"","institution":"Hangzhou Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jia","middleName":"","lastName":"Lu","suffix":""},{"id":464588720,"identity":"ef91e8af-91fb-4eaa-b714-8ceb7ac9ed07","order_by":5,"name":"Qin Qiu","email":"","orcid":"","institution":"Jiangsu Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Qin","middleName":"","lastName":"Qiu","suffix":""},{"id":464588721,"identity":"4de65f1a-2d44-4f48-b785-105b316bbdfa","order_by":6,"name":"Lin Ma","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIiWNgGAWjYBACfmbGxgcJBjZy/AwHiNQi2d582OBDRZqxZAOxWgzOHEuTnHHmcOIGYnUwMNzIMZPmbWNO3Hzw8MOPPxjs5BnYz+LXzTgjx9iat43NeNuBY8bSPAzJhg08eQl4tTBL5Bje5m3jkd124ICBNJCfwCDBY4BXC5tEjgHQYRKMmxuOf/75g6GesBYenmNJQO8bKG5gOGMmwcNwmLAWCXZwICcYSxw4U2bNY3DcsI0nB78W+8PgqPwvxz/j+OabPyqq5fnZz+DXgmTfASABVMxGpHog4G8gXu0oGAWjYBSMLAAAj5FGl+t6iG8AAAAASUVORK5CYII=","orcid":"","institution":"Jiangsu Academy of Agricultural Sciences","correspondingAuthor":true,"prefix":"","firstName":"Lin","middleName":"","lastName":"Ma","suffix":""},{"id":464588722,"identity":"a788b4d8-dd77-4014-9050-4b51cd17d74f","order_by":7,"name":"Jiling Song","email":"","orcid":"","institution":"Hangzhou Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jiling","middleName":"","lastName":"Song","suffix":""}],"badges":[],"createdAt":"2025-05-14 06:38:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6661042/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6661042/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83862892,"identity":"9a61229b-6174-4f9c-98cd-485c08bf2bd6","added_by":"auto","created_at":"2025-06-03 20:10:50","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":512050,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of mushroom residues returned to the field on soil nutrients, physico-chemical properties, extracellular enzyme activities, and rice yield.\u003c/p\u003e","description":"","filename":"Figure1a.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6661042/v1/e483dd290add40ac8ebbb2fb.jpg"},{"id":83863476,"identity":"4af9659e-0955-43d7-a96d-5680f4335964","added_by":"auto","created_at":"2025-06-03 20:26:50","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":491283,"visible":true,"origin":"","legend":"\u003cp\u003eSpecies richness and diversity of soil bacteria and fungi after returning mushroom residues to the field. \u003cstrong\u003e(a):\u003c/strong\u003eACE, Chao1, Shannon, and Simpson indices for bacteria; \u003cstrong\u003e(b):\u003c/strong\u003e ACE, Chao1, Shannon, and Simpson for fungi.\u003c/p\u003e","description":"","filename":"Figure2a.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6661042/v1/5ae24bba7117e5b41163c615.jpg"},{"id":83862895,"identity":"794c00fc-476d-48c8-9740-1a102dded3df","added_by":"auto","created_at":"2025-06-03 20:10:50","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":742050,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of mushroom residues returned to the field on soil bacterial and fungal species composition. \u003cstrong\u003e(a): \u003c/strong\u003eSpecies composition at the bacterial phylum level; \u003cstrong\u003e(b): \u003c/strong\u003eSpecies composition at the bacterial genus level; \u003cstrong\u003e(c):\u003c/strong\u003e Species composition at the fungal phylum level; \u003cstrong\u003e(d): \u003c/strong\u003eSpecies composition at the fungal genus level.\u003c/p\u003e","description":"","filename":"Figure3a.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6661042/v1/93723fbbe73999ed30350806.jpg"},{"id":83862536,"identity":"46c3aadd-7fdb-47d8-9295-7b84c8a50f17","added_by":"auto","created_at":"2025-06-03 20:02:50","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":229215,"visible":true,"origin":"","legend":"\u003cp\u003eRedundancy analysis of soil nutrients, enzyme activities, and microbial community for bacteria (a) and fungi (b).\u003c/p\u003e","description":"","filename":"Figure4a.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6661042/v1/6aa9aed9aa34c2b64f4b1248.jpg"},{"id":83862539,"identity":"3ab4b9b3-a3c4-48d1-92f8-d5f58f69c2ee","added_by":"auto","created_at":"2025-06-03 20:02:50","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1198276,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation of dominant soil microbial species with indicators of soil fertility and enzyme activity for bacteria (a) and fungi (b). Red indicates a significant positive correlation and blue indicates a significant negative correlation. * p \u0026lt; 0.05; ** p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure5a.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6661042/v1/63ec093da1ca5a1d8c7694b3.jpg"},{"id":90173835,"identity":"91d332bb-a12e-42ee-91e1-48caf4c45f1c","added_by":"auto","created_at":"2025-08-29 11:54:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4112802,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6661042/v1/ddcdafb8-dac1-4ebd-928b-a7ab26f2a8da.pdf"},{"id":83862533,"identity":"2d31c5fc-9807-4695-b430-c2b1eeb061cb","added_by":"auto","created_at":"2025-06-03 20:02:50","extension":"doc","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":50688,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata.doc","url":"https://assets-eu.researchsquare.com/files/rs-6661042/v1/bd290dcf60d75b2067c08eaf.doc"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of Auricularia auricula residue on soil physicochemical properties, microbial community composition and diversity, and rice yield","fulltext":[{"header":"Introduction","content":"\u003cp\u003eChina has a high species diversity and abundant fungal resources, making it one of the world’s largest producers of edible mushrooms. Specifically, China’s annual production of edible mushrooms accounts for more than 70% of the total global production\u003csup\u003e1\u003c/sup\u003e. However, with rapid development of the edible mushroom industry, the production of mushroom residue by-products has also shown a sharp increase. According to Hu et al.\u003csup\u003e2\u003c/sup\u003e, the annual output of mushroom residue in China has exceeded 8.0 × 10\u003csup\u003e7\u003c/sup\u003e tons.\u0026nbsp;As an accompanying by-product of the production process of edible fungi, mushroom residue is rich in organic matter (45.3–67.8%), total nitrogen (TN; 1.2–2.5%), total phosphorus (TK; 0.4–0.8%), polysaccharides, amino acids, and other nutrients\u003csup\u003e3\u003c/sup\u003e. From the perspective of resource recycling and reuse, mushroom residue has significant potential value. Multiple studies have shown that properly processed mushroom residue can be converted into high-quality organic fertilizer\u003csup\u003e4\u003c/sup\u003e. The use of these fertilizers has proven to significantly enhance the soil structure, improve water retention and aeration, and increase microbial activity in the soil. In addition, polysaccharides in mushroom residue promote plant growth and enhance resistance\u003csup\u003e5\u003c/sup\u003e. Therefore, the use of mushroom residue not only recycles agricultural waste but also provides a new solution for enhancing soil and promoting sustainable agricultural development.\u003c/p\u003e\n\u003cp\u003eThe return of mushroom residue to the field has been widely used in global agricultural production practices as an effective measure to improve the soil environment and enhance fertility\u003csup\u003e6\u003c/sup\u003e. It is now well-established that the return of mushroom residue to the field not only provides the necessary nutrients for crop growth\u003csup\u003e7\u003c/sup\u003e, but also has the effect of regulating the physical shelf\u003csup\u003e8\u003c/sup\u003e and optimizing the biological function\u003csup\u003e9\u003c/sup\u003e of the soil. Tang et al.\u003csup\u003e10\u003c/sup\u003e showed that the return of mushroom residue to the field can significantly increase the TN, TK, and humus contents in the soil of single-season planted crops, thereby effectively improving soil fertility. Zhang et al.\u003csup\u003e11\u003c/sup\u003e found that the nitrogen and organic matter contents of soil were significantly increased after the implementation of shiitake and \u003cem\u003eAgaricus bisporu\u003c/em\u003es mushroom residue in a rice–wheat rotation farmland.\u003c/p\u003e\n\u003cp\u003eSoil enzymes and microorganisms are essential indicators of soil biological properties, significantly influencing processes such as soil nutrient cycling and organic matter decomposition\u003csup\u003e12\u003c/sup\u003e. The decomposition and transformation processes of mushroom residue returned to the field are largely driven by soil microorganisms. The incorporation of mushroom residue into the soil provides a rich and diverse carbon source, promoting the proliferation and growth of soil microbial communities\u003csup\u003e13\u003c/sup\u003e. Wang et al.\u003csup\u003e14\u003c/sup\u003e demonstrated that returning mushroom residue to the field alters the community structure of Proteobacteria and increases the abundance of bacteria associated with complex organic matter degradation.\u0026nbsp;Peng et al.\u003csup\u003e15\u003c/sup\u003e revealed that combining mushroom residue with chemical fertilizer significantly enhanced soil fertility, improved soil enzyme activity and bacterial abundance, and reshaped the bacterial community structure compared to the impacts of chemical fertilizer alone. Therefore, studying soil microorganisms is highly beneficial for maintaining and optimizing soil ecosystem functions.\u003c/p\u003e\n\u003cp\u003eRice–\u003cem\u003eAuricularia auricula\u003c/em\u003e rotation\u0026nbsp;is a new eco-efficient cultivation mode that can fully utilize the land, light, and temperature resources in the autumn and winter after rice production. This rotation system can also reduce the occurrence of diseases and pests in the cultivation process of \u003cem\u003eA. auricula\u003c/em\u003e while increasing the yield of rice, thereby achieving significant ecological and economic benefits\u003csup\u003e16\u003c/sup\u003e. Given this background, the aim of this study was to investigate the effects of \u003cem\u003eA. auricula\u003c/em\u003e residue returned to the field on soil nutrients, enzyme activities, bacterial and fungal diversity, and microbial community structure under a rice–\u003cem\u003eA. auricula\u003c/em\u003e rotation system. Toward this end, we analyzed the patterns of soil nutrient and microbial community changes and their intrinsic relationships with soil enzyme activity. This research seeks to provide a scientific foundation for the rational utilization of \u003cem\u003eA. auricula\u003c/em\u003e residue and establishment of a practice for the precise regulation of soil nutrients to ultimately realize an enhanced crop yield and improved soil biological functions.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eEffects of mushroom residue application on soil nutrients, physicochemical properties, extracellular enzyme activities, and rice yield\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe application of mushroom residue significantly influenced the soil nutrient content. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-b and Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, with increasing amounts of mushroom residue, the levels of AN, TN, TK, TP, QK, QP, and organic matter exhibited a gradual increase, reaching their highest values in the F2 treatment. In contrast, the NN content was the highest in the K0 treatment at 1.90 mg/kg. Therefore, mushroom residue application effectively promoted the accumulation of organic matter and nutrients in the soil. Additionally, the pH, WAT, and EC showed gradual increases with higher mushroom residue application levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec and Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). These results demonstrate that using \u003cem\u003eA. auricula\u003c/em\u003e residue enhances soil nutrient levels and influences soil physico-chemical properties.\u003c/p\u003e \u003cp\u003eSoil enzymes play a crucial role in nearly all chemical reactions within the soil and are highly sensitive to environmental changes. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed-e and Table S2, the activities of UE, SC, β-GC, and NAG exhibited gradual increases with higher mushroom residue application levels, reaching their peak values in the F2 treatment. In contrast, the activities of CAT and LAP showed an initial increase followed by a decrease, peaking in the F1 treatment. This suggests that an appropriate amount of mushroom residue is more conducive to the expression of oxidative stress\u0026ndash;related enzymes. Conversely, NR activity displayed an opposite trend, initially decreasing and then increasing with a higher level of mushroom residue application, reaching its highest value in the K0 treatment at 5.06 U/g. These results indicate that the addition of \u003cem\u003eA. auricula\u003c/em\u003e residue significantly influences the activities of various extracellular enzymes in the soil. However, different enzymes exhibited distinct response patterns to mushroom residue application, which is attributed to their unique characteristics and functional roles in soil ecological processes.\u003c/p\u003e \u003cp\u003eThe application of \u003cem\u003eA. auricula\u003c/em\u003e residue also significantly influenced rice yield. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and Supplementary Table S3, rice yield exhibited a gradual increase with higher mushroom residue application levels, reaching its peak value in the F2 treatment at 646 kg/667 m\u0026sup2;, representing a significant increase of 21.57% compared to that of the K0 treatment. The F1 treatment followed, with a yield of 623.36 kg/667 m\u0026sup2;, which was 17.14% higher than that of the K0 treatment. These results demonstrate that the rice\u0026ndash;\u003cem\u003eA. auricula\u003c/em\u003e rotation system significantly enhances rice yield.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eEffects of mushroom residue application on soil microbial diversity\u003c/h3\u003e\n\u003cp\u003eSignificant differences in bacterial and fungal diversity were observed among the treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Alpha-diversity analysis of the bacterial communities (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) revealed that the ACE, Chao1, Shannon, and Simpson indices were the highest in the F2 treatment, with no significant differences between the F2 and F1 treatments; compared to those of the K0 group, these indices increased by 9.26%, 9.2%, 1.65%, and 0.03%, respectively. In contrast, fungal communities (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) exhibited the lowest ACE, Chao1, and Shannon indices in the F2 treatment, which decreased by 52.68%, 22.61%, and 51.81%, respectively, relative to those of K0. The Simpson index was the lowest in the F1 treatment (no significant difference from F2), showing a 3.05% reduction compared to that of K0. These results indicate that mushroom residue application effectively enhances bacterial richness (Chao1), evenness (ACE), and diversity (Shannon and Simpson), but reduces fungal richness, evenness, and diversity to some extent.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe composition of the soil bacteria communities at the phylum and genus levels differed significantly among treatments after the return of the mushroom residue to the field. At the phylum level (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), dominant taxa (mean relative abundance\u0026thinsp;\u0026ge;\u0026thinsp;2.4%) included Acidobacteriota (29.13\u0026ndash;38.13%), Proteobacteria (26.41\u0026ndash;30.7%), Chloroflexi (5.99\u0026ndash;6.47%), Myxococcota (4.13\u0026ndash;9.09%), Gemmatimonadota (3.28\u0026ndash;5.13%), Methylomirabilota (1.13\u0026ndash;4.25%), Desulfobacterota (1.61\u0026ndash;3.96%), Nitrospirota (2.07\u0026ndash;3.96%), and Bacteroidota (1.85\u0026ndash;2.68%). The relative abundances of Myxococcota and Desulfobacterota increased with higher mushroom residue application levels, while the relative abundances of Gemmatimonadota, Methylomirabilota, Nitrospirota, and Bacteroidota peaked in the F1 treatment (with moderate residue application).\u003c/p\u003e \u003cp\u003eTo analyze the relevant data more accurately, unclassified data were removed at the genus level (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). The top 10 genera were \u003cem\u003eCandidatus_Solibacter\u003c/em\u003e (2.64\u0026ndash;4.23%), \u003cem\u003eAnaeromyxobacter\u003c/em\u003e (1.7\u0026ndash;4.25%), \u003cem\u003eBryobacter\u003c/em\u003e (1.61\u0026ndash;2.92%), \u003cem\u003eCandidatus_Koribacter\u003c/em\u003e (1.04\u0026ndash;2.38%), \u003cem\u003eRhodanobacter\u003c/em\u003e (0.62\u0026ndash;3.15%), \u003cem\u003eHaliangium\u003c/em\u003e (0.62\u0026ndash;2.25%), and \u003cem\u003eAcidibacter\u003c/em\u003e (0.62\u0026ndash;1.58%). The relative abundances of \u003cem\u003eAnaeromyxobacter\u003c/em\u003e and \u003cem\u003eHaliangium\u003c/em\u003e significantly increased with higher levels of residue application, whereas those of \u003cem\u003eCandidatus_Koribacter\u003c/em\u003e and \u003cem\u003eRhodanobacter\u003c/em\u003e decreased.\u003c/p\u003e \u003cp\u003eSignificant differences in fungal community composition at the phylum and genus levels were also observed among the treatments. At the phylum level (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), the dominant taxa (mean relative abundance\u0026thinsp;\u0026ge;\u0026thinsp;1%) included Ascomycota (51.47\u0026ndash;63.73%), Basidiomycota (4.26\u0026ndash;31.69%), Mortierellomycota (1.69\u0026ndash;11.63%), Rozellomycota (2.03\u0026ndash;5.35%), Chytridiomycota (0.62\u0026ndash;4.61%), and Olpidiomycota (0.08\u0026ndash;5.13%). Compared to those of the K0 treatment, the relative abundances of Ascomycota and Basidiomycota significantly increased with mushroom residue application.\u003c/p\u003e \u003cp\u003eFor a more precise analysis, unclassified taxa were excluded at the genus level (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). The top 10 fungal genera were \u003cem\u003eAgrocybe\u003c/em\u003e (0.04\u0026ndash;26.0%), \u003cem\u003eMortierella\u003c/em\u003e (1.66\u0026ndash;11.27%), \u003cem\u003eSchizothecium\u003c/em\u003e (1.49\u0026ndash;7.19%), \u003cem\u003ePyrenochaetopsis\u003c/em\u003e (1.14\u0026ndash;5.69%), \u003cem\u003eFusarium\u003c/em\u003e (0.13\u0026ndash;7.53%), \u003cem\u003eTrichoderma\u003c/em\u003e (0.15\u0026ndash;6.7%), \u003cem\u003ePodospora\u003c/em\u003e (0.2\u0026ndash;5.93%), \u003cem\u003eEchria\u003c/em\u003e (0.47\u0026ndash;1.57%), \u003cem\u003eSarocladium\u003c/em\u003e (0.09\u0026ndash;2.53%), and \u003cem\u003eFusicolla\u003c/em\u003e (0.11\u0026ndash;1.96%). Compared to those of the K0 treatment, the relative abundances of \u003cem\u003eAgrocybe, Schizothecium\u003c/em\u003e, \u003cem\u003ePyrenochaetopsis, Fusarium, Podospora\u003c/em\u003e, \u003cem\u003eEchria\u003c/em\u003e, and \u003cem\u003eSarocladium\u003c/em\u003e significantly increased. Notably, the relative abundance of \u003cem\u003eSarocladium\u003c/em\u003e increased with higher levels of mushroom residue returned to the field, while \u003cem\u003eTrichoderma, Mortierella\u003c/em\u003e, and \u003cem\u003eFusicolla\u003c/em\u003e had their highest relative abundances in the treatment without the application of mushroom residue.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eRDA of soil nutrients, enzyme activities, and microbial diversity\u003c/h2\u003e \u003cp\u003eRDA revealed significant correlations between soil nutrients, enzyme activities, and bacterial and fungal communities following the return of \u003cem\u003eA. auricula\u003c/em\u003e residue to the field. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, RDA1 explained 98.94% of the total variance between soil nutrients, enzyme activities, and bacterial communities, with QK, β-GC, and urease identified as the primary factors influencing the bacterial community composition. Similarly, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb demonstrates that RDA1 accounted for 92.21% of the variance between soil nutrients, enzyme activities, and fungal communities, with QK, β-GC, and urease also being the dominant drivers of fungal community changes. These results indicate that under the rice\u0026ndash;\u003cem\u003eAuricularia\u003c/em\u003e rotation system, the return of mushroom residue to the field significantly alters soil nutrients and enzyme activities, which are closely associated with shifts in microbial community structure.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCorrelation analysis of soil microbial diversity with soil nutrients, enzyme activities, and rice yield\u003c/h3\u003e\n\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, significant correlations were observed between soil bacteria and soil fertility as well as rice yield. Specifically, the relative abundances of the genera \u003cem\u003eAnaeromyxobacter, Geothrix, Haliangium\u003c/em\u003e, and \u003cem\u003eSideroxydans\u003c/em\u003e exhibited highly significant positive correlations with AN and yield, while the relative abundances of the genera \u003cem\u003eDokdonella, Holophaga, Nitrosospira, Pseudolabrys, Rhodanobacter\u003c/em\u003e, and \u003cem\u003eCandidatus_Koribacter\u003c/em\u003e showed highly significant negative correlations with AN and yield. Additionally, \u003cem\u003eGeothrix\u003c/em\u003e and \u003cem\u003eNovosphingobium\u003c/em\u003e were positively correlated with TP; \u003cem\u003eSideroxydans\u003c/em\u003e was positively correlated with TK, TP, and yield; \u003cem\u003eAnaeromyxobacter\u003c/em\u003e was positively correlated with TK, QK, and yield; and \u003cem\u003eHaliangium\u003c/em\u003e was positively correlated with TK, TP, QK, and yield. In contrast, \u003cem\u003eCandidatus_Koribacter, Dokdonella, Holophaga\u003c/em\u003e, \u003cem\u003eNitrosospira, Rhodanobacter\u003c/em\u003e, and \u003cem\u003eRhodoferax\u003c/em\u003e were negatively correlated with QK, while the relative abundances of \u003cem\u003eDokdonella, Holophaga, Nitrosospira\u003c/em\u003e, and \u003cem\u003eRhodanobacter\u003c/em\u003e showed significant negative correlations with TP.\u003c/p\u003e \u003cp\u003eSignificant correlations were also found between soil bacteria and enzyme activities. \u003cem\u003eAnaeromyxobacter, Haliangium\u003c/em\u003e, and \u003cem\u003eSideroxydans\u003c/em\u003e were positively correlated with β-GC and sucrase, \u003cem\u003eGeothrix\u003c/em\u003e was positively correlated with NAG and urease, and \u003cem\u003eNovosphingobium\u003c/em\u003e was positively correlated with β-GC and urease. Conversely, \u003cem\u003eDokdonella, Holophaga\u003c/em\u003e, \u003cem\u003eNitrosospira\u003c/em\u003e, and \u003cem\u003eRhodanobacter\u003c/em\u003e were negatively correlated with β-GC, NAG, and sucrase; \u003cem\u003eCandidatus_Koribacter\u003c/em\u003e was negatively correlated with β-GC and sucrase; \u003cem\u003ePseudolabrys\u003c/em\u003e was negatively correlated with β-GC; and \u003cem\u003eRhodoferax\u003c/em\u003e was negatively correlated with NAG and NR.\u003c/p\u003e \u003cp\u003eAs illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, significant correlations were observed between soil fungi, soil fertility, and rice yield. Specifically, \u003cem\u003eHypholoma, Nigrospora\u003c/em\u003e, and \u003cem\u003eTetraplosphaeria\u003c/em\u003e exhibited highly significant positive correlations with QK, AN, and yield; \u003cem\u003eLecythophora\u003c/em\u003e and \u003cem\u003eTetraploa\u003c/em\u003e were positively correlated with QK, QP, and yield; \u003cem\u003ePhaeosphaeria\u003c/em\u003e was positively correlated with AN, TK, TP, and yield; and \u003cem\u003eSarocladium\u003c/em\u003e was positively correlated with AN and yield. In contrast, \u003cem\u003eArticulospora, Cystofilobasidium, Oidiodendron\u003c/em\u003e, and \u003cem\u003ePenicillium\u003c/em\u003e showed highly significant negative correlations with AN and TK, while \u003cem\u003eFusicolla, Mortierella, Talaromyces\u003c/em\u003e, and \u003cem\u003eParaphaeosphaeria\u003c/em\u003e were negatively correlated with AN, TP, and yield.\u003c/p\u003e \u003cp\u003eSignificant correlations were also found between soil fungi and enzyme activities. \u003cem\u003eFuniliomyces, Hypholoma, Tetraplosphaeria\u003c/em\u003e, and \u003cem\u003eNigrospora\u003c/em\u003e were positively correlated with SC and β-GC, while \u003cem\u003eApiosordaria, Articulospora, Fusicolla, Mortierella, Paraphaeosphaeria\u003c/em\u003e, \u003cem\u003ePenicillium\u003c/em\u003e, and \u003cem\u003eTalaromyces\u003c/em\u003e were negatively correlated with sucrase and β-GC activities. Additionally, positive correlations were found for \u003cem\u003eHypholoma\u003c/em\u003e with NAG; \u003cem\u003eLecythophora\u003c/em\u003e with sucrase; \u003cem\u003eOchroconis\u003c/em\u003e with urease, NAG, and β-GC; and \u003cem\u003eTetraploa\u003c/em\u003e with sucrase. Conversely, negative correlations were found for \u003cem\u003eCystofilobasidium\u003c/em\u003e with NAG and β-GC; \u003cem\u003eMortierella\u003c/em\u003e with NAG; \u003cem\u003eOidiodendron\u003c/em\u003e with NAG and TN; and \u003cem\u003eParaphaeosphaeria\u003c/em\u003e, \u003cem\u003ePenicillium\u003c/em\u003e, and \u003cem\u003eTalaromyces\u003c/em\u003e with NAG.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eChanges in soil physico-chemical properties and nutrient content are closely associated with the return of mushroom residue to the field. Following mushroom residue application, significant increases were observed in soil organic matter, TN, TP, TK, QK, QP, and AN. Hao et al.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e demonstrated that the return of \u003cem\u003eStropharia rugosoannulata\u003c/em\u003e residue effectively enhanced soil organic carbon, nitrogen, and potassium contents, aligning with our findings. Additionally, Chen et al.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e noted that mushroom residue, which is rich in lignin and polysaccharides, can slowly release nutrients through microbial degradation, consistent with the 59.29% increase in organic matter observed in the F2 treatment in this study. Mushroom residue contains substantial organic matter and nutrients, and its appropriate application not only significantly improves the soil nutrient content and structure but also enhances the water retention capacity and mitigates soil acidity or alkalinity\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. The soil pH in the F1 and F2 treatments was significantly higher than that in the K0 treatment, consistent with findings reported by Frąc et al.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e This indicates that mushroom residue application effectively alleviates soil acidification and its adverse effects, thereby promoting nutrient uptake and improving crop yield and nutritional status. Overall, the results of this study highlight that the rational application of \u003cem\u003eA. auricula\u003c/em\u003e residue in rice-cultivated soils significantly enhances the soil nutrient content, offering substantial benefits for improving soil quality and increasing rice yield.\u003c/p\u003e \u003cp\u003eEnzyme activity has been proposed as a biological indicator of soil quality and is closely linked to soil functions. Specifically, soil urease catalyzes the hydrolysis of urea, generating ammonia, a vital nitrogen source for crop growth\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. β-GC converts cellobiose into glucose, a nutrient essential for plants\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Sucrase facilitates the hydrolysis of organic matter into glucose and fructose, playing a critical role in increasing soluble nutrients in the soil\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. NAG is crucial for promoting soil carbon and nitrogen cycling and is closely associated with soil microorganisms, indirectly reflecting the intensity and direction of biochemical processes in the soil\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The significant increases in β-GC, urease, and NAG activities found in this study indicate that mushroom residue input accelerates carbon and nitrogen cycling\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDuring the decomposition of mushroom residue returned to the field, soil aggregate and pore structures are altered, enhancing the buffering capacity and water retention, thereby providing carriers for various enzymes. Additionally, the mushroom residue supplies abundant energy substrates for soil microorganisms, optimizing their habitat and stimulating metabolic activities. This in turn promotes the secretion of enzymes involved in carbon, nitrogen, and phosphorus cycling, which enter the soil through vigorous root exudation and microbial activity, leading to increased enzyme activity and faster carbon and nitrogen turnover\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Nannipieri et al.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e emphasized that soil enzyme activity is directly related to substrate availability, and suggested that the cellulose and chitin in mushroom residue may provide specific substrates for β-GC. Furthermore, in paddy field experiments, Zhang et al.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e found that sucrase activity was significantly positively correlated with the QP content, supporting the synergistic effect observed in this study, where the sucrase activity increased by 407.78% alongside a 38.84% rise in QP.\u003c/p\u003e \u003cp\u003eIn this study, the activities of sucrase, NAG, catalase, urease, LAP, and β-GC all increased after \u003cem\u003eA. auricula\u003c/em\u003e residue application, with urease, β-GC, sucrase, and NAG activities rising with higher mushroom residue application levels. These findings further demonstrate that the return of \u003cem\u003eA. auricula\u003c/em\u003e residue to the field effectively promotes the transformation of enzyme activities related to carbon and nitrogen cycling, facilitating the decomposition of slow-release nutrients into readily available forms for plant uptake. This process provides additional nutrients for plant growth, playing a significant role in soil nutrient cycling and ecosystem stability.\u003c/p\u003e \u003cp\u003eSoil microorganisms play a pivotal role in soil ecosystems, driving nutrient cycling and organic matter degradation; therefore, soil microbes are recognized as key indicators of soil health\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. The results of this study demonstrate that the return of mushroom residue to the field significantly influences the diversity and community structure of soil bacteria and fungi. Specifically, mushroom residue application notably enhanced bacterial diversity and richness while reducing fungal diversity and richness, consistent with the findings of Su et al.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e In addition, Six et al.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e suggested that bacteria, compared to fungi, preferentially utilize readily decomposable resources. The addition of mushroom residue provides heterotrophic bacteria with exogenous organic materials and nutrients, promoting their proliferation, reducing intercommunity competition, and thereby enhancing bacterial diversity in paddy soils\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Although the return of \u003cem\u003eA. auricula\u003c/em\u003e residue to the field impacted fungal diversity and richness, it significantly increased the relative abundance of \u003cem\u003eSarocladium\u003c/em\u003e while reducing the relative abundances of \u003cem\u003eTrichoderma\u003c/em\u003e, \u003cem\u003eMortierella\u003c/em\u003e, and \u003cem\u003eFusicolla\u003c/em\u003e, thereby modulating the fungal community structure and composition.\u003c/p\u003e \u003cp\u003eFurthermore, the composition of bacterial and fungal communities varied among treatments\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. In this study, the return of \u003cem\u003eA. auricula\u003c/em\u003e residue to the field increased the relative abundances of beneficial bacterial phyla such as Acidobacteriota, Proteobacteria, Chloroflexi, Myxococcota, and Desulfobacterota. Proteobacteria play a crucial role in soil carbon, nitrogen, and sulfur cycling and exhibit strong resistance to heavily polluted soils\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, particularly under heavy metal stress\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Acidobacteriota can degrade cellulose\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e and participate in iron cycling and other ecosystem functions\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Chloroflexi generates energy through photosynthesis and facilitates carbon transformation and utilization under low nutrient availability\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. With mushroom residue addition, the relative abundances of Myxococcota and Desulfobacterota significantly increased, accelerating organic matter decomposition\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, providing more nutrients for plant growth, and preventing sulfide accumulation that could harm plants. Chen et al.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e also found that organic waste application significantly increased the relative abundances of Proteobacteria and Acidobacteriota, which was directly linked to the input of carbon and nitrogen sources from the residue. These microorganisms play key roles in carbon and nitrogen cycling, further validating the promotion of functional microbes by mushroom residue return to the field.\u003c/p\u003e \u003cp\u003eCompared to those found in the K0 treatment, the F1 and F2 treatments exhibited higher relative abundances of the genera \u003cem\u003eAnaeromyxobacter\u003c/em\u003e, \u003cem\u003eHaliangium\u003c/em\u003e, \u003cem\u003eCandidatus_Koribacter\u003c/em\u003e, and \u003cem\u003eRhodanobacter\u003c/em\u003e. \u003cem\u003eAnaeromyxobacter\u003c/em\u003e, an anaerobic bacterium, participates in the redox processes of multivalent heavy metals, stabilizes radioactive elements, and metabolizes organic halides\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eHaliangium\u003c/em\u003e enhances soil carbon cycling, accelerates organic matter decomposition, and provides nutrients for plants\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eCandidatus_Koribacter\u003c/em\u003e is essential for degrading various organic compounds, including cellulose, hemicellulose, starch, and chitin\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eRhodanobacter\u003c/em\u003e promotes nitrification pathways and utilizes Fe\u0026sup2;⁺ for denitrification\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Li et al.\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e reported that return of organic material to the field can suppress the proliferation of pathogenic fungi (e.g., \u003cem\u003eFusarium\u003c/em\u003e) while promoting the colonization of beneficial fungi (e.g., \u003cem\u003eTrichoderma\u003c/em\u003e) by regulating the soil pH and carbon-to-nitrogen ratio, consistent with the observed trends in fungal community structure in this study. The increase in beneficial microorganisms in the soil can promote plant growth and enhance yield\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Therefore, the diverse microbial community formed by the return of \u003cem\u003eA. auricula\u003c/em\u003e residue to the field contributes to soil improvement and the maintenance of high soil functionality.\u003c/p\u003e\n\u003ch3\u003eInfluence of the soil environment on microbial communities\u003c/h3\u003e\n\u003cp\u003eSoil nutrients, enzyme activities, and microbial communities are key factors maintaining the rhizosphere microenvironment, interacting with and constraining one another. RDA revealed that QK, β-GC, and urease were the primary drivers of the shifts observed in bacterial and fungal community structures after the return of mushroom residue to the field. In a life-cycle assessment, Dorr et al.\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e highlighted that the return of mushroom residue to the field reduces the risk of nitrogen leaching by regulating microbial functions, further validating the positive correlation between AN and bacterial abundance observed in this study. Spearman correlation heatmaps indicated that after the return of \u003cem\u003eA. auricula\u003c/em\u003e residue to the field, bacterial genera such as \u003cem\u003eGeothrix, Novosphingobium\u003c/em\u003e, \u003cem\u003ePromicromonospora, Anaeromyxobacter, Haliangium\u003c/em\u003e, and \u003cem\u003eSideroxydans\u003c/em\u003e significantly and positively influenced soil properties, including QP, TP, pH, QK, AN, TK, and organic matter, as well as the activities of soil enzymes such as urease, sucrase, β-GC, and NAG.\u003c/p\u003e \u003cp\u003eA systematic review\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e emphasized the central role of soil enzymes in carbon, nitrogen, and phosphorus cycling, noting that β-GC and urease activities are significantly positively correlated with organic matter decomposition rates, providing a theoretical basis for the observed increase in enzyme activities observed in this study. Soil organic matter supplies ample substrates for enzymes such as urease and sucrase, thereby enhancing their activities. Urease catalyzes urea hydrolysis to produce ammonia and carbon dioxide, providing nitrogen and carbon sources for \u003cem\u003eGeothrix\u003c/em\u003e. During metabolism, \u003cem\u003eGeothrix\u003c/em\u003e generates organic acids, whose carboxyl and hydroxyl groups bind with calcium ions in calcium phosphate, forming soluble complexes that promote phosphate dissolution and increase soil-available phosphorus. Sucrase catalyzes the hydrolysis of sucrose into glucose and fructose; therefore, the enhanced activity of sucrase increases available carbon sources in the soil, providing more energy for bacterial growth. In a paddy field experiment, Huang et al.\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e found that increased sucrase activity is closely linked to higher soil-available phosphorus, indicating that enzyme activities improve phosphorus availability by promoting organic phosphorus mineralization, corroborating the findings of the present study. Bacteria such as \u003cem\u003eGeothrix\u003c/em\u003e and \u003cem\u003eNovosphingobium\u003c/em\u003e utilize nutrients released by the activities of these enzymes for their growth and metabolism. Additionally, the return of mushroom residue to the field effectively reduced the abundance of pathogenic fungi such as \u003cem\u003eCeratocystis\u003c/em\u003e and \u003cem\u003eFusarium\u003c/em\u003e. In summary, the return of \u003cem\u003eA. auricula\u003c/em\u003e residue to the field can foster sustainable and healthy soil ecosystems through interactions between microorganisms and soil physicochemical properties.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eUnder the rice\u0026ndash;\u003cem\u003eA. auricula\u003c/em\u003e rotation mode, the return of mushroom residue to the field can effectively increase the soil nutrient content, soil enzyme activity, and the diversity and abundance of soil microorganisms. High-throughput sequencing technology showed that the return of mushroom residue to the field could significantly increase the Chao1 and Shannon diversity indices of soil bacteria and decrease those of fungi. The dominant bacterial phyla were Acidobacteria and Proteobacteria, while Ascomycota and Basidiomycota were identified as the major fungal taxa, with soil organic matter, QK, β-GC, and urease activity being the main factors affecting the composition of soil bacterial and fungal communities in the farmland under rice\u0026ndash;\u003cem\u003eA. auricula\u003c/em\u003e rotation. In conclusion, the return of \u003cem\u003eA. auricula\u003c/em\u003e residue to the field has significant potential for achieving soil improvement under the rice\u0026ndash;\u003cem\u003eA. auricula\u003c/em\u003e rotation pattern, which is important for improving soil quality and agricultural production.\u003c/p\u003e"},{"header":"Methods","content":" \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eExperimental site\u003c/h2\u003e \u003cp\u003eThe experiment was carried out at the Hecheng Edible Fungi Cooperative, located in Tonglu County, Hangzhou, Zhejiang Province, China. In mid-October 2022, after the rice harvest, \u003cem\u003eA. auricula\u003c/em\u003e cultivation was initiated using sawdust and bran as the primary substrates, with each bag weighing approximately 1.6\u0026ndash;1.7 kg. Approximately 8,000 or 10,000 bags were deployed per mu (667 m\u0026sup2;). Harvesting was completed by mid-April 2023, yielding an average dry mushroom residue of 450 g per bag. The mushroom residue was then directly returned to the field after bag removal. Rice (variety Yongyou 1540) was transplanted in early June 2023, with a fertilization rate of 30 kg/667 m\u0026sup2; compound fertilizer and 10 kg/667 m\u0026sup2; urea. Field management followed standard cultivation practices.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eExperimental sampling and design\u003c/h3\u003e\n\u003cp\u003eThe experiment included three treatments: treatment 1 (K0) involved no application of mushroom residue, treatment 2 (F1) involved the application of mushroom residue at 3,600 kg/667 m\u0026sup2; (8,000 bags), and treatment 3 (F2) involved the application of mushroom residue at 4,500 kg/667 m\u0026sup2; (10,000 bags). Each treatment was set up with three replicates and each replicate covered 1 mu (667 m\u0026sup2;). Rice yields were measured in all paddies for each replicate. Rice was threshed and sun-dried (adjusted to a 14% moisture content) to obtain the yield. After the rice harvest, soil samples were collected from the plough layer (0\u0026ndash;20 cm) using an \u0026ldquo;S\u0026rdquo;-shaped five-point sampling method. Five random soil samples were mixed, sieved through a 1-mm mesh, and divided into two portions: one portion was air-dried for soil fertility and enzyme activity analysis and the other was stored at \u0026minus;\u0026thinsp;20\u0026deg;C for soil microbial sequencing.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSoil fertility analysis\u003c/h2\u003e \u003cp\u003eNitrate nitrogen (NN) was determined by the phenol disulfonic acid colorimetric method\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e and ammonium nitrogen (AN) was determined by the potassium chloride leaching\u0026ndash;indophenol blue colorimetric method\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Available potassium (QK) and available phosphorus (QP) were measured using the flame photometry and Olsen-P method, respectively\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. The total nitrogen (TN) was determined by the Kjeldahl method\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, total potassium (TK) was determined by flame photometry\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, total phosphorus (TP) was determined by the molybdenum-antimony anti-colorimetric method\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, and organic matter (OMT) was analyzed using the potassium dichromate oxidation method\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Soil water content (WAT) was determined by the drying method\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Soil pH and electrical conductivity (EC) were measured using a pH meter (Sartorius PB-10, Germany) and a conductivity meter (DDSJ-308F, Leici, China), respectively, after extraction with deionized water (soil:water\u0026thinsp;=\u0026thinsp;1:5)\u003csup\u003e6,51\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSoil enzyme activity analysis\u003c/h2\u003e \u003cp\u003eLeucine aminopeptidase (LAP) activity was quantified using microplate fluorometric analysis\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e and sucrase (SC) activity was assessed using the 3,5-dinitrosalicylic acid colorimetric method\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Nitrate reductase (NR) activity was determined by the sulfanilamide colorimetric method\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Catalase (CAT) activity was measured by potassium permanganate titration\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Soil urease (UE) activity was determined using the sodium phenolate\u0026ndash;sodium hypochlorite colorimetric method\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. β-Glucosidase (β-GC) and acetyl-β-D-glucosaminidase (NAG) activities were analyzed via the nitrophenol colorimetric method\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eDNA extraction, polymerase chain reaction (PCR) amplification, and sequencing of soil samples\u003c/h2\u003e \u003cp\u003eFor each treatment, a 0.5 g soil sample was accurately weighed and genomic DNA was extracted using the TGuide Magnetic Bead Soil DNA Kit (Tiangen Biotech, DP812). Using the extracted DNA as a template, bacterial 16S rRNA genes were amplified at the V4 region with primers 515F (5\u0026prime;-GTGYCAGCMGCCGCGGTAA-3\u0026prime;) and 806R2 (5\u0026prime;-GGACTACNVGGGTWTCTTAAT-3\u0026prime;), while fungal ITS genes were amplified in the ITS1 region with primers ITS1F (5\u0026prime;-CTTGGTCATTTAGAGGAAGTAA-3\u0026prime;) and ITS2 (5\u0026prime;-GCTGCGTTCTTCATCGATGC-3\u0026prime;).\u003c/p\u003e \u003cp\u003eThe PCR system consisted of 50 ng DNA, 0.3 \u0026micro;L each forward and reverse primer (10 \u0026micro;M), 5 \u0026micro;L KOD FX Neo Buffer, 2 \u0026micro;L dNTPs (2 mM each), 0.2 \u0026micro;L KOD FX Neo, and ddH\u003csub\u003e2\u003c/sub\u003eO to a final volume of 10 \u0026micro;L. PCR conditions were as follows: initial denaturation at 95\u0026deg;C for 3 min; 25 cycles of 95\u0026deg;C for 30 s, 50\u0026deg;C for 30 s, and 72\u0026deg;C for 40 s; final extension at 72\u0026deg;C for 5 min; and hold at 4\u0026deg;C. PCR products were purified using VAHTSTM DNA Clean Beads and recovered by 1.8% agarose gel electrophoresis. High-throughput sequencing was performed on the Illumina NovaSeq 6000 platform.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eData processing and statistical analyses\u003c/h2\u003e \u003cp\u003eStatistical analyses were conducted using SPSS 27.0. Differences among treatments in soil fertility indicators and enzyme activities were compared with one-way analysis of variance and Duncan\u0026rsquo;s test (significance level P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Primer sequences were identified and trimmed using cutadapt v2.7.8, low-quality sequences were filtered using Trimmomatic v0.33, and paired-end reads were assembled using FLASH v1.2.11. The spliced data were denoised and chimeric sequences were removed using QIIME2 v2020.6 to obtain final valid data (non-chimeric reads). Alpha diversity indices, including the Chao1, Simpson, Shannon, and ACE indices, were determined by QIIME2 v2020.6. Taxonomic classification of bacterial and fungal communities was achieved by aligning feature sequences to the respective Silva 138 and UNITE reference databases, with dominant species identified at the genus and phylum levels based on relative abundance. Redundancy analysis (RDA) using origin 21.0 and heatmap correlation analysis by the Spearman Rank Correlation Coefficient Algorithm were employed to elucidate the interactions between environmental factors and microbial community structures.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eTN \u0026nbsp;total nitrogen\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTK \u0026nbsp;total phosphorus\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNN \u0026nbsp;nitrate nitrogen\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAN \u0026nbsp;ammonium nitrogen\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eQK \u0026nbsp;available\u0026nbsp;potassium\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eQP \u0026nbsp;available\u0026nbsp;phosphorus\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTP \u0026nbsp;total phosphorus\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOMT \u0026nbsp;organic matter\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWAT \u0026nbsp;water content\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEC \u0026nbsp;electrical conductivity\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLAP \u0026nbsp;leucine aminopeptidase\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSC \u0026nbsp;sucrase\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNR \u0026nbsp;nitrate reductase\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCAT \u0026nbsp;catalase\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eUE \u0026nbsp;urease\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eβ-GC \u0026nbsp;β-Glucosidase\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNAG \u0026nbsp;acetyl-β-D-glucosaminidase\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRDA \u0026nbsp;redundancy analysis\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eW.Y., J.S., and L.M. conceived and designed the experiments; T.Z., B.Y., and Y.X. collected the samples; W.Y., J.L. and Q.Q. analyzed the data and wrote the manuscript; and J.S. and L.M. revised and approved the final version of the paper. W.Y., T.Z., and B.Y. contributed to the work equally and should be regarded as co-first authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProject of Collaborative Extension of Major Agricultural Technologies of Zhejiang (No. \u0026nbsp;2021XTTGSYJ02-1) and the China Agriculture Research System of MOF and MARA (No. CARS-20).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data of this study are included in the article or the Supplementary Materials. The raw sequence data of microorganisms reported in this paper have been deposited in the Genome Sequence Archive (GSA) database under accession numbers CRA******. All the data have been submitted. The application is currently undergoing review and the number allocation is pending. The Project number is PRJCA040766, the biological Biosample number is subSAM142064, and the GSA application number is subCRA042610. The details will be added to the manuscript promptly after the number is assigned.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrespondence\u003c/strong\u003e and requests for materials should be addressed to L.M. or J.S.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eQiao, Y. et al. Comprehensive evaluation on effect of planting and breeding waste composts on the yield, nutrient utilization, and soil environment of baby cabbage. \u003cem\u003eJ. Environ. Manage.\u003c/em\u003e \u003cb\u003e341\u003c/b\u003e, 117941 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu, L. et al. Fungi residue returning: effects on soil physicochemical characters, microorganisms and enzyme activity in vegetable field. \u003cem\u003eChin. Agric. Sci. Bull.\u003c/em\u003e \u003cb\u003e36\u003c/b\u003e (1), 98\u0026ndash;104 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, L. \u0026amp; Sun, X. Changes in physical, chemical, and microbiological properties during the two-stage co-composting of green waste with spent mushroom compost and biochar. \u003cem\u003eBioresource Technol.\u003c/em\u003e \u003cb\u003e171\u003c/b\u003e, 274\u0026ndash;284 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMajchrowska-Safaryan, A., Pakuła, K. \u0026amp; Becher, M. The influence of spent mushroom substrate fertilization on the selected properties of arable soil. \u003cem\u003eEnviron. Prot. Nat. Resour.\u003c/em\u003e \u003cb\u003e31\u003c/b\u003e, 28\u0026ndash;34 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXiao, W. et al. Effect of polysaccharides of nine edible mushroom seed bodies on the growth of rice seedlings. \u003cem\u003eActa Edulis Fungi\u003c/em\u003e. \u003cb\u003e27\u003c/b\u003e (1), 69\u0026ndash;74 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, L. et al. Short-term responses of soil nutrients, heavy metals and microbial community to partial substitution of chemical fertilizer with spent mushroom substrates (SMS). \u003cem\u003eSci. Total Environ.\u003c/em\u003e \u003cb\u003e844\u003c/b\u003e, 157064 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXiao, J. et al. Meta-analysis of biochar application effects on soil fertility and yieldsof fruit and vegetables in greenhouse. \u003cem\u003eJ. Plant. Nutr. Fertilizers\u003c/em\u003e. \u003cb\u003e24\u003c/b\u003e (1), 228\u0026ndash;236 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, X. et al. Effects of fertilizer with gypsum application on growing development and nutrient uptake of potting corn in coastal saline soil. \u003cem\u003eJ. Anhui Sci. Technol. Univ.\u003c/em\u003e \u003cb\u003e25\u003c/b\u003e (6), 23\u0026ndash;28 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, N. et al. Response of soil nutrients and microbial communities to increased application of mushroom dreg in albic soil. \u003cem\u003eChin. Agric. Sci. Bull.\u003c/em\u003e \u003cb\u003e41\u003c/b\u003e (2), 49\u0026ndash;55 (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang, Q., Liu, W., Huang, H., Peng, Z. \u0026amp; Deng, L. Responses of crop yield, soil fertility, and heavy metals to spent mushroom residues application. \u003cem\u003ePlants\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e (5), 663 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, H. et al. Effects of combined application of chemical fertilizers with different mushroom residues on soil fertility and enzymes activities. \u003cem\u003eJ. Soil. Water Conserv.\u003c/em\u003e \u003cb\u003e36\u003c/b\u003e (6), 364\u0026ndash;370 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, Y., Zhang, L., Fang, S., Tian, Y. \u0026amp; Guo, J. Variation of soil enzyme activity and microbial biomass in poplar plantations of different genotypes and stem spacings. \u003cem\u003eJ. Forestry Res.\u003c/em\u003e \u003cb\u003e29\u003c/b\u003e (4), 963\u0026ndash;972 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, F. et al. Effect of mushroom residue on soil property and crop and research progress in its recycling. \u003cem\u003eJ. Agr Sci. Tech.\u003c/em\u003e \u003cb\u003e17\u003c/b\u003e (3), 100\u0026ndash;106 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, Q. et al. Long-term fertilization changes bacterial diversity and bacterial communities in the maize rhizosphere of Chinese Mollisols. \u003cem\u003eAppl. Soil. Ecol.\u003c/em\u003e \u003cb\u003e125\u003c/b\u003e, 88\u0026ndash;96 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeng, X. et al. Effects of returning \u003cem\u003eStropharia rugosoannulata\u003c/em\u003e cultivation residue to paddy fields on rice growth and soil environment. \u003cem\u003eActa Agriculturae Universitatis Jiangxiensis\u003c/em\u003e. \u003cb\u003e45\u003c/b\u003e (2), 349\u0026ndash;360 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu, Y., Ma, J., Ye, Z., Di, C. \u0026amp; Wang, X. Effects of continous application of edibe fungus residue on soil fertility preperties under rice-edible fungus rotation system. \u003cem\u003eJ. Soil. Water Conserv.\u003c/em\u003e \u003cb\u003e27\u003c/b\u003e (6), 172\u0026ndash;176 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHao, H. et al. Effects of the rice-mushroom rotation pattern on soil properties and microbial community succession in paddy fields. \u003cem\u003eFront. Microbiol.\u003c/em\u003e \u003cb\u003e15\u003c/b\u003e, 1449922 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, Y. et al. Organic amendments shift the phosphorus-correlated microbial co-occurrence pattern in the peanut rhizosphere network during long-term fertilization regimes. \u003cem\u003eAppl. Soil. Ecol.\u003c/em\u003e \u003cb\u003e124\u003c/b\u003e, 229\u0026ndash;239 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFrąc, M. et al. Mycobiome composition and diversity under the long-term application of spent mushroom substrate and chicken manure. \u003cem\u003eAdv. Agron.\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e (3), 410 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNannipieri, P., Trasar-Cepeda, C. \u0026amp; Dick, R. P. Soil enzyme activity: a brief history and biochemistry as a basis for appropriate interpretations and meta-analysis. \u003cem\u003eBiol. Fert Soils\u003c/em\u003e. \u003cb\u003e54\u003c/b\u003e (1), 11\u0026ndash;19 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEivazi, F. \u0026amp; Tabatabai, M. A. Factors affecting glucosidase and galactosidase activities in soils. \u003cem\u003eSoil Biology Biochemistry\u003c/em\u003e. \u003cb\u003e22\u003c/b\u003e (7), 891\u0026ndash;897 (1990).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu, J. G., Chu, H., Xie, Z. B. \u0026amp; Yagi, K. Effects of lanthanum on nitrification and ammonification in three Chinese soils. \u003cem\u003eNutr. Cycl. Agroecosys\u003c/em\u003e. \u003cb\u003e63\u003c/b\u003e, 309\u0026ndash;314 (2002).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBurns, R. G. et al. Soil enzymes in a changing environment: current knowledge and future directions. \u003cem\u003eSoil Biology Biochemistry\u003c/em\u003e. \u003cb\u003e58\u003c/b\u003e, 216\u0026ndash;234 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBorase, D. N. et al. Long-term impact of diversified crop rotations and nutrient management practices on soil microbial functions and soil enzymes activity. \u003cem\u003eEcol. Indic.\u003c/em\u003e \u003cb\u003e114\u003c/b\u003e, 106322 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, W. et al. Effects of adding vermicompost on soil fertility and enzyme activity of acidic paddy soil. \u003cem\u003eJ. Nuclear Agricultural Sci.\u003c/em\u003e \u003cb\u003e39\u003c/b\u003e (3), 615\u0026ndash;623 (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao, J. et al. Manipulation of the rhizosphere microbial community through application of a new bio-organic fertilizer improves watermelon quality and health. \u003cem\u003ePLoS One\u003c/em\u003e. \u003cb\u003e13\u003c/b\u003e (2), e0192967 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSu, Y. et al. Linking soil microbial community dynamics to straw-carbon distribution in soil organic carbon. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e (1), 5526 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSix, J., Frey, S. D., Thiet, R. K. \u0026amp; Batten, K. M. Bacterial and fungal contributions to carbon sequestration in agroecosystems. \u003cem\u003eSoil Sci. Soc. Am. J.\u003c/em\u003e \u003cb\u003e70\u003c/b\u003e, 555\u0026ndash;569 (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSubhashini, D. Growth promotion and increased potassium uptake of tobacco by potassium-mobilizing bacterium \u003cem\u003eFrateuria aurantia\u003c/em\u003e grown at different potassium levels in vertisols. \u003cem\u003eCommun. Soil. Sci. Plan.\u003c/em\u003e \u003cb\u003e46\u003c/b\u003e, 210\u0026ndash;220 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrenzinger, K. et al. Steering microbiomes by organic amendments towards climate-smart agricultural soils. \u003cem\u003eBio Fert Soils\u003c/em\u003e. \u003cb\u003e57\u003c/b\u003e (8), 1053\u0026ndash;1074 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRampelotto, P. H., de Siqueira Ferreira, A., Barboza, A. D. \u0026amp; Roesch, L. F. Changes in diversity, abundance, and structure of soil bacterial communities in Brazilian Savanna under different land use systems. \u003cem\u003eMicrob. Ecol.\u003c/em\u003e \u003cb\u003e66\u003c/b\u003e (3), 593\u0026ndash;607 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, X. et al. Changes in the composition and diversity of microbial communities during anaerobic nitrate reduction and Fe(II) oxidation at circumneutral pH in paddy soil. \u003cem\u003eSoil Biology Biochemistry\u003c/em\u003e. \u003cb\u003e94\u003c/b\u003e, 70\u0026ndash;79 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee, S. H., Ka, J. O. \u0026amp; Cho, J. C. Members of the phylum Acidobacteria are dominant and metabolically active in rhizosphere soil. \u003cem\u003eFEMS Microbiol. Lett.\u003c/em\u003e \u003cb\u003e285\u003c/b\u003e (2), 263\u0026ndash;269 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, Y. et al. Comparison of the levels of bacterial diversity in freshwater, intertidal wetland, and marine sediments by using millions of illumina tags. \u003cem\u003eAppl. Environ. Microb.\u003c/em\u003e \u003cb\u003e78\u003c/b\u003e (23), 8264\u0026ndash;8271 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFullerton, H. \u0026amp; Moyer, C. L. Comparative single-cell genomics of \u003cem\u003eChloroflexi\u003c/em\u003e from the Okinawa trough deep-subsurface biosphere. \u003cem\u003eAppl. Environ. Microb.\u003c/em\u003e \u003cb\u003e82\u003c/b\u003e (10), 3000\u0026ndash;3008 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuan, Y. et al. Effects of continuous straw returning on soil functional microorganisms and microbial communities. \u003cem\u003eJ. Microbiol.\u003c/em\u003e \u003cb\u003e61\u003c/b\u003e (1), 49\u0026ndash;62 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePrakash, O. et al. \u003cem\u003eGeobacter daltonii\u003c/em\u003e sp. nov., an Fe(III)- and uranium(VI)-reducing bacterium isolated from a shallow subsurface exposed to mixed heavy metal and hydrocarbon contamination. \u003cem\u003eInternatonal J. Syst. Evolutionary Microbiol.\u003c/em\u003e \u003cb\u003e60\u003c/b\u003e (3), 546\u0026ndash;553 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDai, W. et al. Phylogenetic diversity of stochasticity-dominated predatory myxobacterial community drives multi-nutrient cycling in typical farmland soils. \u003cem\u003eSci. Total Environ.\u003c/em\u003e \u003cb\u003e871\u003c/b\u003e, 161680 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, X. et al. Responses of soil nutrients and microbial communities to intercropping medicinal plants in moso bamboo plantations in subtropical China. \u003cem\u003eEnviron. Sci. Pollut R\u003c/em\u003e. \u003cb\u003e27\u003c/b\u003e (2), 2301\u0026ndash;2310 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCao, X. et al. Effects of iron-based substrate on coupling of nitrification, aerobic denitrification and Fe(II) autotrophic denitrification in tidal flow constructed wetlands. \u003cem\u003eBioresource Technol.\u003c/em\u003e \u003cb\u003e361\u003c/b\u003e, 127657 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, J., Yang, Y., Wen, J., Mo, F. \u0026amp; Liu, Y. Continuous manure application strengthens the associations between soil microbial function and crop production: Evidence from a 7-year multisite field experiment on the Guanzhong Plain. Agriculture, \u003cem\u003eEcosystems\u003c/em\u003e \u0026amp; \u003cem\u003eEnvironment\u003c/em\u003e 338, 108082 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGarbeva, P., van Veen, J. A. \u0026amp; van Elsas, J. D. Microbial diversity in soil: selection microbial populations by plant and soil type and implications for disease suppressiveness. \u003cem\u003eAnnu. Rev. Phytopathol.\u003c/em\u003e \u003cb\u003e42\u003c/b\u003e, 243\u0026ndash;270 (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDorr, E., Koegler, M., Gabrielle, B. \u0026amp; Aubry, C. Life cycle assessment of a circular, urban mushroom farm. \u003cem\u003eJ. Clean. Prod.\u003c/em\u003e \u003cb\u003e288\u003c/b\u003e, 125668 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang, M. et al. Effects of different tillage measures during summer fallow seasonontents and enzyme activities in dryland wheat field. \u003cem\u003eSoil. Fertilizer Sci. China\u003c/em\u003e. \u003cb\u003e8\u003c/b\u003e, 30\u0026ndash;42 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNorman, R. J. \u0026amp; Stucki, J. W. The determination of nitrate and nitrite in soil extracts by ultraviolet spectrophotometry. \u003cem\u003eSoil Sci. Soc. Am. J.\u003c/em\u003e \u003cb\u003e45\u003c/b\u003e (2), 347\u0026ndash;353 (1981).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRhine, E. D., Mulvaney, R. L., Pratt, E. J. \u0026amp; Sims, G. K. Improving the Berthelot reaction for determining ammonium in soil extracts and water. \u003cem\u003eSoil Sci. Soc. Am. J.\u003c/em\u003e \u003cb\u003e62\u003c/b\u003e, 473\u0026ndash;480 (1998).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYing, B., Xiao, S., Xiong, K., Cheng, Q. \u0026amp; Luo, J. Comparative studies of the distribution characteristics of rocky desertification and land use/land cover classes in typical areas of Guizhou province, China. \u003cem\u003eEnviron. Earth Sci.\u003c/em\u003e \u003cb\u003e71\u003c/b\u003e (2), 631\u0026ndash;645 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVance, E. D., Brookes, P. C. \u0026amp; Jenkinson, D. S. An extraction method for measuring soil microbial biomass C. \u003cem\u003eSoil Biology Biochemistry\u003c/em\u003e. \u003cb\u003e19\u003c/b\u003e (6), 703\u0026ndash;707 (1987).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCambardella, C. A. \u0026amp; Elliott, E. T. Particulate soil organic-matter changes across a grassland cultivation sequence. \u003cem\u003eSoil Sci. Soc. Am. J.\u003c/em\u003e \u003cb\u003e56\u003c/b\u003e, 777\u0026ndash;783 (1992).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDai, L. et al. Organic matter regulates ammonia-oxidizing bacterial and archaeal communities in the surface sediments of \u003cem\u003eCtenopharyngodon idellus\u003c/em\u003e aquaculture ponds. \u003cem\u003eFront. Microbiol.\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e, 2290 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, Q. et al. Effects of different irrigation levels on the yield and quality of the \u003cem\u003eCyperus esculentus\u003c/em\u003e L. at tuber stage. \u003cem\u003eActa Agrestia Sinica\u003c/em\u003e. \u003cb\u003e32\u003c/b\u003e (11), 3636\u0026ndash;3645 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDu, Y., Bao, Y., Liu, X. \u0026amp; Zhang, X. Effects of tartary buckwheat rotation on enzyme activities and microorganisms in rhizosphere soil of cultivated potato in Yunnan Province. \u003cem\u003eJ. Agr Sci. Tech.\u003c/em\u003e \u003cb\u003e26\u003c/b\u003e (5), 192\u0026ndash;200 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, L. et al. Effects of bio-organic fertilizer with microbial inoculants on the growth, physiological characteristics, yield and quality of garlic seedlings. \u003cem\u003eJ. Gansu Agricultural Univ.\u003c/em\u003e \u003cb\u003e58\u003c/b\u003e (5), 63\u0026ndash;70 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, D. et al. Effects of crop rotation on the growth, quality and soil environment of \u003cem\u003eEuryale ferox\u003c/em\u003e. \u003cem\u003eJ. Chin. Med. Mater.\u003c/em\u003e \u003cb\u003e47\u003c/b\u003e (1), 12\u0026ndash;21 (2024).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Mushroom residue, Rice–Auricularia auricula rotation, Soil fertility, Enzyme activity, Microbial diversity","lastPublishedDoi":"10.21203/rs.3.rs-6661042/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6661042/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe return of mushroom residue to the field is an effective measure to improve soil fertility and maintain agroecosystem productivity. We investigated the effects of returning \u003cem\u003eAuricularia auricula\u003c/em\u003e residue to the field on the soil nutrients, enzyme activities, and microbial communities in a rice\u0026ndash;\u003cem\u003eA. auricula\u003c/em\u003e rotation farmland. The trial was designed with three treatments: K0 (no mushroom residue), F1 (3600 kg/667 m\u0026sup2; residue), and F2 (4500 kg/667 m\u0026sup2; residue). The return of \u003cem\u003eA. auricula\u003c/em\u003e residue to the field significantly increased the soil nutrient content. The contents of ammonium nitrogen, total nitrogen, total potassium, total phosphorus, available potassium (QK), available phosphorus, and organic matter in the F2 treatment increased by 180.49%, 70.41%, 16.3%, 54.35%, 137.33%, 38.84%, and 59.29%, respectively, compared to those of the K0 treatment. The activities of the soil enzymes urease, sucrase, β-glucosidase (β-GC), and acetyl-β-\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ed\u003c/span\u003e-glucosidase significantly increased by 32.98%, 407.78%, 206.85%, and 186.26%, respectively, in the F2 treatment compared to those of K0; catalase and leucine aminopeptidase activities were increased by 244.42% and 130.90% in the F1 treatment compared to those of K0. Moreover, \u003cem\u003eA. auricula\u003c/em\u003e residue return to the field increased the Chao1 and Shannon indices of the bacterial community, but decreased the diversity of the fungal community. Redundancy analysis showed that QK, β-GC, and urease were the main factors driving the changes in microbial communities. In conclusion, returning \u003cem\u003eA. auricula\u003c/em\u003e mushroom residue to the field can enhance soil ecological functions by improving soil nutrients, enzyme activities, and microbial community structure.\u003c/p\u003e","manuscriptTitle":"Effects of Auricularia auricula residue on soil physicochemical properties, microbial community composition and diversity, and rice yield","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-03 20:02:45","doi":"10.21203/rs.3.rs-6661042/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c8946e2f-f894-4130-b0c3-b97214bf90ba","owner":[],"postedDate":"June 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":49330169,"name":"Earth and environmental sciences/Environmental sciences"},{"id":49330170,"name":"Earth and environmental sciences/Environmental sciences/Environmental impact"}],"tags":[],"updatedAt":"2025-08-29T11:53:52+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-03 20:02:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6661042","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6661042","identity":"rs-6661042","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}