Effects of Hypsizygus marmoreus spent substrate on prevention and control potential of continuous cropping obstacle in Dictyophora indusiata cultivation

Effects of Hypsizygus marmoreus spent substrate on prevention and control potential of continuous cropping obstacle in Dictyophora indusiata cultivation | 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 Research Article Effects of Hypsizygus marmoreus spent substrate on prevention and control potential of continuous cropping obstacle in Dictyophora indusiata cultivation Xiao Yue Di, Yinghao Sun, Fengju Jiang, Xianai Huang, Jiale Feng, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8761948/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Background Continuous cropping poses a significant challenge to the sustainable cultivation of Dictyphora indusiata , often leading to soil degradation, microbial imbalance, and the accumulation of soil-borne pathogens, which severely compromise yield and quality. Conventional mitigation strategies, raise environmental and safety concerns. This two-year study investigated the efficacy of using fermented Hypsizygus marmoreus (seafood mushroom) spent substrate (SMS) as a sustainable, biocontrol-based approach to remediate soil, suppress pathogen communities, and alleviate the continuous cropping obstacles affecting D. indusiata. Results By varying SMS application timing, we assessed it effects on yield, nutritional quality, soil physicochemical properties, enzyme activities, and microbial community diversity. Applying SMS three months before planting (S3-24) stabilized soil pH in the second year. Soil available phosphorus increased by 87.34%, total nitrogen by 14.14%, and organic matter declined less (14.14% decrease vs. 29.95% in control). Soil microbial diversity increased within 1-2 months, with phosphate-solubilizing bacteria reaching 7.52%. Soil phenolic acids increased, especially p -coumaric acid (up 179.14% in S3-24). In vitro tests showed that at 250 mg/L phenolic acids, both D. indusiata mycelium and pathogens grew faster, but pathogens had a stronger growth advantage. Fruiting body weight peaked at 19.61 g per fruit in S3-24, and fresh yield reached 12,758.4 kg/hm², representing a 745.07% higher than control. Crude polysaccharide and protein increased by 32.42% and 1.60%. Phenolic acid also increased mycelial diameter to 2.4-4.8 μm, improving mycelium structure and supporting higher yield. Moderate phenolic acid levels benefit both D. indusiata and pathogens, but pathogens competitiveness may drive cropping obstacles. PLS-PM showed that S3-24 strengthened key pathways-from soil properties to enzyme activity (path coefficient: 16.29) and fungal communities (4.77)-and the enzyme-phenolic acid-microbe cascade effect (19.58), improving coordination and stability in the soil microecological network (GoF=0.78) and alleviating continuous cropping obstacles. Conclusions Appropriate SMS use mitigated these challenges, stabilizes yield, and improves quality. This approach recycled agricultural waste and offers practical solutions for future D. indusiata continuous cultivation. Dictyophora indusiata Seafood mushroom spent substrate Phenolic acid Soil environment Sustainable development Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Dictyphora indusiata (commonly known as bamboo mushroom), a saprophytic fungal belonging to the family Phallaceae within the phylum Basidiomycota, is predominantly distributed in subtropical regions of China, particularly in the southwestern provinces. This species processes significant nutritional and pharmacological value owing to its high content of protein, essential amino acids, polysaccharides, and lipids [ 1 , 2 ] Pharmacological studies have demonstrated its bioactive properties, including inhibition of tumor growth and broad-specutrum antimicrobial activity [ 3 , 4 ]. However, the rapid expansion of D. indusiata cultivation has led to sever continuous cropping obstacles, characterized by yield instability, pathogen proliferation, quality decline, and soil microbiome dysbiosis [ 5 ]. Continuous cropping obstacle, defined as the progressive decline in crop productivity and quality under repeated monoculture regimes [ 6 ], arise from three primary hazard following as soil physicochemical degradation, allelopathic autotoxicity, and microbial community shifts. Current research frameworks recognized these factors as interdependent divers, where alterations in soil microbial communities exacerbate disruptions in nutrient cycling [ 7 – 9 ]. Yin et al. identified significant autotoxicity in Morchella cultivation soils [ 10 ], where phenolic acid extracts exhibited concentration-dependent inhibition of mycelial growth. In contrast, Ji et al. documented progressive rhizosphere degradation in Ganoderma lucidum monoculture systems [ 11 ], characterized by deterioration of the soil environment. Most research focuses on implementing targeted agronomic interventions to mitigate continuous cropping obstacles, with emerging strategies demonstrating multifaceted efficacy. Intercropping tea and oyster mushrooms ( Pleuroutus ostreatus ) significantly improved soil physicochemical properties [ 12 ], while Huang et al. demonstrated that Imazalil application effectively suppressed pathogenic bacterial proliferation in Ganoderma lucidum cultivation by disrupting biosynthesis pathway [ 13 ]. Additionally, pre-cultivation dazomet fumigation in Morchella systems reconfigured rhizosphere microbiomes by reducing pathogenic fungi and enhancing beneficial bacteria, ultimately improving yield under continuous cropping yield [ 14 ]. China dominates global edible mushroom production, accounting for 76.8% of total output. As a result, over 1.2×10 8 tons of spent mushroom substrate are generated annually, primarily composed of lignocellulosic biomass, residual proteins, and bioactive compounds [ 15 ]. Conventional disposal methods such as landfilling and incineration contribute to environmental pollution through greenhouse gas emissions. Recent studies have identified multiple valorization pathways for spent mushroom substrates, including secondary fungal cultivation, conversion into organic fertilizers, use as ruminant feed supplements, and bioenergy production via anaerobic digestion [ 16 – 18 ]. This study explored the application of Hypsizygus marmoreus (seafood mushroom) spent substrate (SMS) to alleviate continuous cropping obstacles in D. indusiata cultivation through controlled filed trials. We systematically evaluated four key aspects: (1) the temporal dynamics of SMS application on phenolic acid degradation rates in D. indusiata rhizosphere soil over a 0–3 months period; (2) treatment effects on fruiting body yield and nutritional quality; (3) recovery of soil physicochemical parameter under continuous cropping systems; and (4) modulation of rhizosphere microbial diversity following SMS amendments. These results finding offer both mechanistic insights and practical strategies to support sustainable D. indusiata cultivation. Materials and Methods Experimental design and sample collection The field experiment was conducted at the Shunchang Juncao Technology Base Yard (26°45’7’’ N, 117°48’20’’ E), situated in a mid-subtropical marine monsoon climate zone characterized by a mean annual temperature of 18.5 ℃, annual precipitation of 1,756 mm, and annual sunshine duration of 1740.7 h. A 266 m 2 experimental plot (19 × 14 m) was divided into four equal treatment zones. The D. indusiata strain (GenBank accession number: AF324167.2) was provided by the National Engineering Research Center of Juncao Technology at Fujian Agricultural and Forestry University, while SMS sourced from Fujian Shunchang Xinjundu Mushroom Industry Development Co., Ltd. Each treatment zone, representing a one-year continuous D. indusiata cropping system, was arranged in triplicate mushroom beds (0.8 m wide × 0.5 m high), separated by 0.6 m drainage channels. Soil volumetric water content was maintained at 55%-60% using automated irrigation systems (Fig. 1). In traditional D.indusiata cultivation, post-harvest fungal residue is not removed or recycled but is instead directly incorporated into the soil prior to the following year's planting, enabling continuous cultivation, production and experimentation trials of D.indusiata or other crops. Based on varying application times, each test-initiated composting of seafood mushroom residue for 45 days before the experiment commenced, following the fermentation protocol described by Li et al. [19]. treatments are no SMS application (S0-24), application 1 month prior (S1-24), 2 months prior (S2-24) and 3 months prior (S3-24). The soil sampled before cultivation (S0-23) served as the baseline control. Amendments were applied uniformly at a rate of 1 ton per plot and mechanical mixed into the topsoil (20 cm depth) using a rotary tiller. Following established D. indusiata cultivation protocols [20], the substrate was prepared through controlled fermentation initiated on 30 th December 2023, consisting 49% Cenchrus fungigraminus , 49% sawdust, 1% urea, and 1% light calcium carbonate. The thermophilic phase (core temperature > 65 ℃) was maintained via three turning at 10-day intervals. Fermentation was deemed complete upon achieving stable mesophilic conditions (35 °C sustained for one week) and complete volatilization of ammonia [19]. Field inoculation of D. indusiata was carried out on 25 th February 2024, with fruiting body development and management occurring from June to July 2024. Georeferenced soil samples (5 per plot) were collected monthly throughout the growth cycle, starting from primordia initiation to final harvest. Surface soil (0-20 cm depth) was aseptically collected using stainless steel augers, immediately placed in sterile bags, and transported on dry ice to the laboratory. Post-collection processing included: (1) homogenization of composite samples, followed by dark-air-drying at 15 ± 2 °C and sieving through a 2-mm mesh for physicochemical analysis and enzymatic assays (urease, dehydrogenase); and (2) cryopreservation of microbial subsamples at -80 °C for metagenomic sequencing. To ensure data accurate, D. indusiata fruiting bodies were randomly sampled across all treatment groups. The experimental protocol spanned from 2023 to 2025, encompassing two complete cultivation cycles. Assessment of Yield, Agronomic Characteristic and Nutritional Composition in D. indusiata The biological yield of D. indusiata fruiting bodies over the production cycle was calculated The biological yield of D. indusiata fruiting bodies over the production cycle was calculated based on fresh mushroom output per experimental plot: total yield (kg/hm 2 /day) = daily output (kg) × 150.38 (hm 2 ) (conversion factor for 66.5 m 2 to hm 2 ) × 60 d (harvesting period). Agronomic characterization of D. indusiata basidiocarps was performed using precision instruments: individual fresh weight was measured using an analytical balance, while cap diameter and thickness, as well as stipe dimensions, were determined with digital calipers. Fresh fruiting bodies were dried in a forced-air oven (DHG-9240A, Shanghai Yiheng, China) at 40 °C for 2 h, followed by desiccation at 55 °C for 2 h, and finally stabilized at 40 °C for 24 h. The dried samples were ground under low temperature conditions and stored at low temperature prior to analysis for all target indices. For compositional analysis, the gravimetric combustion method described Jia et al. [21] was employed, involving incineration in muffle furnaces at 550 °C until ash mass reached constant weight. Total soluble sugars and crude polysaccharide were quantified using the phenol-sulfuric acid spectrophotometric method [22], and crude protein content was determined via the Kjeldahl method based on nitrogen content, using an automated distillation system (SKD-1800, Shanghai Peiou, China). Physical and Chemical Properties of Soil for D. indusiata Cultivation Total nitrogen (TN) content was determined using the digestion method [23]. Detection of ammonium nitrogen according to the indoxyl blue colorimetric method [24] Available phosphorus was extracted with Mehlich 3 solution and quantified colorimetrically at 880 nm following the Murphy and Riley method, while available potassium was measured by flame photometry at 766.5 nm as described by Knudsen. Organic carbon content was determined using the Walkley-Black wet oxidation procedure [25]. And soil organic matter (SOM) was estimated by multiplying organic carbon content by a conversion factor of 1.724. For pH determination, a standardized soil-to-water ratio of 1:2.5 (w/v) was prepared, followed vigorous stirring at 200 rpm for 30 min and a settling period for 10 min. The supernatant pH was then measured using a calibrated digital pH meter (PB-10, Beijing Sartorius Science, China). Determination of Soil Enzyme Activities in D. indusiata Cultivation Soil enzymatic activities were quantified using standardized colorimetric methods. Sucrase activity (S-SC) was determined by 3,5-dinitrosalicylic acid colorimetry (DNS) according to Li et al. [26]. Catalase activity (S-CAT) was measured using UV spectrophotometry as described by Du et al. [27]. Soil urease activity (S-UE) was assessed via the indophenol blue method [28], with enzyme activity expressed in units of μg NH 3 -N per gram of soil per day (U/g). Acid phosphatase activity (S-ACP) was quantified using the p-nitrophenyl phosphate (PNPP) method [29], and results were reported as μmol p-nitrophenol released per gram of soil per hour (μmol/g/h). Analysis of Soil Phenolic Acid Metabolites in D. indusiata Cultivation Six phenolic acid standards ( p -hydroxybenzoic acid, vanillic acid, ferulic acid, p -coumaric acid, syringic acid, and coumalic acid) were dissolved in 10 mL brown volumetric flasks and diluted with methanol to prepare standard solution with varying concentration gradients. The solutions were analyzed using a Waters high-performance liquid chromatography system (E2695, Waters, Milford, USA) equipped with a UranusC18 column (250 mm × 4.6 mm × 5 μm) and a guard column (20 mm × 4.6 mm × 5 μm). Detection was performed at 280 nm for the identification and quantification of phenolic acid in soil samples. Each analytical run lasted 90 min, with a 10-minute equilibration period between injections [30-31]. Analysis of Soil Microbial Diversity in D. indusiata Cultivation High-throughput sequencing According to the manufacturer 's instructions, TGuide S96 magnetic soil DNA kit (Tiangen, Beijing, China) was used to extract genomic DNA (gDNA) from soil samples S0-23 in October 2023 before planting in the second year and soil samples S3-24, S2-24, S1-24, and S0-24 in June 2024 after planting in the second year. The integrity of gDNA was verified by 1.8% agarose gel electrophoresis, and concentration was determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, USA). The bacterial 16S rRNA gene hypervariable V3-V4 were amplified using primer pairs 338F (5'-ACTCCTACGGGAGGCAGCA-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). For fungal community analysis, the ITS1 region employed primers ITS1F(5'-CTTGGTCATTTAGAGGAAGTAA-3') and ITS2R ITS2(5'-GCTGCGTTCTTCATCGATGC-3'). The Polymerase Chain Reaction (PCR) reactions consisted of 5-50 ng DNA template, 0.3 μmol/L of each primers, 5 μL KOD FX Neo Buffer, 2 μL dNTP (2 mmol/L each), 0.2 μL KOD FX Neopolymerase, and ddH 2 O to a final volume of 20 μL. Thermal cycling conditions included an initial denaturation at 95 °C for 5 min, followed by 20 cycles of 95 °C for 30 s (denaturation), 50 °C for 30 s (annealing), and 72 °C for 40 s (extension), with a final extension at 72 °C for 7 min. Amplified products were purified (Omega Inc., Norcross, GA, USA), quantified using Qsep-400 fragment analyzer (BiOptic, Taiwan, China), and sequenced on the Illumina novaseq6000 platform (Biomarker, Beijing, China). Raw sequencing data were submitted to the National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/) with accession number PRJNA1234400 (publicly accessible on December 31 st , 2026). Bioinformatics Analysis Bioinformatics analysis was conducted on BMK Cloud platform (http://www.biocloud.net/). Raw sequencing data were subjected to quality filtering using Trimmomatic (v0.33) [32]. Primer sequences were removed using Cutadapt [33] (v1.9.1). Paired-end reads were assembled in denovo mode using USEARCH [34] (v10), followed by chimera detection and removal with UCHIME. Final clean reads were clustered into operational taxonomic units (OTUs) at a 97% sequence similarity threshold using USEARCH [35] (v8.1). Taxonomy annotation of the OTUs was performed in QIIME2 (v2021) [36] using a Bayesian classifier aligned against the SILVA database (v138.1) [37] with a minimum confidence threshold of 70%. Alpha diversity indices (including Shannon, Chao1, Observed Species) were calculated in QIIME2 and visualized using phylogeny (v1.34.0) in R software (v4.1.2). The top 20 species with the highest abundance are selected, and the heatmap is generated using R's pheatmap package. Each color block in the heatmap represents the abundance of a genus in a sample. Samples are arranged horizontally, while species are arranged vertically. Clustering in the heatmap allows us to understand the similarity between samples and the similarity in community composition across different taxonomic levels. The species distribution histogram was drawn by python2 (matplotlib-v1.5.1). FUNGuild fungal function prediction analysis was conducted using the Funguild (1.0) software and database, while bacterial phenotypic traits were predicted using Bugbase (0.1.0). Correlation network analysis is conducted based on the abundance and variation of species across samples. Spearman's rank correlation analysis is performed (using the default method) to identify correlations with a magnitude greater than 0.1 and a p-value less than 0.05. These data are used to construct a correlation network. In vitro Co-culture of D. indusiata Mycelium with Soil Pathogens According to Lei et al. [31], p -coumaric acid at a concentration of 250 mg/L can promote the growth of D. indusiata mycelium. This experiment evaluated the growth response of D. indusiata mycelium and major pathogenic microorganisms at this concentration. The top 50 microorganisms by abundance were screened from the continuous cropping soil of D. indusiata , including two fungi ( Aspergillus fumigatus and Penicillium simplicissimum ) and two bacteria ( Bacillus sp. and Acinetobacter baumannii ), which were selected for co-culture assays. After autoclaving, the solid PDA medium was cooled to 60-70 °C, then supplemented with 250 mg/L of p -coumaric acid that had been filter through a 0.22 μm membrane, mixed thoroughly, and poured into Petri dishes. Once the medium had cooled and solidified, mycelial plugs of equal size were inoculated onto the plates using a sterile inoculation tool; for bacteria strains, a small volume of standardized bacterial suspension was used for spot inoculation. The control group consisted of PDA plates without p -coumaric acid, inoculated with the same D. indusiata and pathogenic microorganisms under identical conditions. All treatments were incubated at 26 °C for 21 d with six replicates per treatment, and fungal growth was monitored regularly throughout the incubation period. In addition, single colonies of Aspergillus fumigatus , Penicillium simplicissimum , Bacillus sp. and Acinetobacter baumannii were individually picked from solid PDA medium and inoculated into 100 mL of liquid PDA medium. Cultures were incubation in a shaker at 25 °C and 200 rpm for 3 d. Upon completion of incubation, the bacterial or fungal suspensions were filtered through eight layers of sterile gauze to remove mycelial clumps or cellular aggregates. Subsequently, 2 g agar powder was added to every 100 mL of the filtered suspension, followed by autoclaving at high-temperature to prepare solidified plates. After cooling and solidification, mycelial plugs of D. indusiata were inoculated onto the plates and cultured at 26 °C for 21 d, with fungal growth monitored regularly. Each treatment was performed in six replicates. Fresh mycelium of D. indusiata was collected under different treatment conditions and immediately immersed in 5% glutaraldehyde solution, followed by fixation at 4 °C for 4 h. The samples were then rinsed three times with 0.2 mol/L phosphate buffer, 10-15 min per rinse. Post-fixation was carried out with 1% tart acid at 4 °C for 4 h. After completion of fixation, the samples were rinsed three times with distilled water, each for 10-15 min. Gradient dehydration was performed sequentially using 50%, 70%, 80% and 90% ethanol solutions, with 10-15 min incubation at each concentration; this was followed by two changes of 100% ethanol, each lasting 10-15 min. Subsequently, the samples were treated twice with propylene oxide to facilitate resin infiltration. Finally, the specimens were processed in a freeze-dryer (HCP-2, HITACHI, Japan). Dried samples were mounted on stubs and coated with a thin conductive layer using an ion sputter coater (IB-5, EIKO, Japan), after which they were examined and imaged under a scanning electron microscope (JSM-6380LV, JEOL, Japan). Data Analysis Statistical analysis was conducted using Microsoft Office Excel 2019 (Microsoft Corporation, WA, USA) for data organization, while significance testing was performed using Turkey’s test within a one-way ANOVA (Analysis of Variance) framework in SPSS 26.0 (IBM Corporation, New York, USA). Data visualization was achieved through an integrated pipeline combining Graph Pad Prism 9 (GraphPad Software, CA, USA) and Adobe Photoshop CC 2018 (Adobe, CA, USA) to generate publication-quality figures. R language was used for bacterial and fungal metabolic pathway statistics and partial least squares path modeling (PLS-PM). Result Effects of Different Treatments on Yield and Quality of Continuous D. indusiata Fruiting Bodies According to Table 1, the S3-24 treatment with SMS achieved the highest D. indusiata yield indices , with both single fruit weight and acreage yield following the order S3-24 > S2-24 > S1-24 > S0-24. The acreage yields of S3-24, S2-24 and S1-24 increased by 745.07%, 496.99% and 387.05%, respectively, compared to S0-24 (P < 0.05). However, relative to the previous year’s baseline (S0-23), the acreage yields of S3-24, S2-24, S1-24 and S0-24 decreased by 4.48%, 32.52%, 44.95%, and 88.70%, respectively. Nutrient analysis revealed that S3-24 and S1-24 exhibited elevated total sugar contents (4.11% and 4.84%), while the samples from S3-24, S2-24, and S1-24 demonstrated significant increases in crude polysaccharide (32.42%, 6.99%, and 3.09%) and crude protein (1.60%, 1.60%, and 0.57%) relative to untreated controls. Notably, S3-24 displayed the lowest ash content, indicating higher organic matter and reduced heavy metal accumulation [38]. A three-month continuous treatment effectively alleviated second-year continuous cropping obstacles, significantly enhancing both the yield and quality of D. indusiata . Table 1 Yield and quality variations in D. indusiata fruiting bodies under second-year continuous cropping conditions Treatments First year total yield (kg/hm 2 ) Second year total yield (kg/hm 2 ) Individual Substrate Weight (g) TS (mg/g) CPS (g/100 g) CP (g/100 g) Ash (%) S3-24 13357.50 12758.40 19.61±0.69 a 547.66±4.11 a 11.56±1.09 a 24.81±0.01 a 7.85±0.36 c S2-24 9013.05 17.66±0.94 ab 523.56±1.84 b 9.34±0.29 b 24.81±0.01 a 10.73±0.29 a S1-24 7353.30 18.37±0.42 ab 551.43±6.47 a 9.00±0.03 b 24.56±0.13 b 10.22±0.15 a S0-24 1509.75 17.04±0.55 b 526.05±7.06 b 8.73±0.54 b 24.42±0.09 b 8.64±0.17 b Notes: TS denotes total sugars; CPS denotes crude polysaccharides; and CP denotes crude proteins. Different lowercase letters within the same column indicate significant differences (P < 0.05). This notation applies to all subsequent tables. Soil Physicochemical Dynamics Under Continuous D. indusiata Cropping Systems As a predominant inorganic nitrogen species, soil ammonium nitrogen (NH 4 + -N) governs nitrogen availability and crop productivity. Fig. 2A shown that NH 4 + -N level increased following SMS amendments during the initial phase, with increases of +120.62% (S3-24), 20.86% (S2-24) and 15.65% (S1-24) relative to the original soil (S0-23). Notably, subsequent depletion of NH 4 + -N coincided with D. indusiata growth cycles, resulting in concentrations below baseline levels across all treatments-most markedly in S3-24, which exhibited 41.2% reduction-suggesting rhizosphere feedback mechanisms contribute to continuous cropping constraints. Fig. 2B revealed paradoxical potassium dynamics in soils amended with SMS. Although available potassium increased in June across all treatments compared to the control (S3-24: +120.62%; S2-24: +20.86%; S1-24: +15.65%), D. indusiata cultivation triggered progressive K depletion thereafter, with high-yield groups showing particularly severe losses (S3-24: -41.2% by harvest). This bidirectional nutrient flux indicated initial amendment-mediated K mobilization followed by crop-driven depletion exceeding soil replenishment capacity. Plant-available phosphorus, a critical component of soil fertility, plays essential roles in energy metabolism and root development. As shown in Fig. 2C, available phosphorus content increased over time in all SMS-treated soil compared to S0-23, whereas it declined progressively in S0-24 decreased monthly. By June 2024, available phosphorus levels in the treated soils (S3-24, S2-24, and S1-24) had increased by 87.34%, 79.90%, and 110.08%, significantly higher than in S0-23. These findings suggested that D. indusiate cultivation enhanced the mobilization and stabilization of soil phosphorus pools. Total nitrogen is a key indicator of soil fertility. Fig. 2D shown that, in June, total nitrogen increased by 14.14% (S3-24) and 0.27% (S1-24) compared to the control, but decreased by 28.09% (S2-24) and 29.95% (S0-24). Over time, nitrogen content initially declined and then recovered, indicating consumption during D. indusiata growth. Supplementing SMS in the second year of cultivation, depending on duration and dosage, improves soil nitrogen status and supports balanced nutrient management. Soil organic matter plays a critical role in supplying essential nutrients, improving soil structure and enhancing water retention capacity. However, as shown in Fig. 2E, at the end of D. indusiata growth cycle (June 2024), organic matter content in SMS-treated soils were lower than in S0-23, with the extent of reduction following the order: S0-24 > S1-24 > S2-24 > S3-24. These results suggested that D. indusiata treatments, particularly when combined with SMS, can mitigate the decline in soil organic matter under continuous cropping. Soil acidification is a major constraint in D. indusiata continuous cropping obstacle. However, as illustrated in Fig. 2F, SMS incorporation effectively stabilizes pH in continuously cropping soil and alleviated acidification, with S3-24 exhibiting the most pronounced effect. Effects of Treatments on Enzyme Activities in D. indusiata Continuous Cropping Soil Fig. 2G-J illustrated the variations in enzymatic activity across all treatments. Among the four enzymes analyzed, only acid phosphatase in S3-24 exhibited increased activity compared to S0-23. Specially, soil acid phosphatase activity in S3-24 during June increased significantly by 37.84% relative to S0-23. In contrast, the enzyme activities in all other experimental groups were lower than those in S0-23. Notably, except for available phosphorus, the S3-24 showed higher enzyme activity than the other treatments at the end of entire growth cycle. Fig. 2H and I showed that sucrase decreased as the growth cycle of continuous D. indusiate extended. In June, catalase activities in S3-24, S2-24, S1-24 and S0-24 were reduced by 17.02%, 32.85%, 20.4% and 36.58%, respectively, compared to S0-23. Sucrase activity also declined in S3-24, S2-24 and S1-24 by 31.04%, 31.6% and 41.66%, respectively. These results indicated that the continuous cropping obstacle significantly affected catalase and sucrase activities, thereby impairing organic carbon decomposition and energy supply for D. indusiata mycelium and fruiting body development. However, monthly comparisons revealed that sucrase activity in S3-24 remained consistently higher throughout the cultivation period compared to S0-23, suggesting SMS application positively enhance soil enzymatic activity. Urease plays a key role in enhancing soil nitrogen availability. As showed in Fig. 2J, changes in urease activity during continuous D. indusiata cultivation were not insignificant across treatments. Overall, urease activities in S3-24, S2-24, S1-24 and S0-24 decreased by 0.34%, 19.22%, 10.29% and 2.74% compared to the previous year's S0-23. Nevertheless, after completion of the full growth cycle, S3-24 exhibited urease activity closest to that of the original soil levels. In summary, compared with S0-23, soil enzyme activity declined at the end of D. indusiata fruiting body period due to continuous cropping obstacles . The S3-24 showed the smallest reduction in enzyme activity, whereas the S0-24 exhibited significantly lower activities of acid phosphatase, catalase and sucrase. Therefore, the addition of SMS promoted soil enzyme activity under continuous cropping condition, with the most pronounced positive effect observed in S3-24. Effects of Different Treatments on Phenolic Acid of D. indusiata Continuous Cropping Soil After D. indusiata continuous soil treated with SMS, at the end of the full growth cycle (in June 2024), phenolic acid levels in all experimental groups were significantly higher than in S0-23 (P < 0.05). Specifically, total phenolic acid content increased by 2.07-, 2.23-, 1.08-, and 1.77- fold in S3-24, S2-24, S1-24, and S0-24, respectively, compared to S0-23. In February 2024, SMS-treated groups (S1-24, S2-24, S3-24) showed lower total phenolic acid than S0-23, with p -coumaric acid decreasing by 33.51%, 37.01 %, and 54.50%, respectively. After three months, phenolic acid accumulation was higher in the SMS-treated compared to S0-24, with p -coumaric acid increasing by 48.63%, 11.59%, and 179.14% in S1-24, S2-24, and S3-24, respectively (Fig. 3). From February to June 2024, the dynamic changes in phenolic acids and p -coumaric acid suggested that distiller's grains improve D. indusiata growth conditions by modulating phenolic acid content, with p -coumaric acid potentially being a key factor influencing continuous cropping. Effects of Different Treatments on Bacterial and Fungal Diversity in D. indusiata Continuous Cropping Soil OUT analysis of soil microorganisms The five treatments (S0-23, S3-24, S2-24, S1-24, and S0-24) each consisted of five replicates, yielding a total of 373,690 original sequences. After filtering out low-quality and short sequences, 318,701 high-quality sequences were retained, with respective counts of 57,479, 64,597, 66,271, 65,277 and 65,076, accounting for 17.91%, 20.27%, 20.80%, 20.48% and 20.42% of the total. The bacterial classification analysis showed 50 phyla, 137 classes, 403 orders, 907 families and 2069 genera. The classification of fungi identified 14 phyla, 46 classes, 95 orders, 203 families and 369 genera (Table S1 and S2). A venn diagram showed that 18 bacterial OUTs across all treatments (Fig. S1A). In fungal ITS sequence analysis, a total of 359,800 original sequences were generated from the five treatments. Following quality filtering, 316,007 high-quality sequences remained, with counts of 67,363, 62,733, 64,702, 67,352 and 53,856, representing 21.32%, 19.85%, 20.47%, 21.31% and 17.04% of the total. The Venn diagram revealed that 13 shared fungal OUTs across treatments (Fig. S1B). For soil microbial sequencing, random sampling was used to generate dilution curve (Wang et al., 2012) and species accumulation curves (Specaccum). According to Fig.S2 A and B, the rarefaction curves approached saturation with increasing sequencing depth, indicating sufficient sequencing data and that the data adequately represent microbial community changes in the samples. Analysis of bacterial and fungi diversity in D. indusiata continuous cropping soil As showed in Table 2, Table S3 and S4), compared to S0-23, bacterial and fungal richness, diversity and evenness were significantly enhanced in all treatments (S3-24, S2-24, S1-24, S0-24) during the second year. Specifically, Chao1, ACE and Shannon indices were higher than those of S0-23. For bacteria, the abundance and evenness in S3-24, S2-24, S1-24 and S0-24 increased by 41.93%, 39.66%, 45.75% and 44.62%, relative to S0-23. The Ace and Chao1 indices indicated that bacterial diversity was highest in S2-24, followed by S1-24 > S3-24 > S0-24 > S2-24. For fungi, the Chao1, ACE and Shannon indices were higher in all treated samples compared to S0-23, with increases in abundance and evenness of 11.19% (S3-24), 1.88% (S2-24), 7.85% (S1-24) and 11.43% (S0-24). Notably, fungal diversity as reflected by the Ace and Chao1 indices was the highest in S2-24, followed by S2-24 > S1-24 > S0-24 > S3-24. These results suggested that applying SMS 1-2 months in advance can significantly enhance bacteria and fungi diversity in sustainable cropping soil of D. indusiata . Table 2 The impact of different treatments on soil microbial diversity for cultivated D. indusiata Treatments Chaol Simpson Shannon ACE Bacterial S0-23 2292.48±371.88 a 0.93±0.0035 c 7.06±0.12 d 2295.24±371.18 a S3-24 2616.81±59.45 a 1±0.0005 ab 10.02±0.074 bc 2623.83±60.53 a S2-24 2523.87±367.08 a 1±0.0008 b 9.86±0.17 c 2533.03±368.31 a S1-24 2687.57±361.73 a 1±0.0014 ab 10.29±0.27 a 2694.08±363.19 a S0-24 2541.68±160.33 a 1±0.0002 a 10.21±0.12 ab 2546.34±159.65 a Fungal S0-23 103.4±97.30 b 0.97±0.01 a 5.86±1.08 a 103.49±97.26 b S3-24 181.5±31.78 ab 0.97±0.02 a 6.18±0.29 a 181.52±31.77 ab S2-24 249.48±41.19 a 0.94±0.02 b 5.97±0.27 a 249.98±41.54 a S1-24 211.22±88.14 a 0.97±0.01 a 6.32±0.53 a 211.33±88.43 a S0-24 186.44±23.28 ab 0.98±0.00 a 6.53±0.23 a 186.69±23.34 ab Notes: Each index is expressed as the mean ± standard deviation. Different lowercase letters within the same data column indicate significant differences (P < 0.05; one-way ANOVA). Analysis of The Abundance of Species in Soil Bacterial and Fungal Community Structure Phylum-level abundance of soil microbial communities As shown in Fig. 4A, the dominant bacterial phyla include Proteobacteria (21.58%-35.47%), Acidobacteriota (2.07%-23.39%), Actinobacteriota (5.41%-11.99%), Firmicutes (2.84%-9.29%), Bacteroidota (3.30%-6.07%), Chloroflexi (2.80%-5.68%). Among these, Proteobacteria, Acetobacteria and Actinomycete are the predominant bacterial groups with relatively high abundance. The relative abundance of Proteobacteria is S3-24 > S1-24 > S2-24 > S0-24 > S0-23; the Acetobacteria as S2-24 > S0-24 > S3-24 > S1-24 > S0-23; the Actinomycete as S1-24 > S2-24 > S0-24 > S3-24 > S0-23. Following SMS treatment, the abundance of the top three dominant bacterial phyla (Proteobacteria, Acetobacteria and Actinomycete) in the second-year experimental treatments were higher than that in S0-23. As shown in Fig. 4B, the fungal community is primarily composed of Ascomycota (41.52%-50.49%), Mortierellomycota (18.13%-24.59%), Basidiomycota (10.76%-24.02%), Rozellomycota (0.31%-24.02%), and Chytridiomycota (1.19%-2.30%). Among these, Ascomycetes is the most abundant fungal phylum, with relative abundance decreasing in the order as S0-23 > S1-24 > S0-24 > S3-24 > S2-24, and plays a critical role in soil organic matter decomposition, crop disease suppression, and soil restoration. Basidiomycota includes saprophytic, symbiotic and parasitic fungi, many of which from beneficial associations with crop root system by enhancing minerals uptake. Saprophytic fungi contribute to increase soil phosphatase activity and reduce pest incidence. However, certain species within Chytridiomycota may be pathogenic and potentially contribute to crop diseases. Genus-level abundance of soil microbial communities The dominant bacterial genera include Bacillus (0.59%-7.52%), Pseudarthrobacter (0.08%-6.14%), Ellin (0.03%-1.71%), Bryobacter (0.21%-1.51%), and Acinetobacter (0.052%-1.58%). Among these, Bacillus showed the highest relative abundance, with levels decreasing in S0-24 > S1-24 > S2-24 > S3-24 > S0-23 (Fig.4C). The longer the SMS treatment duration, the more closely the soil bacterial community composition approaches that of S0-23 following D. indusiata cultivation. As shown in Fig. 4D, the predominant fungal genera include Mortierella (17.66%-28.36%), Fusarium (1.69%-7.06%), Botryotrichum (0.71%-8.02%). Compared to S0-23, the abundance of Thermomyces in S1-24 and S2-24 increased by 21.3% and 24.5%, respectively, indicating a significant enhancement in beneficial fungal population with 1-2 months after SMS treatment. Meanwhile, the abundance of Fusarium decreased by 48.4%, 76.1%, 15.8% and 21.3% in S0-24, S1-24, S2-24, and S3-24, respectively, suggesting that SMS exerts an inhibitory effect on this genus, potentially reducing plant diseases incidence and contributing to improve soil microbial ecological balance [39]. Functional Prediction of Microbial Communities BugBase predicts functional pathway coverage and biologically interpretable phenotypes in complex microbial communities by normalizing OUT abundances based on 16S rRNA gene copy numbers and using pre-calculated reference files for phenotype prediction [40]. Bacterial phenotype results after two years of continuous cropping are shown in Fig. S3A. Among the top 10 phenotypes, ranked from lowest to highest abundance: facultative anaerobes (1.3%-3.5%), Gram-positive bacteria (1.7%-4.7%), anaerobic bacteria (1.9%-6.0%), aerobic bacteria (9.4%-12.1%), potential pathogens (9.8%-17.8%), stress-tolerant bacteria (10.9%-17.4%), mobile element-containing bacteria (11.3%-19.8%), biofilm-forming bacteria (15.4%-20.2%), and Gram-negative bacteria (16.9%-21.4%). This order shows the clear abundance hierarchy of bacterial phenotypes in the samples. FUNGuild is a bioinformatics tool that classifies fungal communities by linking taxonomy to ecological functions [41]. Fungi are grouped into three nutritional modes: pathotrophic, symbiotrophic, and saprotrophic. Based on these, ten functional groups are identified in Fig. S3B. Ranked by increasing abundance: litter saprotrophs (0-20.73%), plant saprotrophs (0.07%-6.82%), fungal parasites (0.08%-6.06%), animal pathogens (0.09%-10.29%), plant pathogens (0.53%-23.13%), endophytes (0.88%-24.34%), dung saprotrophs (0.91%-8.13%), soil saprotrophs (1.37%-12.25%), wood saprotrophs (12.59%-26.23%), and undefined saprotrophs (19.85%-59.55%). Undefined saprotrophs are the most abundant group. Analysis of the Correlation between Environmental Factors and Soil Microbial Communities Based on the Pearson correlation coefficient, the relationships between the relative abundances of top 20 bacterial and fungal genera and soil environmental factors were analyzed. As showed in Fig.5A-C, Bacillus , Nitrospira , and Pseudarthrobacter exhibited significantly positively correlation with ferulic acid, vanillic acid and syringic acid, but negative correlations with AN, pH, SUC, ACP and URE (P < 0.05). Acinetobacter showed significant positive correlations with AN, pH, TN, CAT, SUC and URE, while significant negative correlations with coumalic acid and p -coumaric acid (P < 0.05). Pseudolabrys was significantly positively correlated with p -coumaric acid and AP, while negatively correlated with AN, OM, pH, TN, CAT, SUC, ACP and URE (P < 0.05). In Fig. 5D-F, Botryotrichum , Agaricomycetes , Gelasinospora and Pseudaleuria were significantly positively correlated with vanillic acid, ferulic acid and syringic acid, but negatively correlated with pH, ACP and SUC (P < 0.05). Thermomyces exhibited significant positive correlations with coumalic acid, ferulic acid and AP, while showing a significant negative correlation with URE (P < 0.05). Fusarium was significantly positively correlated with pH, ACP and SUC, but negatively correlated with vanillic acid, ferulic acid and syringic acid (P < 0.05). Penicillium showed a significant positive correlation with OM, and significant negative correlations with vanillic acid, ferulic acid and syringic acid (P < 0.05). Humicola was significantly positively correlated with AP, but negatively correlated with pH, AN, CAT, SUC, ACP and URE (P < 0.05). A multi-index network model in R was used to assess how application timing of SMS affects soil ecosystem structure, incorporating soil physicochemical properties, enzyme activity, phenolic acids, and fungal and bacterial communities. All treatments had goodness-of-fit (GoF) values > 0.65, confirming reliable model fit and strong representation of direct interactions among variables (Fig. 6). In the no-SMS control (S0-24; Fig. 6A), enzyme activity strongly promoted phenolic acid accumulation (path coefficient: 11.67) and moderately increased fungal growth (1.89). Soil properties boosted phenolic acids (1.78) but suppressed enzyme activity (-0.93). With one-month SMS application (S1-24; Fig. 6B), phenolic acids inhibited bacteria (-11.38) but stimulated fungi (3.63), while enzyme activity drove phenolic acid buildup (2.62). Two months before planting (S2-24; Fig. 6C), soil properties strongly enhanced fungal growth (114.26), and phenolic acids continued to suppress bacteria (-2.61). Three months before planting (S3-24; Fig. 6D), soil properties boosted enzyme activity (16.29) and fungi (4.77), with minimal effect on bacteria (-0.98). Enzyme activity strongly increased phenolic acids (19.58), which promoted fungi (1.16) and suppressed bacteria (-4.37), this result coincides with the findings of the in vitro culture experiments. Bacteria enhanced fungal growth (5.25), while fungi reduced phenolic acid accumulation (-3.84), indicating feedback. After D. indusiata cultivation (Fig. 6E), enzyme activity decreased (-0.86) and fungal diversity rose (1.04), showing cropping reshapes microbial communities. Effects of D. indusiata Continuous Cropping Soil on Microbial Network Structure According to Fig. 7A-E, the dominant bacterial taxa in S0-23 were Proteobacteria , Escherichia , and Shigella (Crenarchaeota). Chronic rhizobacteria and salt-tolerant rhizobacteria showed significantly negatively correlations, whereas Lactobacillus and Alistipes exhibited positive association. In S0-24, key bacterial genera included Cupriavidus , Lysobacter , Nitrospirota , and Pseudomonas , among which Nitrospiromonas was negatively correlated with Pseudomonas . In S1-24, Ellin 6067 (an ammonia-oxidizing bacterium) was linked to 19 other genera and played a pivotal role in the nitrogen cycle. PseudoArganomali and Bacillus displayed the highest relative abundances. In S2-24, Dongia (within the Proteobacteria), Actinobacteriota, and Sideroxydans were positively correlated. In S3-24, predominant genera included Dongia, Gemmatimonadota and Candidatus rokubactera bacterium (phylum Methylomirabilota). Flavobacterium was positively associated with Phenylbacterium, while Viagra showed a negative correlation with Acidbacillus. As shown in Fig. 7F-J, in S0-23, Basidiomycota was negatively correlated with Ascomycota and Mortierellomycota. In S0-24, Mortierellomycota was primarily negatively correlated with Ascomycota and Chytridiomycota, whereas Ascomycota showed a positive correlation with Olpidiomycota . Nodes 36 and 42 ( Fusarium ), representing typical plant pathogens associated with blight and root rot, were negatively correlated with regulatory fungal taxa (Nodes 8-13). The Curvularia influenced the fungal community through positively correlated nodes 22-25. In S1-24, Solicoccozyma (within Ascomycota) exhibited a significant positive correlation with nodes 8-15. In S2-24, Mortierellomycota and Ascomycota dominated and exerted predominantly negative regulatory effects. In S3-24, Mortierellomycota displayed widespread negative correlations, while Hannaella (under Ascomycota and Chytridiomycota) was positively correlated with Gibellulopsis , including pathogenic members within this clade. In vitro coculture experimental of D. indusiata with pathogenic microorganisms Under 250 mg/L phenolic acid conditions, the mycelial growth of D. indusiata was enhanced with hyphal diameters ranging from 2.4 to 4.8 μm (Fig. 8M), larger than those in the control (2.1-4.1 μm, Fig. 8N). The mycelium appeared thicker and more robust following phenolic acid treatment. In co-cultivation with Acinetobacter baumannii and D. indusiata exhibited rapid growth and formed white hyphae of phenolic acid presence (Fig. 8B and C). In contrast, when cultured with heat-inactivated A. baumannii , mycelial growth was severely inhibited, showing sparse and underdeveloped hyphae (Fig. 8A). These findings demonstrated that substances associated with heated-inactivated bacterial cells retain strong inhibitory effects on D. indusiata development. In the co-culture of D. indusiata and Bacillus sp., mycelium growth was significantly inhibited in the absence of phenolic acid and without Bacillus sp. inactivation, with minimal visible growth (diameter 1.14-3.41 μm, Fig. 8D and E). Upon addition of phenolic acid to the system, growth remained strongly suppressed by Bacillus sp., exhibiting even narrower hypal diameters (0.68-3.18 μm, Fig. 8F). Under both inactivated and co-culture conditions with Penicillium simplicissimum , exerted a significant inhibitory effect on D. indusiata mycelium growth (2-4.5 μm, Fig. 8G-I). Notably, in the absence of phenolic acid demonstrated a stronger inhibitory effect, resulting in severe fragmentation of D. indusiata mycelia, which impaired their ability to extend outward and absorb nutrients effectively (1-4.5 μm, Fig. 8H). In the inactivated medium of Aspergillus fumigatus , the hyphal growth of D. indusiata was significantly inhibited, as evidenced by a reduced diameter (2-2.5 μm) and irregular morphology (Fig. 8J). In dual cultures of A. fumigatus and D. indusiata without phenolic acid, mycelial development of D. indusiata was severely restricted, characterized by limited spatial coverage, pale pigmentation, sparse distribution, and a loosely organized, compressed, and deformed hyphal network (2.8-3.2 μm, Fig. 8K). Upon addition of phenolic acid, the inhibitory effects on D. indusiata were partially alleviated, with improved colony coverage, enhanced whiteness, and partial restoration of hyphal diameter to 3.3-3.6 μm (Fig. 8L). Nevertheless, under these conditions, the mycelial growth of A. fumigatus remained markedly superior to that of D. indusiata . Discussion SMS Improved Yield and Quality of Continuous D. indusiata Fruiting Body Production The yield and quality of D. indusiata fruiting bodies partially reflect the nutrient and supply capacity of the culture substrate and soil. Mushroom spent substrate, a byproduct of mushroom cultivation, is rich in organic nutrients and primarily composed of cellulose, hemicellulose, lignin, carbohydrates, proteins and residual fungal mycelium [ 42 ]. The recycling of mushroom spent substrate in agriculture has emerged as a key strategy for advancing sustainable and green agriculture systems. Studies have demonstrated that, compared to traditional planting methods, substituting conventional nursery soil with SMS can increase soluble protein concentration in rice seedlings, enhance superoxide dismutase (SOD) activity, and improve total porosity and structural stability, thereby creating a more favorable environment for crop growth [ 43 – 45 ] reported that supplementing bacterial manure with D. indusiata fruiting bodies can modulate the abundance and structure of soil microbial communities, enhance soil fertility, and stabilize the soil environment, while simultaneously improving the yield and quality of subsequently cultivated D. indusiat a. This study showed that continuous production of D. indusiata fruiting body in the second year was significantly enhanced by mushroom spent substrate application. Notably, the S3-24 treatment group (inoculated for 3 months) exhibited the highest performance, achieving a fresh yield of 12,758.4 kg/hm approximately 7 times higher than the untreated control group (S0-24). The results indicated that the production of D. indusiata was nearly equivalent to that of the previous year. Quality analysis results revealed that the S3-24 group had 32.42% higher crude polysaccharide content and 1.60% higher crude protein content compared to the control, with total sugar content reaching 547.66 ± 4.11 mg/g. These findings indicated that amending soil with SMS significantly promotes both yield and quality of continuous D. indusiata fruiting body production. A Novel Technology for Continuous D. indusiata Cultivation Improves Soil Fertility The partially substitution of chemical fertilizers with SMS can improve soil nutrients and reduces the accumulation of heavy metals in soil. Compared to control soil, Agaricus bisporus in SMS-amended soil has been shown to increase organic nitrogen and available phosphorus content [ 46 ]. Peregrina et al. [ 47 ] demonstrated that applying bacterial granules improved labile organic matter and microbial activity in vineyard soils, while also enhancing nitrogen, and available phosphorus, concomitantly elevating soil respiration rates and phosphatase activity [ 46 ]. Phosphorus is a critical nutrient that determines plant growth and productivity, limiting crop yields on over 40% of the world's arable land. Increased phosphorus availability has been consistently shown to enhance crop yield [ 48 – 50 ]. Under low-phosphorus conditions, crops and soil microorganisms secrete acid phosphatases within a pH range of 5.73–7.11 to mobilize inorganic phosphorus and meet physiological demands [ 51 ]. In this study, soil treated with bacterial sterilization over a three-month period (S3-24 group) exhibited an 87.34% increase in available phosphorus content (Fig. 2 C), a 14.14% rise in total nitrogen (Fig. 2 D), and only a marginal decline in organic matter (14.14% lower than S0-23, Fig. 2 E), demonstrating that this treatment significantly enhances soil fertility. A Novel Technology for Cultivating D. indusiata Improves Soil Microbial Diversity and Composition Research showed that long-term monoculture and continuous cropping disorder can alter the dynamic evolution of microbial assembly mechanism. Imbalance of soil microbial structure and accumulation of pathogenic microorganisms are the main reasons for continuous cropping obstacles [ 52 ]. In both soil and root system under monoculture conditions, bacteria exhibit greater responsiveness to environmental filtering factors, whereas fungi are more strongly influenced by dispersal limitations and stochastic processes. These dynamic changes highlight the importance of monitoring microbial population shifts following bacterial treatments [ 53 ]. Use of discarded mushroom waste in cucumber cultivation has been found to suppress Fusarium wilt disease [ 54 ]. Incorporating spent cucumber residues into growth substrates enhances seedling vigor and developmental progression without in vitro fertilization, while also improving microbial activity, promoting rice seedlings growth, the facilitating rhizosphere microbial recruitment [ 55 – 56 ]. Some studies have shown that the combination of biofertilizers and cover crops can improve soil health while actively regulating the structure and function of the rhizosphere microbial community, improving the growth of Apium graveolens under continuous cropping, and ultimately supporting high yield and high-quality crops [ 57 ]. Here, SMS treatment technology was employed to regulate the microbial community structure in D. indusiata continuous cropping soil. Microbial co-occurrence network analysis clearly illustrates that sterilization fosters a more tightly interconnected soil microbial community, enriching the overall microbial environment. Second-year diversity analysis reveals that the bacterial sterilization treatment significantly altered the soil microbial composition in the experimental group, with a notable increase in the abundance of Gemmatimonadaceae, Acetobacteria, Basidiomyceae, Botryotrichum, Fusarium. Conversely, the abundance of Firmicutes, Bacillus and Botryotrichum decreased following the treatment. Notably, several taxa associated with enhanced soil fertility were enriched. For instance, Gemmatimonadaceae, belonging to the phylum Gemmatimonadota, played an important role in the phosphorus cycling [ 58 ]. Basidiomycetes are among the primary decomposers of lignin and cellulose in nature, ecosystems, contributing fundamentally to the formation of stable soil organic carbon and long-term soil fertility [ 59 ]. These microbial shifts indicated that bacteria sterilization can positively influence ecosystem health and soil stability by reshaping the soil microecology and promoting organic matter decomposition. However, the concurrent increased in Fusarium caution due to its potential as a plant pathogen. Thus, while the treatment effectively modulates soil microbial dynamics, further research is need to optimize its application—maximizing benefits for soil health while mitigating risk associated with pathogenic taxa. Among all PLS-PM models, S0-23 had the highest GoF (0.81), indicating a relatively stable soil system before the second year of continuous D. indusiata cultivation. In contrast, after two years of continuous cropping and SMS application, S3-24 showed the highest GoF (0.78) among treatment groups, suggesting that longer substrate pre-treatment improves coordination among soil indicators and enhances ecological network stability. SMS use significantly altered interactions among soil factors. With longer pre-treatment, positive linkages between microbial communities (especially bacteria and fungi) and soil physicochemical properties or enzyme activities strengthened. Multiple high-weight pathways (path coefficient > 10) emerged, indicating that SMS addition promotes nutrient cycling and microbial activity, boosting material and energy transfer. The negative associations between phenolic acid compounds and microbial taxa also weakened after substrate application. Notably, the S3-24 network showed greater complexity and integration, confirming that three-month pre-treatment stabilizes the soil micro-ecological network. In contrast, S1-24 and S2-24 showed only moderate improvements, with many weak or inhibitory connections remaining. Overall, starting SMS treatment three months in advance is more effective for building a stable, coordinated cultivation environment to overcome continuous cropping obstacles, supporting sustainable D. indusiata cultivation. Effects of Continuous Cropping of D. indusiata on Soil Organic Acids Phenolic acids are secondary metabolites widely present in higher plant tissues and closely associated with plant growth and development. For decades, it has been hypothesized that the autotoxin effects of phenolic acid acting as allelochemicals are a key factor contributing to continuous cropping disorders. Prolonged monoculture has been shown to correlated with a significant accumulation of phenolic acids and a marked decline in soil pH [ 60 ]. Soil acidification may promote the buildup of phenolic acid and is positively correlated with shifts in bacteria community composition, suggesting that the interplay between phenolic acid accumulation and decreasing soil pH could be a critical driver in reshaping soil microbial communities [ 61 ]. Furthermore, Qu and Wang [ 62 ] demonstrated that phenolic acid significantly influence microbial biomass and activity. Specifically, low concentrations of phenolic compounds such as 2,4-di-tert-butylphenol and vanillic acid enhanced soil microbial biomass, whereas higher concentrations exert inhibitory effect. In this study, the soil phenolic acid content in the second year of continuous D. indusiata cultivation was significantly higher than that in the first year and compared to previous research findings [ 31 ]. The accumulation of phenolic acid substances in soil increased markedly, with coumaric acid showing the most pronounced change—its concentration in S3-24 increased by 179.14% relative to the other five phenolic acid compounds. Based on the analysis of soil phenolic acid, optimal inactivated conditions for the mycelium of D. indusiate were identified through controlled culture experiments. The metabolites produced by the pathogen significantly inhibited the D. indusiata mycelium growth. Even in coculture systems without exogenous phenolic acid addition, pathogen-induced inhibition of mycelial growth was evident. Notably, the addition of phenolic acid promoted the growth of both D. indusiata and pathogenic bacteria; however, the stimulatory effect was more pronounced for pathogenic bacteria, which outcompeted D. indusiata mycelium. Taken together, changes in phenolic acid concentrations and results from plate assays indicated that shifts in soil phenolic acid levels are closely associated with the occurrence of continuous cropping disorders of D. indusiata . This study improved the soil by applying bacterial granules to investigate their alleviating effect on continuous cropping obstacles of D. indusiata . Field trial results demonstrated that following application of a specific dose of SMS, D. indusiata yield in the second year (S3-24) closely approached that of the first year and was significantly higher than the control group in the same period. Nutritional analysis of the fruiting body revealed that all major nutritional indicators in the treated group were superior to those in the control, indicating that bacterial granule application positively enhanced the nutritional quality of D. indusiata . Furthermore, the treatment markedly improved soil quality, particularly in terms of physical and chemical properties and enzyme activities. It effectively mitigated soil acidification associated with continuous cropping, stabilized soil pH, and increased availability of key nutrients such as nitrogen and phosphorus. Microbial community analysis indicated a significantly increase in beneficial populations, reflecting an overall improvement in soil fertility and microbial balance, thereby confirming the microecological regulatory potential of this approach. Compared with conventional methods for managing continuous cropping obstacles, the SMS pretreatment method exhibited distinct advantages. Traditional high-temperature sterilization only partially reduces pathogens and is associated with high energy consumption and operational complexity; field rotation can alleviate soil degradation but compromises land use efficiency and production continuity; while chemical fumigation is effective against soil-borne diseases, it lacks selectivity, often harming beneficial microbes [ 63 – 64 ], and poses risks of environmental pollution and residue accumulation. In contrast, pre-planting soil amendment with bacterial granules not only effectively alleviates yield decline due to continuous cropping but also directly enhances soil nutrients status and optimizes microbial community structure through a simple and practical operation. PLS-PM further confirmed that application of SMS distiller’s grains significantly increased the positive path coefficients between microorganisms and environmental factors and enhanced the model’s explanatory power, thereby improving the overall stability and coordination of the soil micro-ecological network. Moreover, this strategy enables high-value utilization of agricultural waste-derived bacterial granules, significantly reducing management costs and offering both ecological and economic benefits. In conclusion, this green, efficient, and cost-effective continuous cropping pathway, enhancing soil sustainability, and promoting high-yield, high-quality crop production. Conclusion Over two years of field experiments, this study evaluated fermented SMS for alleviating continuous cropping obstacles and improving soil microecology properties. Analysis of soil physicochemical properties, enzyme activities, microbial diversity, and agronomic and nutritional indicators showed that SMS significantly improved soil fertility. The 3-month treatment had the greatest effect: it stabilized soil pH, reduced acidification, and increased available phosphorus and total nitrogen, indicating enhanced soil nutrient availability. All SMS treatments suppressed excessive organic matter degradation, most effectively in 3-month group. These improvements were link to enrich functional microbes involved in phosphorus cycling, especially increased Bacillus abundance, which enhances phosphorus solubilization. Phenolic acid analysis revealed accumulation under continuous cropping, but SMS reduced coumaric acid levels. Notably, moderate phenolic acid promoted growth of D. indusiata and some pathogens. SMS also enhanced bacterial-fungal network complexity and diversity by shaping microbial community structure and enriching beneficial bacteria associated with soil fertility and stability. This mechanism alleviated key soil dysfunctions from continuous cropping, such as microbial imbalance and reduced decomposition capacity. Crucially, SMS improved D. indusiata yield and quality. Crude polysaccharides and protein also improved. Microscopy showed thicker, more robust hyphae in treated mycelia—supporting high productivity. This study demonstrated successful SMS resource recovery, providing practical and theoretical solutions for D. indusiate continuous cropping. Future work should optimize microbial strains and application methods, developed inoculants, and advance sustainable agriculture. Declarations Acknowledgements We thank Guirong Lin from the Xinjundu Company for his technical guidance on D. indusiata cultivation and Shaoqing Shu from the Xingwang family farm for managing the experimental site. Furthermore, we also thank the Shunchang Juncao Science and Technology Backyard for providing accommodation to our technicians and students. Author Contributions Jing Li and Xiongjie Lin: Conceptualization, Funding acquisition, Supervision, Writing–original draft, Writing–review & editing, Project administration. Xiaoyue Di: Conceptualization, Data curation, Formal analysis, Investigation, Writing – original draft, Writing – review & editing, Visualization. Yinghao Sun, Xianai Huang, Fengju Jiang, and Jiale Feng: Data curation, Formal analysis, Investigation. Dongmei Lin, and Zhanxi Lin: Writing – review & editing. Funding This study was supported by the National Key Research and Development Program of China “Research and Application Demonstration of Key Technologies for Juncao Medicinal and Edible Mushrooms and Orchids Cultivation” (2023YFD1000502); The Project for the Enhancement of the First-Class Discipline of Forestry (Juncao Science) at Fujian Agriculture and Forestry University (725025010A). Data availability The original dataset on microbial diversity for this study has been submitted to the National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/), along with the accession number PRJNA1234400 (this data will be made publicly available on December 31, 2026). Ethics approval and consent to participate Not applicable. Consent for publication All authors agreed to the publication. Competing interests The authors declare that they have no competing financial interests or personal relationships that may have influenced the work reported in this study. 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Rep. 2019;9:12499. https://doi.org/10.1038/s41598-019-48611-5. Qu XH, Wang J. Effect of amendments with different phenolic acids on soil microbial biomass, activity, and community diversity. Appl. Soil Ecol. 2008;39:172-179. https://doi.org/10.1016/j.apsoil.2007.12.007. Jayaraman S, Naorem A, Lal R, Dalal RC, Sinha NK, Patra AK, et al. Disease-suppressive soils-beyond food production: a critical review. J. Soil Sci. Plant Nutr. 2021;21:1437-1465. https://doi.org/10.1007/s42729-021-00451-x. Lopes EA, Canedo EJ, Gomes VA, Vieira BS, Parreira DF, Neves WS. Anaerobic soil disinfestation for the management of soilborne pathogens: A review. Appl. Soil Ecol. 2022;174:104408. http:// doi.org/10.1016/j.apsoil.2022.104408. Additional Declarations No competing interests reported. Supplementary Files GraphicalAbstract.jpeg Fig.S1.jpeg Fig. S1. Venn diagram of bacterial Notes: (A) and fungal (B) OUTs in D. indusiata continuous cropping soil. Fig.S2.jpeg Fig. S2. Dilution curve. Notes: (A) Bacterial dilution curve; (B) Fungal dilution curve. Fig.S3.jpeg Fig. S3. Predicted functional profiles of microbial communities. Notes: (A) Bacterial phenotypic predictions using Bugbase; (B) Fungal functional predictions using FUNGuild. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 02 Mar, 2026 Reviews received at journal 25 Feb, 2026 Reviews received at journal 18 Feb, 2026 Reviewers agreed at journal 17 Feb, 2026 Reviewers agreed at journal 11 Feb, 2026 Reviewers invited by journal 11 Feb, 2026 Editor assigned by journal 06 Feb, 2026 Submission checks completed at journal 06 Feb, 2026 First submitted to journal 02 Feb, 2026 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. 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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-8761948","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":591088523,"identity":"12346dc9-b048-41b7-a98e-91389d74e7ef","order_by":0,"name":"Xiao Yue Di","email":"","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"Yue","lastName":"Di","suffix":""},{"id":591088524,"identity":"1dcfbefc-6208-4269-b29c-444a2e8b1c53","order_by":1,"name":"Yinghao Sun","email":"","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Yinghao","middleName":"","lastName":"Sun","suffix":""},{"id":591088525,"identity":"e22a597e-cf9b-459d-b16b-f51afb47990d","order_by":2,"name":"Fengju Jiang","email":"","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Fengju","middleName":"","lastName":"Jiang","suffix":""},{"id":591088526,"identity":"6f959477-28df-4e0b-aaac-c3245178d094","order_by":3,"name":"Xianai Huang","email":"","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Xianai","middleName":"","lastName":"Huang","suffix":""},{"id":591088527,"identity":"3575b8eb-62c6-4341-a0d6-c68b1a2ef268","order_by":4,"name":"Jiale Feng","email":"","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Jiale","middleName":"","lastName":"Feng","suffix":""},{"id":591088528,"identity":"495b7849-ff19-41e4-8993-5951fec2644f","order_by":5,"name":"Dongmei Lin","email":"","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Dongmei","middleName":"","lastName":"Lin","suffix":""},{"id":591088529,"identity":"0c4801d7-5212-4cd1-8be4-e259b303cba7","order_by":6,"name":"Zhanxi Lin","email":"","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Zhanxi","middleName":"","lastName":"Lin","suffix":""},{"id":591088530,"identity":"09647a33-34cf-4ed8-ad2c-b86b59e670c0","order_by":7,"name":"Xiongjie Lin","email":"","orcid":"","institution":"Fujian Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Xiongjie","middleName":"","lastName":"Lin","suffix":""},{"id":591088531,"identity":"1ed4a572-6a37-400d-bbbb-7c5dd7e507d3","order_by":8,"name":"Jing Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIiWNgGAWjYBAC9gbGBwwPDNh4GBiYD3xgsAGJJeDXwnOA2YAhAayFLXEGQxrRWiBMQyK1sB9mk0go4JMx51/zseFHwmEGfvYcA4afO/Bo4UkGagE6zHLG242NPUAtkj1vDBh7z+DWYs+QfwysxeDG2e0PeH8cZjC4kWPAzNiGxxb+x2xQLWceNv4B2mJPUIsE1GEG53sYm3mAWgwkCGp5zGwBsYXNsFkmIZ1H4syzgoO9eB2WzHjjw59j9gbnDz9sfJNgLcffnrzxwU88WqDgGAODRALEDBBxgKAGBoYaBgZ+YtSNglEwCkbBiAQAuBBO4+jJdXMAAAAASUVORK5CYII=","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":true,"prefix":"","firstName":"Jing","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2026-02-02 07:53:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8761948/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8761948/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102833237,"identity":"a8e6441c-4419-4e57-a1a4-74b93fddd37b","added_by":"auto","created_at":"2026-02-17 10:26:30","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":897980,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the experimental site.\u003c/p\u003e\n\u003cp\u003eNotes: (A) Layout of the field trail; (B) Detailed design of \u003cem\u003eD. indusiata\u003c/em\u003e cultivation area.\u003c/p\u003e","description":"","filename":"Fig.1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8761948/v1/b904c4d05f70f67a0c989815.jpeg"},{"id":102833239,"identity":"3941896e-d0fd-4998-bd74-bc1f4ef0ab40","added_by":"auto","created_at":"2026-02-17 10:26:31","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2697957,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of SMS on physicochemical properties and enzyme activities in\u003cem\u003e D. indusiata\u003c/em\u003econtinuous cropping soil.\u003c/p\u003e\n\u003cp\u003eNotes: Including ammonium nitrogen (A), available potassium (B), available phosphorus (C), total nitrogen (D), organic matter (E), pH (F), acid phosphatase (G), catalase (H), sucrase (I), urease (J).\u003c/p\u003e","description":"","filename":"Fig.2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8761948/v1/447b3c259011c2b01db27f01.jpeg"},{"id":102833224,"identity":"d3c58a0b-49d4-4df6-beb8-8a61490b6f22","added_by":"auto","created_at":"2026-02-17 10:26:29","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2626206,"visible":true,"origin":"","legend":"\u003cp\u003ePhenolic acid content across different sampling periods under various treatment groups.\u003c/p\u003e\n\u003cp\u003eNotes: (A) Total phenolic acid content in the soil of each treatment group at different periods; (B-F) Distribution profiles of six phenolic acids in the soil of each treatment group in February (B), March (C), April (D), May (E) and June (F) in 2025.\u003c/p\u003e","description":"","filename":"Fig.3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8761948/v1/356398ba136cc8465a14c287.jpeg"},{"id":102833219,"identity":"49cfefa7-9f48-4c25-956b-72418f69827c","added_by":"auto","created_at":"2026-02-17 10:26:28","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4486012,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of taxonomy composition of microbial communities.\u003c/p\u003e\n\u003cp\u003eNotes: At the phylum level: (A) bacterial community composition; (B) fungal community composition. At the genus level: (C) bacterial community composition; (D) fungal community composition.\u003c/p\u003e","description":"","filename":"Fig.4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8761948/v1/0fcf53219dbf7a4574f8ace4.jpeg"},{"id":102962633,"identity":"370bff10-4eba-4a67-b6ef-053c40b2a707","added_by":"auto","created_at":"2026-02-19 04:10:12","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":613264,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap of Pearson Correlation Between Top 20 Microbial Genera and Soil Environmental Factors.\u003c/p\u003e\n\u003cp\u003eNotes: (A) Correlation analysis between soil phenolic acids and bacterial genera; (B) soil physicochemical properties and bacterial genera; (C) soil enzyme activities and bacterial genera; (D) soil phenolic acids and fungal genera; (E) soil physicochemical properties and fungal genera; (F) soil enzyme activities and fungal genera. The abbreviations of phenolic acid in the figure are FA (ferulic acid), VA (vanillic acid), SA (syringic acid), pHBA (\u003cem\u003ep\u003c/em\u003e-hydroxybenzoic acid), CA (coumalic acid), and p-CA (\u003cem\u003ep\u003c/em\u003e-coumaric acid). The vertical dendrogram represents clustering of microbial taxa, the horizontal dendrogram represents sample clustering, and the central heatmap displays the correlation coefficients. Red indicates positive correlations, blue indicates negative correlations, with darker shades representing stronger correlations magnitudes. Significance levels are denoted by asterisks (* P \u0026lt; 0.05, ** P \u0026lt; 0.001, *** P \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Fig.5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8761948/v1/88dd850e7dc5617ff84dbb95.jpeg"},{"id":102833215,"identity":"d28cfb58-f856-4e27-bde5-f65bd2892f7b","added_by":"auto","created_at":"2026-02-17 10:26:24","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":526986,"visible":true,"origin":"","legend":"\u003cp\u003ePLS-PM analysis of different treatment indicators and microbial genera.\u003c/p\u003e\n\u003cp\u003eNotes: (A) S0-24 (GoF=0.66); (B) S1-24 (GoF=0.76); (C) S2-24 (GoF=0.74); (D) S3-24 (GoF=0.78); (E) S0-23 (GoF=0.81).PLS-PM diagram illustrates the causal relationships among fungi, bacteria, physicochemical properties, phenolic acids and enzyme activities. Orange arrows represent positive effects (path coefficient \u0026gt; 0), blue arrow denote negative effects (path coefficient \u0026lt; 0), and the values adjacent to the arrows indicate the standardized path coefficients, reflecting the magnitude and direction of the direct effects between variables. (Model of different treatments during the M6 period).\u003c/p\u003e","description":"","filename":"Fig.6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8761948/v1/1b5b6b02797cac13089419d8.jpeg"},{"id":102833243,"identity":"33333a4f-6746-4899-aad6-55059b154a7e","added_by":"auto","created_at":"2026-02-17 10:26:31","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3306662,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation network diagram of \u003cem\u003eD. indusiata\u003c/em\u003e continuous cropping soil on microbial network structure.\u003c/p\u003e\n\u003cp\u003eNotes: The bacteria co-occurrence network includes panels A-E: (A) S0-23; (B) S0-24; (C) S1-24; (D) S2-24; (E) S3-24. The fungal co-occurrence network includes panels F-J: (F) S0-23; (G) S0-24; (H) S1-24; (I) S2-24; (J) S3-24. Spherical nodes represent microbial species, with node size indicating relative abundance and note color representing the corresponding phylum. Lines between nodes indicated significant correlations: line thickness reflects the strength of the correlation, while color denotes the correlation type-red for positive and green for negative correlations.\u003c/p\u003e","description":"","filename":"Fig.7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8761948/v1/2b8c2e50e51dfb927067b004.jpeg"},{"id":102833235,"identity":"baec3f74-7b3c-4145-91d0-022e550817cb","added_by":"auto","created_at":"2026-02-17 10:26:30","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":3162847,"visible":true,"origin":"","legend":"\u003cp\u003eIn vitro co-culture results of \u003cem\u003eD. indusiata\u003c/em\u003e with continuous cropping soil-borne pathogens.\u003c/p\u003e\n\u003cp\u003eNotes: Each panel shows plate images (left) followed by corresponding scanning electron micrographs of \u003cem\u003eD. indusiata\u003c/em\u003e (right). (A) \u003cem\u003eD. indusiata\u003c/em\u003e on \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e-inactivated plate; scanning electron micrograph showed mycelium interacting with inactivated bacteria; (B) \u003cem\u003eA. baumannii\u003c/em\u003e-\u003cem\u003eD. indusiata\u003c/em\u003e antagonistic plate; scanning electron micrograph of \u003cem\u003eD. indusiata\u003c/em\u003e mycelium; (C) \u003cem\u003eA. baumannii\u003c/em\u003e-\u003cem\u003eD. indusiata\u003c/em\u003e co-culture plate with phenolic acid and scanning electron micrograph of \u003cem\u003eD. indusiata\u003c/em\u003e mycelium; (D) \u003cem\u003eBacillus\u003c/em\u003esp. inactivation and \u003cem\u003eD. indusiata\u003c/em\u003e antagonistic plate and scanning electron micrograph of \u003cem\u003eD. indusiata\u003c/em\u003e mycelium; (E) \u003cem\u003eBacillus \u003c/em\u003esp.-\u003cem\u003eD. indusiata\u003c/em\u003e co-culture antagonistic plate and scanning electron micrograph of \u003cem\u003eD. indusiata\u003c/em\u003e mycelium; (F) \u003cem\u003eBacillus \u003c/em\u003esp.-\u003cem\u003eD. indusiata\u003c/em\u003e co-culture plate with phenolic acid and scanning electron micrograph of \u003cem\u003eD. indusiata\u003c/em\u003e mycelium; (G) \u003cem\u003eP. simplicissimum\u003c/em\u003e inactivation and \u003cem\u003eD. indusiata\u003c/em\u003e antagonistic plate and scanning electron micrograph of \u003cem\u003eD. indusiata\u003c/em\u003e mycelium; (H) \u003cem\u003eP. simplicissimum\u003c/em\u003e-\u003cem\u003eD. indusiata\u003c/em\u003e co-culture antagonistic plate and scanning electron micrograph of \u003cem\u003eD. indusiata\u003c/em\u003e mycelium; (I) \u003cem\u003eP. simplicissimum\u003c/em\u003e-\u003cem\u003eD. indusiata\u003c/em\u003e co-culture plate with phenolic acid plate and scanning electron micrograph of \u003cem\u003eD. indusiate\u003c/em\u003emycelium; (J) \u003cem\u003eA. fumigatius\u003c/em\u003e inactivation and \u003cem\u003eD. indusiata\u003c/em\u003e antagonistic plate and scanning electron micrograph of \u003cem\u003eD. indusiate \u003c/em\u003emycelium; (K) \u003cem\u003eA. fumigatus\u003c/em\u003e-\u003cem\u003eD. indusiata\u003c/em\u003e co-culture plate and scanning electron micrograph of \u003cem\u003eD. indusiata\u003c/em\u003emycelium; (L) \u003cem\u003eA. fumigatus-D. indusiata\u003c/em\u003e co-culture plate with phenolic acid and scanning electron micrograph of \u003cem\u003eD. indusiata \u003c/em\u003emycelium grown; (M) \u003cem\u003eD. indusiata\u003c/em\u003e cultured on PDA medium supplemented with phenolic acid and scanning electron microscope of \u003cem\u003eD. indusiata\u003c/em\u003e mycelium; (N) \u003cem\u003eD. indusiata\u003c/em\u003e cultured on PDA medium without phenolic acid and\u003cem\u003e \u003c/em\u003escanning electron micrograph of \u003cem\u003eD. indusiate\u003c/em\u003emycelium.\u003c/p\u003e","description":"","filename":"Fig.8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8761948/v1/8ff9455f14b563d435fa12a4.jpeg"},{"id":102965018,"identity":"95bddfc4-7bad-4d6e-a7b9-b7ca394a8d55","added_by":"auto","created_at":"2026-02-19 04:29:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":19954634,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8761948/v1/549dd164-3a56-41ce-82c9-370fa000f27c.pdf"},{"id":102833210,"identity":"8fbe6add-0102-4aaf-a84e-a449e86a6de3","added_by":"auto","created_at":"2026-02-17 10:26:20","extension":"jpeg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":495639,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8761948/v1/acfbb015a49b5cc08e7f763a.jpeg"},{"id":102833241,"identity":"c6492ecd-bf61-405f-bdf4-b50c297bbe3b","added_by":"auto","created_at":"2026-02-17 10:26:31","extension":"jpeg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":259825,"visible":true,"origin":"","legend":"\u003cp\u003eFig. S1. Venn diagram of bacterial\u003c/p\u003e\n\u003cp\u003eNotes: (A) and fungal (B) OUTs in \u003cem\u003eD. indusiata\u003c/em\u003econtinuous cropping soil.\u003c/p\u003e","description":"","filename":"Fig.S1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8761948/v1/96919763419c2251e10853a2.jpeg"},{"id":102833204,"identity":"5b6b3247-54b9-4046-ba17-d0ab60176300","added_by":"auto","created_at":"2026-02-17 10:26:18","extension":"jpeg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":283102,"visible":true,"origin":"","legend":"\u003cp\u003eFig. S2. Dilution curve.\u003c/p\u003e\n\u003cp\u003eNotes: (A) Bacterial dilution curve; (B) Fungal dilution curve.\u003c/p\u003e","description":"","filename":"Fig.S2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8761948/v1/fa07146916d0b7b8783f71f7.jpeg"},{"id":102833212,"identity":"6abaf305-57dd-4a19-bcbd-b6479800b643","added_by":"auto","created_at":"2026-02-17 10:26:20","extension":"jpeg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1116483,"visible":true,"origin":"","legend":"\u003cp\u003eFig. S3. Predicted functional profiles of microbial communities.\u003c/p\u003e\n\u003cp\u003eNotes: (A) Bacterial phenotypic predictions using Bugbase; (B) Fungal functional predictions using FUNGuild.\u003c/p\u003e","description":"","filename":"Fig.S3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8761948/v1/d937fed07c9633360852e42f.jpeg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of Hypsizygus marmoreus spent substrate on prevention and control potential of continuous cropping obstacle in Dictyophora indusiata cultivation","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eDictyphora indusiata\u003c/em\u003e (commonly known as bamboo mushroom), a saprophytic fungal belonging to the family Phallaceae within the phylum Basidiomycota, is predominantly distributed in subtropical regions of China, particularly in the southwestern provinces. This species processes significant nutritional and pharmacological value owing to its high content of protein, essential amino acids, polysaccharides, and lipids [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] Pharmacological studies have demonstrated its bioactive properties, including inhibition of tumor growth and broad-specutrum antimicrobial activity [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, the rapid expansion of \u003cem\u003eD. indusiata\u003c/em\u003e cultivation has led to sever continuous cropping obstacles, characterized by yield instability, pathogen proliferation, quality decline, and soil microbiome dysbiosis [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eContinuous cropping obstacle, defined as the progressive decline in crop productivity and quality under repeated monoculture regimes [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], arise from three primary hazard following as soil physicochemical degradation, allelopathic autotoxicity, and microbial community shifts. Current research frameworks recognized these factors as interdependent divers, where alterations in soil microbial communities exacerbate disruptions in nutrient cycling [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Yin et al. identified significant autotoxicity in \u003cem\u003eMorchella\u003c/em\u003e cultivation soils [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], where phenolic acid extracts exhibited concentration-dependent inhibition of mycelial growth. In contrast, Ji et al. documented progressive rhizosphere degradation in \u003cem\u003eGanoderma lucidum\u003c/em\u003e monoculture systems [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], characterized by deterioration of the soil environment. Most research focuses on implementing targeted agronomic interventions to mitigate continuous cropping obstacles, with emerging strategies demonstrating multifaceted efficacy. Intercropping tea and oyster mushrooms (\u003cem\u003ePleuroutus ostreatus\u003c/em\u003e) significantly improved soil physicochemical properties [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], while Huang et al. demonstrated that \u003cem\u003eImazalil\u003c/em\u003e application effectively suppressed pathogenic bacterial proliferation in Ganoderma \u003cem\u003elucidum\u003c/em\u003e cultivation by disrupting biosynthesis pathway [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Additionally, pre-cultivation dazomet fumigation in \u003cem\u003eMorchella\u003c/em\u003e systems reconfigured rhizosphere microbiomes by reducing pathogenic fungi and enhancing beneficial bacteria, ultimately improving yield under continuous cropping yield [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eChina dominates global edible mushroom production, accounting for 76.8% of total output. As a result, over 1.2\u0026times;10\u003csup\u003e8\u003c/sup\u003e tons of spent mushroom substrate are generated annually, primarily composed of lignocellulosic biomass, residual proteins, and bioactive compounds [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Conventional disposal methods such as landfilling and incineration contribute to environmental pollution through greenhouse gas emissions. Recent studies have identified multiple valorization pathways for spent mushroom substrates, including secondary fungal cultivation, conversion into organic fertilizers, use as ruminant feed supplements, and bioenergy production via anaerobic digestion [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. This study explored the application of \u003cem\u003eHypsizygus marmoreus\u003c/em\u003e (seafood mushroom) spent substrate (SMS) to alleviate continuous cropping obstacles in \u003cem\u003eD. indusiata\u003c/em\u003e cultivation through controlled filed trials. We systematically evaluated four key aspects: (1) the temporal dynamics of SMS application on phenolic acid degradation rates in \u003cem\u003eD. indusiata\u003c/em\u003e rhizosphere soil over a 0\u0026ndash;3 months period; (2) treatment effects on fruiting body yield and nutritional quality; (3) recovery of soil physicochemical parameter under continuous cropping systems; and (4) modulation of rhizosphere microbial diversity following SMS amendments. These results finding offer both mechanistic insights and practical strategies to support sustainable \u003cem\u003eD. indusiata\u003c/em\u003e cultivation.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eExperimental design and sample collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe field experiment was conducted at the Shunchang Juncao Technology Base Yard (26\u0026deg;45\u0026rsquo;7\u0026rsquo;\u0026rsquo; N, 117\u0026deg;48\u0026rsquo;20\u0026rsquo;\u0026rsquo; E), situated in a mid-subtropical marine monsoon climate zone characterized by a mean annual temperature of 18.5 ℃, annual precipitation of 1,756 mm, and annual sunshine duration of 1740.7 h. A 266 m\u003csup\u003e2\u003c/sup\u003e experimental plot (19 \u0026times; 14 m) was divided into four equal treatment zones. The \u003cem\u003eD. indusiata\u003c/em\u003e strain (GenBank accession number: AF324167.2) was provided by the National Engineering Research Center of Juncao Technology at Fujian Agricultural and Forestry University, while SMS sourced from Fujian Shunchang Xinjundu Mushroom Industry Development Co., Ltd. Each treatment zone, representing a one-year continuous \u003cem\u003eD. indusiata\u003c/em\u003e cropping system, was arranged in triplicate mushroom beds (0.8 m wide \u0026times; 0.5 m high), separated by 0.6 m drainage channels. Soil volumetric water content was maintained at 55%-60% using automated irrigation systems (Fig. 1). In traditional \u003cem\u003eD.indusiata\u003c/em\u003e cultivation, post-harvest fungal residue is not removed or recycled but is instead directly incorporated into the soil prior to the following year\u0026apos;s planting, enabling continuous cultivation, production and experimentation trials of \u003cem\u003eD.indusiata\u003c/em\u003e or other crops. Based on varying application times, each test-initiated composting of seafood mushroom residue for 45 days before the experiment commenced, following the fermentation protocol described by Li et al. [19]. treatments are no SMS application (S0-24), application 1 month prior (S1-24), 2 months prior (S2-24) and 3 months prior (S3-24). The soil sampled before cultivation (S0-23) served as the baseline control. Amendments were applied uniformly at a rate of 1 ton per plot and mechanical mixed into the topsoil (20 cm depth) using a rotary tiller. Following established \u003cem\u003eD. indusiata\u003c/em\u003e cultivation protocols [20], the substrate was prepared through controlled fermentation initiated on 30\u003csup\u003eth\u003c/sup\u003e December 2023, consisting 49% \u003cem\u003eCenchrus fungigraminus\u003c/em\u003e, 49% sawdust, 1% urea, and 1% light calcium carbonate. The thermophilic phase (core temperature \u0026gt; 65 ℃) was maintained via three turning at 10-day intervals. Fermentation was deemed complete upon achieving stable mesophilic conditions (35 \u0026deg;C sustained for one week) and complete volatilization of ammonia [19]. Field inoculation of \u003cem\u003eD. indusiata\u003c/em\u003e was carried out on 25\u003csup\u003eth\u003c/sup\u003e February 2024, with fruiting body development and management occurring from June to July 2024. Georeferenced soil samples (5 per plot) were collected monthly throughout the growth cycle, starting from primordia initiation to final harvest. Surface soil (0-20 cm depth) was aseptically collected using stainless steel augers, immediately placed in sterile bags, and transported on dry ice to the laboratory. Post-collection processing included: (1) homogenization of composite samples, followed by dark-air-drying at 15 \u0026plusmn; 2 \u0026deg;C and sieving through a 2-mm mesh for physicochemical analysis and enzymatic assays (urease, dehydrogenase); and (2) cryopreservation of microbial subsamples at -80 \u0026deg;C for metagenomic sequencing. To ensure data accurate, \u003cem\u003eD. indusiata\u003c/em\u003e fruiting bodies were randomly sampled across all treatment groups. The experimental protocol spanned from 2023 to 2025, encompassing two complete cultivation cycles.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssessment of Yield, Agronomic Characteristic and Nutritional Composition in \u003cem\u003eD. indusiata\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe biological yield of \u003cem\u003eD. indusiata\u003c/em\u003e fruiting bodies over the production cycle was calculated The biological yield of \u003cem\u003eD. indusiata\u003c/em\u003e fruiting bodies over the production cycle was calculated based on fresh mushroom output per experimental plot: total yield (kg/hm\u003csup\u003e2\u003c/sup\u003e/day) = daily output (kg) \u0026times; 150.38 (hm\u003csup\u003e2\u003c/sup\u003e) (conversion factor for 66.5 m\u003csup\u003e2\u003c/sup\u003e to hm\u003csup\u003e2\u003c/sup\u003e) \u0026times; 60 d (harvesting period). Agronomic characterization of \u003cem\u003eD. indusiata\u003c/em\u003e basidiocarps was performed using precision instruments: individual fresh weight was measured using an analytical balance, while cap diameter and thickness, as well as stipe dimensions, were determined with digital calipers. Fresh fruiting bodies were dried in a forced-air oven (DHG-9240A, Shanghai Yiheng, China) at 40 \u0026deg;C for 2 h, followed by desiccation at 55 \u0026deg;C for 2 h, and finally stabilized at 40 \u0026deg;C for 24 h. The dried samples were ground under low temperature conditions and stored at low temperature prior to analysis for all target indices. For compositional analysis, the gravimetric combustion method described Jia et al. [21] was employed, involving incineration in muffle furnaces at 550 \u0026deg;C until ash mass reached constant weight. Total soluble sugars and crude polysaccharide were quantified using the phenol-sulfuric acid spectrophotometric method [22], and crude protein content was determined via the Kjeldahl method based on nitrogen content, using an automated distillation system (SKD-1800, Shanghai Peiou, China).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhysical and Chemical Properties of Soil for \u003cem\u003eD. indusiata\u003c/em\u003e Cultivation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal nitrogen (TN) content was determined using the digestion method [23]. Detection of ammonium nitrogen according to the indoxyl blue colorimetric method [24] Available phosphorus was extracted with Mehlich 3 solution and quantified colorimetrically at 880 nm following the Murphy and Riley method, while available potassium was measured by flame photometry at 766.5 nm as described by Knudsen. Organic carbon content was determined using the Walkley-Black wet oxidation procedure [25]. And soil organic matter (SOM) was estimated by multiplying organic carbon content by a conversion factor of 1.724. For pH determination, a standardized soil-to-water ratio of 1:2.5 (w/v) was prepared, followed vigorous stirring at 200 rpm for 30 min and a settling period for 10 min. The supernatant pH was then measured using a calibrated digital pH meter (PB-10, Beijing Sartorius Science, China).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of Soil Enzyme Activities in \u003cem\u003eD. indusiata\u003c/em\u003e Cultivation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSoil enzymatic activities were quantified using standardized colorimetric methods. Sucrase activity (S-SC) was determined by 3,5-dinitrosalicylic acid colorimetry (DNS) according to Li et al. [26]. Catalase activity (S-CAT) was measured using UV spectrophotometry as described by Du et al. [27]. Soil urease activity (S-UE) was assessed via the indophenol blue method [28], with enzyme activity expressed in units of \u0026mu;g NH\u003csub\u003e3\u003c/sub\u003e-N per gram of soil per day (U/g). Acid phosphatase activity (S-ACP) was quantified using the p-nitrophenyl phosphate (PNPP) method [29], and results were reported as \u0026mu;mol p-nitrophenol released per gram of soil per hour (\u0026mu;mol/g/h).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of Soil Phenolic Acid Metabolites in \u003cem\u003eD. indusiata\u0026nbsp;\u003c/em\u003eCultivation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSix phenolic acid standards (\u003cem\u003ep\u003c/em\u003e-hydroxybenzoic acid, vanillic acid, ferulic acid, \u003cem\u003ep\u003c/em\u003e-coumaric acid, syringic acid, and coumalic acid) were dissolved in 10 mL brown volumetric flasks and diluted with methanol to prepare standard solution with varying concentration gradients. The solutions were analyzed using a Waters high-performance liquid chromatography system (E2695, Waters, Milford, USA) equipped with a UranusC18 column (250 mm \u0026times; 4.6 mm \u0026times; 5 \u0026mu;m) and a guard column (20 mm \u0026times; 4.6 mm \u0026times; 5 \u0026mu;m). Detection was performed at 280 nm for the identification and quantification of phenolic acid in soil samples. Each analytical run lasted 90 min, with a 10-minute equilibration period between injections [30-31].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of Soil Microbial Diversity in \u003cem\u003eD. indusiata\u003c/em\u003e Cultivation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHigh-throughput sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to the manufacturer \u0026apos;s instructions, TGuide S96 magnetic soil DNA kit (Tiangen, Beijing, China) was used to extract genomic DNA (gDNA) from soil samples S0-23 in October 2023 before planting in the second year and soil samples S3-24, S2-24, S1-24, and S0-24 in June 2024 after planting in the second year. The integrity of gDNA was verified by 1.8% agarose gel electrophoresis, and concentration was determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, USA). The bacterial 16S rRNA gene hypervariable V3-V4 were amplified using primer pairs 338F (5\u0026apos;-ACTCCTACGGGAGGCAGCA-3\u0026apos;) and 806R (5\u0026apos;-GGACTACHVGGGTWTCTAAT-3\u0026apos;). For fungal community analysis, the ITS1 region employed primers ITS1F(5\u0026apos;-CTTGGTCATTTAGAGGAAGTAA-3\u0026apos;) and ITS2R ITS2(5\u0026apos;-GCTGCGTTCTTCATCGATGC-3\u0026apos;). The Polymerase Chain Reaction (PCR) reactions consisted of \u0026nbsp;5-50 ng DNA template, 0.3 \u0026mu;mol/L of each primers, 5 \u0026mu;L KOD FX Neo Buffer, 2 \u0026mu;L dNTP (2 mmol/L each), 0.2 \u0026mu;L KOD FX Neopolymerase, and ddH\u003csub\u003e2\u003c/sub\u003eO to a final volume of 20 \u0026mu;L. Thermal cycling conditions included an initial denaturation at 95 \u0026deg;C for 5 min, followed by 20 cycles of 95 \u0026deg;C for 30 s (denaturation), 50 \u0026deg;C for 30 s (annealing), and 72 \u0026deg;C for 40 s (extension), with a final extension at 72 \u0026deg;C for 7 min. Amplified products were purified (Omega Inc., Norcross, GA, USA), quantified using Qsep-400 fragment analyzer (BiOptic, Taiwan, China), and sequenced on the Illumina novaseq6000 platform (Biomarker, Beijing, China). Raw sequencing data were submitted to the National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/) with accession number PRJNA1234400 (publicly accessible on December 31\u003csup\u003est\u003c/sup\u003e, 2026).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBioinformatics Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBioinformatics analysis was conducted on BMK Cloud platform (http://www.biocloud.net/). Raw sequencing data were subjected to quality filtering using Trimmomatic (v0.33) [32]. Primer sequences were removed using Cutadapt [33] (v1.9.1). Paired-end reads were assembled in denovo mode using USEARCH [34] (v10), followed by chimera detection and removal with UCHIME. Final clean reads were clustered into operational taxonomic units (OTUs) at a 97% sequence similarity threshold using USEARCH [35] (v8.1). Taxonomy annotation of the OTUs was performed in QIIME2 (v2021) [36] using a Bayesian classifier aligned against the SILVA database (v138.1) [37] with a minimum confidence threshold of 70%. Alpha diversity indices (including Shannon, Chao1, Observed Species) were calculated in QIIME2 and visualized using phylogeny (v1.34.0) in R software (v4.1.2). The top 20 species with the highest abundance are selected, and the heatmap is generated using R\u0026apos;s pheatmap package. Each color block in the heatmap represents the abundance of a genus in a sample. Samples are arranged horizontally, while species are arranged vertically. Clustering in the heatmap allows us to understand the similarity between samples and the similarity in community composition across different taxonomic levels. The species distribution histogram was drawn by python2 (matplotlib-v1.5.1). FUNGuild fungal function prediction analysis was conducted using the Funguild (1.0) software and database, while bacterial phenotypic traits were predicted using Bugbase (0.1.0). Correlation network analysis is conducted based on the abundance and variation of species across samples. Spearman\u0026apos;s rank correlation analysis is performed (using the default method) to identify correlations with a magnitude greater than 0.1 and a p-value less than 0.05. These data are used to construct a correlation network.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn \u003cem\u003evitro\u003c/em\u003e Co-culture of \u003cem\u003eD. indusiata\u003c/em\u003e Mycelium with Soil Pathogens\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to Lei et al. [31], \u003cem\u003ep\u003c/em\u003e-coumaric acid at a concentration of 250 mg/L can promote the growth of\u003cem\u003e\u0026nbsp;D. indusiata\u003c/em\u003e mycelium. This experiment evaluated the growth response of \u003cem\u003eD. indusiata\u003c/em\u003e mycelium and major pathogenic microorganisms at this concentration. The top 50 microorganisms by abundance were screened from the continuous cropping soil of\u003cem\u003e\u0026nbsp;D. indusiata\u003c/em\u003e, including two fungi (\u003cem\u003eAspergillus fumigatus\u003c/em\u003e and \u003cem\u003ePenicillium simplicissimum\u003c/em\u003e) and two bacteria (\u003cem\u003eBacillus\u003c/em\u003e sp. and \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e), which were selected for co-culture assays. After autoclaving, the solid PDA medium was cooled to 60-70 \u0026deg;C, then supplemented with 250 mg/L of \u003cem\u003ep\u003c/em\u003e-coumaric acid that had been filter through a 0.22 \u0026mu;m membrane, mixed thoroughly, and poured into Petri dishes. Once the medium had cooled and solidified, mycelial plugs of equal size were inoculated onto the plates using a sterile inoculation tool; for bacteria strains, a small volume of standardized bacterial suspension was used for spot inoculation. The control group consisted of PDA plates without \u003cem\u003ep\u003c/em\u003e-coumaric acid, inoculated with the same \u003cem\u003eD. indusiata\u003c/em\u003e and pathogenic microorganisms under identical conditions. All treatments were incubated at 26 \u0026deg;C for 21 d with six replicates per treatment, and fungal growth was monitored regularly throughout the incubation period.\u003c/p\u003e\n\u003cp\u003eIn addition, single colonies of \u003cem\u003eAspergillus fumigatus\u003c/em\u003e, \u003cem\u003ePenicillium simplicissimum\u003c/em\u003e, \u003cem\u003eBacillus\u003c/em\u003e sp.\u003cem\u003e\u0026nbsp;\u003c/em\u003eand \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e were individually picked from solid PDA medium and inoculated into 100 mL of liquid PDA medium. Cultures were incubation in a shaker at 25 \u0026deg;C and 200 rpm for 3 d. Upon completion of incubation, the bacterial or fungal suspensions were filtered through eight layers of sterile gauze to remove mycelial clumps or cellular aggregates. Subsequently, 2 g agar powder was added to every 100 mL of the filtered suspension, followed by autoclaving at high-temperature to prepare solidified plates. After cooling and solidification, mycelial plugs of \u003cem\u003eD. indusiata\u003c/em\u003e were inoculated onto the plates and cultured at 26 \u0026deg;C for 21 d, with fungal growth monitored regularly. Each treatment was performed in six replicates.\u003c/p\u003e\n\u003cp\u003eFresh mycelium of \u003cem\u003eD. indusiata\u003c/em\u003e was collected under different treatment conditions and immediately immersed in 5% glutaraldehyde solution, followed by fixation at 4 \u0026deg;C for 4 h. The samples were then rinsed three times with 0.2 mol/L phosphate buffer, 10-15 min per rinse. Post-fixation was carried out with 1% tart acid at 4 \u0026deg;C for 4 h. After completion of fixation, the samples were rinsed three times with distilled water, each for 10-15 min. Gradient dehydration was performed sequentially using 50%, 70%, 80% and 90% ethanol solutions, with 10-15 min incubation at each concentration; this was followed by two changes of 100% ethanol, each lasting 10-15 min. Subsequently, the samples were treated twice with propylene oxide to facilitate resin infiltration. Finally, the specimens were processed in a freeze-dryer (HCP-2, HITACHI, Japan). Dried samples were mounted on stubs and coated with a thin conductive layer using an ion sputter coater (IB-5, EIKO, Japan), after which they were examined and imaged under a scanning electron microscope (JSM-6380LV, JEOL, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analysis was conducted using Microsoft Office Excel 2019 (Microsoft Corporation, WA, USA) for data organization, while significance testing was performed using Turkey\u0026rsquo;s test within a one-way ANOVA (Analysis of Variance) framework in SPSS 26.0 (IBM Corporation, New York, USA). Data visualization was achieved through an integrated pipeline combining Graph Pad Prism 9 (GraphPad Software, CA, USA) and Adobe Photoshop CC 2018 (Adobe, CA, USA) to generate publication-quality figures. R language was used for bacterial and fungal metabolic pathway statistics and partial least squares path modeling (PLS-PM).\u0026nbsp;\u003c/p\u003e"},{"header":"Result","content":"\u003cp\u003e\u003cstrong\u003eEffects of Different Treatments on Yield and Quality of Continuous \u003cem\u003eD. indusiata\u003c/em\u003e Fruiting Bodies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to Table 1, the S3-24 treatment with SMS achieved the highest \u003cem\u003eD. indusiata\u003c/em\u003e yield indices\u003cem\u003e,\u003c/em\u003e with both single fruit weight and acreage yield following the order S3-24 \u0026gt; S2-24 \u0026gt; S1-24 \u0026gt; S0-24. The acreage yields of S3-24, S2-24 and S1-24 increased by 745.07%, 496.99% and 387.05%, respectively, compared to S0-24 (P \u0026lt; 0.05). However, relative to the previous year\u0026rsquo;s baseline (S0-23), the acreage yields of S3-24, S2-24, S1-24 and S0-24 decreased by 4.48%, 32.52%, 44.95%, and 88.70%, respectively. Nutrient analysis revealed that S3-24 and S1-24 exhibited elevated total sugar contents (4.11% and 4.84%), while the samples from S3-24, S2-24, and S1-24 demonstrated significant increases in crude polysaccharide (32.42%, 6.99%, and 3.09%) and crude protein (1.60%, 1.60%, and 0.57%) relative to untreated controls. Notably, S3-24 displayed the lowest ash content, indicating higher organic matter and reduced heavy metal accumulation [38]. A three-month continuous treatment effectively alleviated second-year continuous cropping obstacles, significantly enhancing both the yield and quality of \u003cem\u003eD. indusiata\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e Yield and quality variations in \u003cem\u003eD. indusiata\u003c/em\u003e fruiting bodies under second-year continuous cropping conditions\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTreatments\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFirst year\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003etotal yield\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(kg/hm\u003csup\u003e2\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSecond year\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003etotal yield\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(kg/hm\u003csup\u003e2\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIndividual Substrate Weight\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(g)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTS\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(mg/g)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCPS\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(g/100 g)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCP\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(g/100 g)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAsh\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003eS3-24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" style=\"width: 85px;\"\u003e\n \u003cp\u003e13357.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e12758.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e19.61\u0026plusmn;0.69\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e547.66\u0026plusmn;4.11\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e11.56\u0026plusmn;1.09\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003e24.81\u0026plusmn;0.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e7.85\u0026plusmn;0.36\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003eS2-24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e9013.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e17.66\u0026plusmn;0.94\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e523.56\u0026plusmn;1.84\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e9.34\u0026plusmn;0.29\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003e24.81\u0026plusmn;0.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e10.73\u0026plusmn;0.29\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003eS1-24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e7353.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e18.37\u0026plusmn;0.42\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e551.43\u0026plusmn;6.47\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e9.00\u0026plusmn;0.03\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003e24.56\u0026plusmn;0.13\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e10.22\u0026plusmn;0.15\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003eS0-24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e1509.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e17.04\u0026plusmn;0.55\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e526.05\u0026plusmn;7.06\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e8.73\u0026plusmn;0.54\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003e24.42\u0026plusmn;0.09\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e8.64\u0026plusmn;0.17\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eNotes: TS denotes total sugars; CPS denotes crude polysaccharides; and CP denotes crude proteins. Different lowercase letters within the same column indicate significant differences (P \u0026lt; 0.05). This notation applies to all subsequent tables.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSoil Physicochemical Dynamics Under Continuous \u003cem\u003eD. indusiata\u003c/em\u003e Cropping Systems\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs a predominant inorganic nitrogen species, soil ammonium nitrogen (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N) governs nitrogen availability and crop productivity. Fig. 2A shown that NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N level increased following SMS amendments during the initial phase, with increases of +120.62% (S3-24), 20.86% (S2-24) and 15.65% (S1-24) relative to the original soil (S0-23). Notably, subsequent depletion of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N coincided with \u003cem\u003eD. indusiata\u003c/em\u003e growth cycles, resulting in concentrations below baseline levels across all treatments-most markedly in S3-24, which exhibited 41.2% reduction-suggesting rhizosphere feedback mechanisms contribute to continuous cropping constraints. Fig. 2B revealed paradoxical potassium dynamics in soils amended with SMS. Although available potassium increased in June across all treatments compared to the control (S3-24: +120.62%; S2-24: +20.86%; S1-24: +15.65%), \u003cem\u003eD. indusiata\u003c/em\u003e cultivation triggered progressive K depletion thereafter, with high-yield groups showing particularly severe losses (S3-24: -41.2% by harvest). This bidirectional nutrient flux indicated initial amendment-mediated K mobilization followed by crop-driven depletion exceeding soil replenishment capacity. Plant-available phosphorus, a critical component of soil fertility, plays essential roles in energy metabolism and root development. As shown in Fig. 2C, available phosphorus content increased over time in all SMS-treated soil compared to S0-23, whereas it declined progressively in S0-24 decreased monthly. By June 2024, available phosphorus levels in the treated soils (S3-24, S2-24, and S1-24) had increased by 87.34%, 79.90%, and 110.08%, significantly higher than in S0-23. These findings suggested that \u003cem\u003eD. indusiate\u0026nbsp;\u003c/em\u003ecultivation enhanced the mobilization and stabilization of soil phosphorus pools. Total nitrogen is a key indicator of soil fertility. Fig. 2D shown that, in June, total nitrogen increased by 14.14% (S3-24) and 0.27% (S1-24) compared to the control, but decreased by 28.09% (S2-24) and 29.95% (S0-24). Over time, nitrogen content initially declined and then recovered, indicating consumption during \u003cem\u003eD. indusiata\u003c/em\u003e growth. Supplementing SMS in the second year of cultivation, depending on duration and dosage, improves soil nitrogen status and supports balanced nutrient management. Soil organic matter plays a critical role in supplying essential nutrients, improving soil structure and enhancing water retention capacity. However, as shown in Fig. 2E, at the end of \u003cem\u003eD. indusiata\u0026nbsp;\u003c/em\u003egrowth cycle (June 2024), organic matter content in SMS-treated soils were lower than in S0-23, with the extent of reduction following the order: S0-24 \u0026gt; S1-24 \u0026gt; S2-24 \u0026gt; S3-24. These results suggested that \u003cem\u003eD. indusiata\u003c/em\u003e treatments, particularly when combined with SMS, can mitigate the decline in soil organic matter under continuous cropping. Soil acidification is a major constraint in \u003cem\u003eD. indusiata\u003c/em\u003e continuous cropping obstacle. However, as illustrated in Fig. 2F, SMS incorporation effectively stabilizes pH in continuously cropping soil and alleviated acidification, with S3-24 exhibiting the most pronounced effect.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of Treatments on Enzyme Activities in \u003cem\u003eD. indusiata\u003c/em\u003e Continuous Cropping Soil\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFig. 2G-J illustrated the variations in enzymatic activity across all treatments. Among the four enzymes analyzed, only acid phosphatase in S3-24 exhibited increased activity compared to S0-23. Specially, soil acid phosphatase activity in S3-24 during June increased significantly by 37.84% relative to S0-23. In contrast, the enzyme activities in all other experimental groups were lower than those in S0-23. Notably, except for available phosphorus, the S3-24 showed higher enzyme activity than the other treatments at the end of entire growth cycle. Fig. 2H and I showed that sucrase decreased as the growth cycle of continuous \u003cem\u003eD. indusiate\u003c/em\u003e extended. In June, catalase activities in S3-24, S2-24, S1-24 and S0-24 were reduced by 17.02%, 32.85%, 20.4% and 36.58%, respectively, compared to S0-23. Sucrase activity also declined in S3-24, S2-24 and S1-24 by 31.04%, 31.6% and 41.66%, respectively. These results indicated that the continuous cropping obstacle significantly affected catalase and sucrase activities, thereby impairing organic carbon decomposition and energy supply for \u003cem\u003eD. indusiata\u003c/em\u003e mycelium and fruiting body development. However, monthly comparisons revealed that sucrase activity in S3-24 remained consistently higher throughout the cultivation period compared to S0-23, suggesting SMS application positively enhance soil enzymatic activity. Urease plays a key role in enhancing soil nitrogen availability. As showed in Fig. 2J, changes in urease activity during continuous \u003cem\u003eD. indusiata\u0026nbsp;\u003c/em\u003ecultivation were\u003cem\u003e\u0026nbsp;\u003c/em\u003enot insignificant across treatments. Overall, urease activities in S3-24, S2-24, S1-24 and S0-24 decreased by 0.34%, 19.22%, 10.29% and 2.74% compared to the previous year\u0026apos;s S0-23. Nevertheless, after completion of the full growth cycle, S3-24 exhibited urease activity closest to that of the original soil levels.\u003c/p\u003e\n\u003cp\u003eIn summary, compared with S0-23, soil enzyme activity declined at the end of \u003cem\u003eD. indusiata\u0026nbsp;\u003c/em\u003efruiting body period due to continuous cropping obstacles\u003cem\u003e.\u0026nbsp;\u003c/em\u003eThe S3-24 showed the smallest reduction in enzyme activity, whereas the S0-24 exhibited significantly lower activities of acid phosphatase, catalase and sucrase. Therefore, the addition of SMS promoted soil enzyme activity under continuous cropping condition, with the most pronounced positive effect observed in S3-24.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of Different Treatments on Phenolic Acid of \u003cem\u003eD. indusiata\u003c/em\u003e Continuous Cropping Soil\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter \u003cem\u003eD. indusiata\u003c/em\u003e continuous soil treated with SMS, at the end of the full growth cycle (in June 2024), phenolic acid levels in all experimental groups were significantly higher than in S0-23 (P \u0026lt; 0.05). Specifically, total phenolic acid content increased by 2.07-, 2.23-, 1.08-, and 1.77- fold in S3-24, S2-24, S1-24, and S0-24, respectively, compared to S0-23. In February 2024, SMS-treated groups (S1-24, S2-24, S3-24) showed lower total phenolic acid than S0-23, with \u003cem\u003ep\u003c/em\u003e-coumaric acid decreasing by 33.51%, 37.01 %, and 54.50%, respectively. After three months, phenolic acid accumulation was higher in the SMS-treated compared to S0-24, with \u003cem\u003ep\u003c/em\u003e-coumaric acid increasing by 48.63%, 11.59%, and 179.14% in S1-24, S2-24, and S3-24, respectively (Fig. 3). From February to June 2024, the dynamic changes in phenolic acids and \u003cem\u003ep\u003c/em\u003e-coumaric acid suggested that distiller\u0026apos;s grains improve \u003cem\u003eD. indusiata\u003c/em\u003e growth conditions by modulating phenolic acid content, with \u003cem\u003ep\u003c/em\u003e-coumaric acid potentially being a key factor influencing continuous cropping.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of Different Treatments on Bacterial and Fungal Diversity in \u003cem\u003eD. indusiata\u003c/em\u003e Continuous\u003c/strong\u003e \u003cstrong\u003eCropping Soil\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOUT analysis of soil microorganisms\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe five treatments (S0-23, S3-24, S2-24, S1-24, and S0-24) each consisted of five replicates, yielding a total of 373,690 original sequences. After filtering out low-quality and short sequences, 318,701 high-quality sequences were retained, with respective counts of 57,479, 64,597, 66,271, 65,277 and 65,076, accounting for 17.91%, 20.27%, 20.80%, 20.48% and 20.42% of the total. The bacterial classification analysis showed 50 phyla, 137 classes, 403 orders, 907 families and 2069 genera. The classification of fungi identified 14 phyla, 46 classes, 95 orders, 203 families and 369 genera (Table S1 and S2). A venn diagram showed that 18 bacterial OUTs across all treatments (Fig. S1A). In fungal ITS sequence analysis, a total of 359,800 original sequences were generated from the five treatments. Following quality filtering, 316,007 high-quality sequences remained, with counts of 67,363, 62,733, 64,702, 67,352 and 53,856, representing 21.32%, 19.85%, 20.47%, 21.31% and 17.04% of the total. The Venn diagram revealed that 13 shared fungal OUTs across treatments (Fig. S1B). For soil microbial sequencing, random sampling was used to generate dilution curve (Wang et al., 2012) and species accumulation curves (Specaccum). According to Fig.S2 A and B, the rarefaction curves approached saturation with increasing sequencing depth, indicating sufficient sequencing data and that the data adequately represent microbial community changes in the samples.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of bacterial and fungi diversity in\u003cem\u003e\u0026nbsp;D. indusiata\u0026nbsp;\u003c/em\u003econtinuous cropping soil\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs showed in Table 2, Table S3 and S4), compared to S0-23, bacterial and fungal richness, diversity and evenness were significantly enhanced in all treatments (S3-24, S2-24, S1-24, S0-24) during the second year. Specifically, Chao1, ACE and Shannon indices were higher than those of S0-23. For bacteria, the abundance and evenness in S3-24, S2-24, S1-24 and S0-24 increased by 41.93%, 39.66%, 45.75% and 44.62%, relative to S0-23. The Ace and Chao1 indices indicated that bacterial diversity was highest in S2-24, followed by S1-24 \u0026gt; S3-24 \u0026gt; S0-24 \u0026gt; S2-24. For fungi, the Chao1, ACE and Shannon indices were higher in all treated samples compared to S0-23, with increases in abundance and evenness of 11.19% (S3-24), 1.88% (S2-24), 7.85% (S1-24) and 11.43% (S0-24). Notably, fungal diversity as reflected by the Ace and Chao1 indices was the highest in S2-24, followed by S2-24 \u0026gt; S1-24 \u0026gt; S0-24 \u0026gt; S3-24. These results suggested that applying SMS 1-2 months in advance can significantly enhance bacteria and fungi diversity in sustainable cropping soil of\u003cem\u003e\u0026nbsp;D. indusiata\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e The impact of different treatments on soil microbial diversity for cultivated \u003cem\u003eD. indusiata\u003c/em\u003e\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"115%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTreatments\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eChaol\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSimpson\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 122px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eShannon\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eACE\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"5\" style=\"width: 65px;\"\u003e\n \u003cp\u003eBacterial\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003eS0-23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e2292.48\u0026plusmn;371.88\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e0.93\u0026plusmn;0.0035\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 122px;\"\u003e\n \u003cp\u003e7.06\u0026plusmn;0.12\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e2295.24\u0026plusmn;371.18\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003eS3-24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e2616.81\u0026plusmn;59.45\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e1\u0026plusmn;0.0005\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 122px;\"\u003e\n \u003cp\u003e10.02\u0026plusmn;0.074\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e2623.83\u0026plusmn;60.53\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003eS2-24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e2523.87\u0026plusmn;367.08\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e1\u0026plusmn;0.0008\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 122px;\"\u003e\n \u003cp\u003e9.86\u0026plusmn;0.17\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e2533.03\u0026plusmn;368.31\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003eS1-24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e2687.57\u0026plusmn;361.73\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e1\u0026plusmn;0.0014\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 122px;\"\u003e\n \u003cp\u003e10.29\u0026plusmn;0.27\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e2694.08\u0026plusmn;363.19\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003eS0-24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e2541.68\u0026plusmn;160.33\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e1\u0026plusmn;0.0002\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 122px;\"\u003e\n \u003cp\u003e10.21\u0026plusmn;0.12\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e2546.34\u0026plusmn;159.65\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"5\" style=\"width: 65px;\"\u003e\n \u003cp\u003eFungal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003eS0-23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e103.4\u0026plusmn;97.30\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e0.97\u0026plusmn;0.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 122px;\"\u003e\n \u003cp\u003e5.86\u0026plusmn;1.08\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e103.49\u0026plusmn;97.26\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003eS3-24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e181.5\u0026plusmn;31.78\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e0.97\u0026plusmn;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 122px;\"\u003e\n \u003cp\u003e6.18\u0026plusmn;0.29\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e181.52\u0026plusmn;31.77\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003eS2-24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e249.48\u0026plusmn;41.19\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e0.94\u0026plusmn;0.02\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 122px;\"\u003e\n \u003cp\u003e5.97\u0026plusmn;0.27\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e249.98\u0026plusmn;41.54\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003eS1-24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e211.22\u0026plusmn;88.14\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e0.97\u0026plusmn;0.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 122px;\"\u003e\n \u003cp\u003e6.32\u0026plusmn;0.53\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e211.33\u0026plusmn;88.43\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003eS0-24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e186.44\u0026plusmn;23.28\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115px;\"\u003e\n \u003cp\u003e0.98\u0026plusmn;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 122px;\"\u003e\n \u003cp\u003e6.53\u0026plusmn;0.23\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e186.69\u0026plusmn;23.34\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eNotes: Each index is expressed as the mean \u0026plusmn; standard deviation. Different lowercase letters within the same data column indicate significant differences (P \u0026lt; 0.05; one-way ANOVA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of The Abundance of Species in Soil Bacterial and Fungal Community Structure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylum-level abundance of soil microbial communities\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Fig. 4A, the dominant bacterial phyla include Proteobacteria (21.58%-35.47%), Acidobacteriota (2.07%-23.39%), Actinobacteriota (5.41%-11.99%), Firmicutes (2.84%-9.29%), Bacteroidota (3.30%-6.07%), Chloroflexi (2.80%-5.68%). Among these, Proteobacteria, Acetobacteria and Actinomycete are the predominant bacterial groups with relatively high abundance. The relative abundance of Proteobacteria is S3-24 \u0026gt; S1-24 \u0026gt; S2-24 \u0026gt; S0-24 \u0026gt; S0-23; the Acetobacteria as S2-24 \u0026gt; S0-24 \u0026gt; S3-24 \u0026gt; S1-24 \u0026gt; S0-23; the Actinomycete as S1-24 \u0026gt; S2-24 \u0026gt; S0-24 \u0026gt; S3-24 \u0026gt; S0-23. Following SMS treatment, the abundance of the top three dominant bacterial phyla (Proteobacteria, Acetobacteria and Actinomycete) in the second-year experimental treatments were higher than that in S0-23. As shown in Fig. 4B, the fungal community is primarily composed of Ascomycota (41.52%-50.49%), Mortierellomycota (18.13%-24.59%), Basidiomycota (10.76%-24.02%), Rozellomycota (0.31%-24.02%), and Chytridiomycota (1.19%-2.30%). Among these, Ascomycetes is the most abundant fungal phylum, with relative abundance decreasing in the order as S0-23 \u0026gt; S1-24 \u0026gt; S0-24 \u0026gt; S3-24 \u0026gt; S2-24, and plays a critical role in soil organic matter decomposition, crop disease suppression, and soil restoration. Basidiomycota includes saprophytic, symbiotic and parasitic fungi, many of which from beneficial associations with crop root system by enhancing minerals uptake. Saprophytic fungi contribute to increase soil phosphatase activity and reduce pest incidence. However, certain species within Chytridiomycota may be pathogenic and potentially contribute to crop diseases.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenus-level abundance of soil microbial communities\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe dominant bacterial genera include \u003cem\u003eBacillus\u003c/em\u003e (0.59%-7.52%), \u003cem\u003ePseudarthrobacter\u003c/em\u003e (0.08%-6.14%), \u003cem\u003eEllin\u003c/em\u003e (0.03%-1.71%), \u003cem\u003eBryobacter\u003c/em\u003e (0.21%-1.51%), and \u003cem\u003eAcinetobacter\u003c/em\u003e (0.052%-1.58%). Among these, \u003cem\u003eBacillus\u003c/em\u003e showed the highest relative abundance, with levels decreasing in S0-24 \u0026gt; S1-24 \u0026gt; S2-24 \u0026gt; S3-24 \u0026gt; S0-23 (Fig.4C). The longer the SMS treatment duration, the more closely the soil bacterial community composition approaches that of S0-23 following \u003cem\u003eD. indusiata\u0026nbsp;\u003c/em\u003ecultivation. As shown in Fig. 4D, the predominant fungal genera include \u003cem\u003eMortierella\u003c/em\u003e (17.66%-28.36%), \u003cem\u003eFusarium\u003c/em\u003e (1.69%-7.06%), \u003cem\u003eBotryotrichum\u003c/em\u003e (0.71%-8.02%). Compared to S0-23, the abundance of \u003cem\u003eThermomyces\u003c/em\u003e in S1-24 and S2-24 increased by 21.3% and 24.5%, respectively, indicating a significant enhancement in beneficial fungal population with 1-2 months after SMS treatment. Meanwhile, the abundance of\u003cem\u003e\u0026nbsp;Fusarium\u003c/em\u003e decreased by 48.4%, 76.1%, 15.8% and 21.3% in S0-24, S1-24, S2-24, and S3-24, respectively, suggesting that SMS exerts an inhibitory effect on this genus, potentially reducing plant diseases incidence and contributing to improve soil microbial ecological balance [39].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunctional Prediction of Microbial Communities\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBugBase predicts functional pathway coverage and biologically interpretable phenotypes in complex microbial communities by normalizing OUT abundances based on 16S rRNA gene copy numbers and using pre-calculated reference files for phenotype prediction [40]. Bacterial phenotype results after two years of continuous cropping are shown in Fig. S3A. Among the top 10 phenotypes, ranked from lowest to highest abundance: facultative anaerobes (1.3%-3.5%), Gram-positive bacteria (1.7%-4.7%), anaerobic bacteria (1.9%-6.0%), aerobic bacteria (9.4%-12.1%), potential pathogens (9.8%-17.8%), stress-tolerant bacteria (10.9%-17.4%), mobile element-containing bacteria (11.3%-19.8%), biofilm-forming bacteria (15.4%-20.2%), and Gram-negative bacteria (16.9%-21.4%). This order shows the clear abundance hierarchy of bacterial phenotypes in the samples.\u003c/p\u003e\n\u003cp\u003eFUNGuild is a bioinformatics tool that classifies fungal communities by linking taxonomy to ecological functions [41]. Fungi are grouped into three nutritional modes: pathotrophic, symbiotrophic, and saprotrophic. Based on these, ten functional groups are identified in Fig. S3B. Ranked by increasing abundance: litter saprotrophs (0-20.73%), plant saprotrophs (0.07%-6.82%), fungal parasites (0.08%-6.06%), animal pathogens (0.09%-10.29%), plant pathogens (0.53%-23.13%), endophytes (0.88%-24.34%), dung saprotrophs (0.91%-8.13%), soil saprotrophs (1.37%-12.25%), wood saprotrophs (12.59%-26.23%), and undefined saprotrophs (19.85%-59.55%). Undefined saprotrophs are the most abundant group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of the Correlation between Environmental Factors and Soil Microbial Communities\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the Pearson correlation coefficient, the relationships between the relative abundances of top 20 bacterial and fungal genera and soil environmental factors were analyzed. As showed in Fig.5A-C, \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003eNitrospira\u003c/em\u003e, and \u003cem\u003ePseudarthrobacter\u003c/em\u003e exhibited significantly positively correlation with ferulic acid, vanillic acid and syringic acid, but negative correlations with AN, pH, SUC, ACP and URE (P \u0026lt; 0.05). Acinetobacter showed significant positive correlations with AN, pH, TN, CAT, SUC and URE, while significant negative correlations with coumalic acid and \u003cem\u003ep\u003c/em\u003e-coumaric acid (P \u0026lt; 0.05). Pseudolabrys was significantly positively correlated with \u003cem\u003ep\u003c/em\u003e-coumaric acid and AP, while negatively correlated with AN, OM, pH, TN, CAT, SUC, ACP and URE (P \u0026lt; 0.05). In Fig. 5D-F, \u003cem\u003eBotryotrichum\u003c/em\u003e, \u003cem\u003eAgaricomycetes\u003c/em\u003e, \u003cem\u003eGelasinospora\u003c/em\u003e and \u003cem\u003ePseudaleuria\u003c/em\u003e were significantly positively correlated with vanillic acid, ferulic acid\u0026nbsp;and syringic acid, but negatively correlated with pH, ACP and SUC (P \u0026lt; 0.05). \u003cem\u003eThermomyces\u003c/em\u003e exhibited significant positive correlations with coumalic acid,\u0026nbsp;ferulic acid\u0026nbsp;and AP, while showing a significant negative correlation with URE (P \u0026lt; 0.05). \u003cem\u003eFusarium\u003c/em\u003e was significantly positively correlated with pH, ACP and SUC, but negatively correlated with vanillic acid, ferulic acid and syringic acid (P \u0026lt; 0.05). Penicillium showed a significant positive correlation with OM, and significant negative correlations with vanillic acid, ferulic acid and syringic acid (P \u0026lt; 0.05). \u003cem\u003eHumicola\u003c/em\u003e was significantly positively correlated with AP, but negatively correlated with pH, AN, CAT, SUC, ACP and URE (P \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003eA multi-index network model in R was used to assess how application timing of SMS affects soil ecosystem structure, incorporating soil physicochemical properties, enzyme activity, phenolic acids, and fungal and bacterial communities. All treatments had goodness-of-fit (GoF) values \u0026gt; 0.65, confirming reliable model fit and strong representation of direct interactions among variables (Fig. 6). In the no-SMS control (S0-24; Fig. 6A), enzyme activity strongly promoted phenolic acid accumulation (path coefficient: 11.67) and moderately increased fungal growth (1.89). Soil properties boosted phenolic acids (1.78) but suppressed enzyme activity (-0.93). With one-month SMS application (S1-24; Fig. 6B), phenolic acids inhibited bacteria (-11.38) but stimulated fungi (3.63), while enzyme activity drove phenolic acid buildup (2.62). Two months before planting (S2-24; Fig. 6C), soil properties strongly enhanced fungal growth (114.26), and phenolic acids continued to suppress bacteria (-2.61). Three months before planting (S3-24; Fig. 6D), soil properties boosted enzyme activity (16.29) and fungi (4.77), with minimal effect on bacteria (-0.98). Enzyme activity strongly increased phenolic acids (19.58), which promoted fungi (1.16) and suppressed bacteria (-4.37), this result coincides with the findings of the in \u003cem\u003evitro\u003c/em\u003e culture experiments. Bacteria enhanced fungal growth (5.25), while fungi reduced phenolic acid accumulation (-3.84), indicating feedback. After \u003cem\u003eD. indusiata\u003c/em\u003e cultivation (Fig. 6E), enzyme activity decreased (-0.86) and fungal diversity rose (1.04), showing cropping reshapes microbial communities.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eD. indusiata\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Continuous Cropping Soil on Microbial Network Structure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to Fig. 7A-E, the dominant bacterial taxa in S0-23 were \u003cem\u003eProteobacteria\u003c/em\u003e, \u003cem\u003eEscherichia\u003c/em\u003e, and \u003cem\u003eShigella\u003c/em\u003e (Crenarchaeota). Chronic rhizobacteria and salt-tolerant rhizobacteria showed significantly negatively correlations, whereas \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eAlistipes\u003c/em\u003e exhibited positive association. In S0-24, key bacterial genera included \u003cem\u003eCupriavidus\u003c/em\u003e, \u003cem\u003eLysobacter\u003c/em\u003e, \u003cem\u003eNitrospirota\u003c/em\u003e, and \u003cem\u003ePseudomonas\u003c/em\u003e, among which \u003cem\u003eNitrospiromonas\u003c/em\u003e was negatively correlated with \u003cem\u003ePseudomonas\u003c/em\u003e. In S1-24,\u003cem\u003e\u0026nbsp;Ellin\u003c/em\u003e6067 (an ammonia-oxidizing bacterium) was linked to 19 other genera and played a pivotal role in the nitrogen cycle. \u003cem\u003ePseudoArganomali\u003c/em\u003e and \u003cem\u003eBacillus\u003c/em\u003e displayed the highest relative abundances. In S2-24, Dongia (within the Proteobacteria), Actinobacteriota, and Sideroxydans were positively correlated. In S3-24, predominant genera included Dongia, Gemmatimonadota and Candidatus rokubactera bacterium (phylum Methylomirabilota). Flavobacterium was positively associated with Phenylbacterium, while Viagra showed a negative correlation with Acidbacillus.\u003c/p\u003e\n\u003cp\u003eAs shown in Fig. 7F-J, in S0-23, Basidiomycota was negatively correlated with Ascomycota and Mortierellomycota. In S0-24, Mortierellomycota was primarily negatively correlated with Ascomycota and Chytridiomycota, whereas Ascomycota showed a positive correlation with \u003cem\u003eOlpidiomycota\u003c/em\u003e. Nodes 36 and 42 (\u003cem\u003eFusarium\u003c/em\u003e), representing typical plant pathogens associated with blight and root rot, were negatively correlated with regulatory fungal taxa (Nodes 8-13). The \u003cem\u003eCurvularia\u0026nbsp;\u003c/em\u003einfluenced the fungal community through positively correlated nodes 22-25. In S1-24, \u003cem\u003eSolicoccozyma\u003c/em\u003e (within Ascomycota) exhibited a significant positive correlation with nodes 8-15. In S2-24, Mortierellomycota and Ascomycota dominated and exerted predominantly negative regulatory effects. In S3-24, Mortierellomycota displayed widespread negative correlations, while \u003cem\u003eHannaella\u003c/em\u003e (under Ascomycota and Chytridiomycota) was positively correlated with \u003cem\u003eGibellulopsis\u003c/em\u003e, including pathogenic members within this clade.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn \u003cem\u003evitro\u003c/em\u003e coculture experimental of \u003cem\u003eD. indusiata\u003c/em\u003e with pathogenic microorganisms\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnder 250 mg/L phenolic acid conditions, the mycelial growth of\u0026nbsp;\u003cem\u003eD. indusiata\u003c/em\u003e was enhanced with hyphal diameters ranging from 2.4 to 4.8 \u0026mu;m (Fig. 8M), larger than those in the control (2.1-4.1 \u0026mu;m, Fig. 8N). The mycelium appeared thicker and more robust following phenolic acid treatment. In co-cultivation with \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e and\u0026nbsp;\u003cem\u003eD. indusiata\u003c/em\u003e exhibited\u0026nbsp;rapid growth and formed white hyphae of phenolic acid presence (Fig. 8B and C). In contrast, when cultured with heat-inactivated \u003cem\u003eA. baumannii\u003c/em\u003e, mycelial growth was severely inhibited, showing sparse and underdeveloped hyphae (Fig. 8A). These findings demonstrated that substances associated with heated-inactivated bacterial cells retain strong inhibitory effects on\u0026nbsp;\u003cem\u003eD. indusiata\u003c/em\u003e development.\u003c/p\u003e\n\u003cp\u003eIn the co-culture of\u0026nbsp;\u003cem\u003eD. indusiata\u003c/em\u003e and \u003cem\u003eBacillus\u0026nbsp;\u003c/em\u003esp., mycelium growth was significantly inhibited in the absence of phenolic acid and without \u003cem\u003eBacillus\u003c/em\u003e sp. inactivation, with minimal visible growth (diameter 1.14-3.41 \u0026mu;m, Fig. 8D and E). Upon addition of phenolic acid to the system, growth remained strongly suppressed by \u003cem\u003eBacillus\u0026nbsp;\u003c/em\u003esp., exhibiting even narrower hypal diameters (0.68-3.18 \u0026mu;m, Fig. 8F).\u003c/p\u003e\n\u003cp\u003eUnder both inactivated and co-culture conditions with \u003cem\u003ePenicillium simplicissimum\u003c/em\u003e, exerted a significant inhibitory effect on \u003cem\u003eD. indusiata\u003c/em\u003e mycelium growth (2-4.5 \u0026mu;m, Fig. 8G-I). Notably, in the absence of phenolic acid demonstrated a stronger inhibitory effect, resulting in severe fragmentation of \u003cem\u003eD. indusiata\u0026nbsp;\u003c/em\u003emycelia, which impaired their ability to extend outward and\u0026nbsp;absorb nutrients effectively (1-4.5 \u0026mu;m, Fig. 8H).\u003c/p\u003e\n\u003cp\u003eIn the inactivated medium of \u003cem\u003eAspergillus fumigatus\u003c/em\u003e, the hyphal growth of \u003cem\u003eD. indusiata\u003c/em\u003e was significantly inhibited, as evidenced by a reduced diameter (2-2.5 \u0026mu;m) and irregular morphology (Fig. 8J). In dual cultures of \u003cem\u003eA. fumigatus\u003c/em\u003e and \u003cem\u003eD. indusiata\u003c/em\u003e without phenolic acid, mycelial development of \u003cem\u003eD. indusiata\u003c/em\u003e was severely restricted, characterized by limited spatial coverage, pale pigmentation, sparse distribution, and a loosely organized, compressed, and deformed hyphal network (2.8-3.2 \u0026mu;m, Fig. 8K). Upon addition of phenolic acid, the inhibitory effects on \u003cem\u003eD. indusiata\u003c/em\u003e were partially alleviated, with improved colony coverage, enhanced whiteness, and partial restoration of hyphal diameter to 3.3-3.6 \u0026mu;m (Fig. 8L). Nevertheless, under these conditions, the mycelial growth of \u003cem\u003eA. fumigatus\u003c/em\u003e remained markedly superior to that of \u003cem\u003eD. indusiata\u003c/em\u003e.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cb\u003eSMS Improved Yield and Quality of Continuous\u003c/b\u003e \u003cb\u003eD. indusiata\u003c/b\u003e \u003cb\u003eFruiting Body Production\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe yield and quality of \u003cem\u003eD. indusiata\u003c/em\u003e fruiting bodies partially reflect the nutrient and supply capacity of the culture substrate and soil. Mushroom spent substrate, a byproduct of mushroom cultivation, is rich in organic nutrients and primarily composed of cellulose, hemicellulose, lignin, carbohydrates, proteins and residual fungal mycelium [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The recycling of mushroom spent substrate in agriculture has emerged as a key strategy for advancing sustainable and green agriculture systems. Studies have demonstrated that, compared to traditional planting methods, substituting conventional nursery soil with SMS can increase soluble protein concentration in rice seedlings, enhance superoxide dismutase (SOD) activity, and improve total porosity and structural stability, thereby creating a more favorable environment for crop growth [\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] reported that supplementing bacterial manure with \u003cem\u003eD. indusiata\u003c/em\u003e fruiting bodies can modulate the abundance and structure of soil microbial communities, enhance soil fertility, and stabilize the soil environment, while simultaneously improving the yield and quality of subsequently cultivated \u003cem\u003eD. indusiat\u003c/em\u003ea. This study showed that continuous production of \u003cem\u003eD. indusiata\u003c/em\u003e fruiting body in the second year was significantly enhanced by mushroom spent substrate application. Notably, the S3-24 treatment group (inoculated for 3 months) exhibited the highest performance, achieving a fresh yield of 12,758.4 kg/hm approximately 7 times higher than the untreated control group (S0-24). The results indicated that the production of \u003cem\u003eD. indusiata\u003c/em\u003e was nearly equivalent to that of the previous year. Quality analysis results revealed that the S3-24 group had 32.42% higher crude polysaccharide content and 1.60% higher crude protein content compared to the control, with total sugar content reaching 547.66\u0026thinsp;\u0026plusmn;\u0026thinsp;4.11 mg/g. These findings indicated that amending soil with SMS significantly promotes both yield and quality of continuous \u003cem\u003eD. indusiata\u003c/em\u003e fruiting body production.\u003c/p\u003e \u003cp\u003e \u003cb\u003eA Novel Technology for Continuous\u003c/b\u003e \u003cb\u003eD. indusiata\u003c/b\u003e \u003cb\u003eCultivation Improves Soil Fertility\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe partially substitution of chemical fertilizers with SMS can improve soil nutrients and reduces the accumulation of heavy metals in soil. Compared to control soil, \u003cem\u003eAgaricus bisporus\u003c/em\u003e in SMS-amended soil has been shown to increase organic nitrogen and available phosphorus content [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Peregrina et al. [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] demonstrated that applying bacterial granules improved labile organic matter and microbial activity in vineyard soils, while also enhancing nitrogen, and available phosphorus, concomitantly elevating soil respiration rates and phosphatase activity [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Phosphorus is a critical nutrient that determines plant growth and productivity, limiting crop yields on over 40% of the world's arable land. Increased phosphorus availability has been consistently shown to enhance crop yield [\u003cspan additionalcitationids=\"CR49\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Under low-phosphorus conditions, crops and soil microorganisms secrete acid phosphatases within a pH range of 5.73\u0026ndash;7.11 to mobilize inorganic phosphorus and meet physiological demands [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. In this study, soil treated with bacterial sterilization over a three-month period (S3-24 group) exhibited an 87.34% increase in available phosphorus content (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), a 14.14% rise in total nitrogen (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), and only a marginal decline in organic matter (14.14% lower than S0-23, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), demonstrating that this treatment significantly enhances soil fertility.\u003c/p\u003e \u003cp\u003e \u003cb\u003eA Novel Technology for Cultivating\u003c/b\u003e \u003cb\u003eD. indusiata\u003c/b\u003e \u003cb\u003eImproves Soil Microbial Diversity and Composition\u003c/b\u003e\u003c/p\u003e \u003cp\u003eResearch showed that long-term monoculture and continuous cropping disorder can alter the dynamic evolution of microbial assembly mechanism. Imbalance of soil microbial structure and accumulation of pathogenic microorganisms are the main reasons for continuous cropping obstacles [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. In both soil and root system under monoculture conditions, bacteria exhibit greater responsiveness to environmental filtering factors, whereas fungi are more strongly influenced by dispersal limitations and stochastic processes. These dynamic changes highlight the importance of monitoring microbial population shifts following bacterial treatments [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Use of discarded mushroom waste in cucumber cultivation has been found to suppress Fusarium wilt disease [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Incorporating spent cucumber residues into growth substrates enhances seedling vigor and developmental progression without in \u003cem\u003evitro\u003c/em\u003e fertilization, while also improving microbial activity, promoting rice seedlings growth, the facilitating rhizosphere microbial recruitment [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Some studies have shown that the combination of biofertilizers and cover crops can improve soil health while actively regulating the structure and function of the rhizosphere microbial community, improving the growth of \u003cem\u003eApium graveolens\u003c/em\u003e under continuous cropping, and ultimately supporting high yield and high-quality crops [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Here, SMS treatment technology was employed to regulate the microbial community structure in \u003cem\u003eD. indusiata\u003c/em\u003e continuous cropping soil. Microbial co-occurrence network analysis clearly illustrates that sterilization fosters a more tightly interconnected soil microbial community, enriching the overall microbial environment. Second-year diversity analysis reveals that the bacterial sterilization treatment significantly altered the soil microbial composition in the experimental group, with a notable increase in the abundance of Gemmatimonadaceae, Acetobacteria, Basidiomyceae, Botryotrichum, Fusarium. Conversely, the abundance of Firmicutes, \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003eBotryotrichum\u003c/em\u003e decreased following the treatment. Notably, several taxa associated with enhanced soil fertility were enriched. For instance, Gemmatimonadaceae, belonging to the phylum Gemmatimonadota, played an important role in the phosphorus cycling [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Basidiomycetes are among the primary decomposers of lignin and cellulose in nature, ecosystems, contributing fundamentally to the formation of stable soil organic carbon and long-term soil fertility [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. These microbial shifts indicated that bacteria sterilization can positively influence ecosystem health and soil stability by reshaping the soil microecology and promoting organic matter decomposition. However, the concurrent increased in Fusarium caution due to its potential as a plant pathogen. Thus, while the treatment effectively modulates soil microbial dynamics, further research is need to optimize its application\u0026mdash;maximizing benefits for soil health while mitigating risk associated with pathogenic taxa.\u003c/p\u003e \u003cp\u003eAmong all PLS-PM models, S0-23 had the highest GoF (0.81), indicating a relatively stable soil system before the second year of continuous \u003cem\u003eD. indusiata\u003c/em\u003e cultivation. In contrast, after two years of continuous cropping and SMS application, S3-24 showed the highest GoF (0.78) among treatment groups, suggesting that longer substrate pre-treatment improves coordination among soil indicators and enhances ecological network stability. SMS use significantly altered interactions among soil factors. With longer pre-treatment, positive linkages between microbial communities (especially bacteria and fungi) and soil physicochemical properties or enzyme activities strengthened. Multiple high-weight pathways (path coefficient\u0026thinsp;\u0026gt;\u0026thinsp;10) emerged, indicating that SMS addition promotes nutrient cycling and microbial activity, boosting material and energy transfer. The negative associations between phenolic acid compounds and microbial taxa also weakened after substrate application. Notably, the S3-24 network showed greater complexity and integration, confirming that three-month pre-treatment stabilizes the soil micro-ecological network. In contrast, S1-24 and S2-24 showed only moderate improvements, with many weak or inhibitory connections remaining. Overall, starting SMS treatment three months in advance is more effective for building a stable, coordinated cultivation environment to overcome continuous cropping obstacles, supporting sustainable \u003cem\u003eD. indusiata\u003c/em\u003e cultivation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of Continuous Cropping of\u003c/b\u003e \u003cb\u003eD. indusiata\u003c/b\u003e \u003cb\u003eon Soil Organic Acids\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePhenolic acids are secondary metabolites widely present in higher plant tissues and closely associated with plant growth and development. For decades, it has been hypothesized that the autotoxin effects of phenolic acid acting as allelochemicals are a key factor contributing to continuous cropping disorders. Prolonged monoculture has been shown to correlated with a significant accumulation of phenolic acids and a marked decline in soil pH [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Soil acidification may promote the buildup of phenolic acid and is positively correlated with shifts in bacteria community composition, suggesting that the interplay between phenolic acid accumulation and decreasing soil pH could be a critical driver in reshaping soil microbial communities [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Furthermore, Qu and Wang [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e] demonstrated that phenolic acid significantly influence microbial biomass and activity. Specifically, low concentrations of phenolic compounds such as 2,4-di-tert-butylphenol and vanillic acid enhanced soil microbial biomass, whereas higher concentrations exert inhibitory effect. In this study, the soil phenolic acid content in the second year of continuous \u003cem\u003eD. indusiata\u003c/em\u003e cultivation was significantly higher than that in the first year and compared to previous research findings [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The accumulation of phenolic acid substances in soil increased markedly, with coumaric acid showing the most pronounced change\u0026mdash;its concentration in S3-24 increased by 179.14% relative to the other five phenolic acid compounds. Based on the analysis of soil phenolic acid, optimal inactivated conditions for the mycelium of \u003cem\u003eD. indusiate\u003c/em\u003e were identified through controlled culture experiments. The metabolites produced by the pathogen significantly inhibited the \u003cem\u003eD. indusiata\u003c/em\u003e mycelium growth. Even in coculture systems without exogenous phenolic acid addition, pathogen-induced inhibition of mycelial growth was evident. Notably, the addition of phenolic acid promoted the growth of both \u003cem\u003eD. indusiata\u003c/em\u003e and pathogenic bacteria; however, the stimulatory effect was more pronounced for pathogenic bacteria, which outcompeted \u003cem\u003eD. indusiata\u003c/em\u003e mycelium. Taken together, changes in phenolic acid concentrations and results from plate assays indicated that shifts in soil phenolic acid levels are closely associated with the occurrence of continuous cropping disorders of \u003cem\u003eD. indusiata\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThis study improved the soil by applying bacterial granules to investigate their alleviating effect on continuous cropping obstacles of \u003cem\u003eD. indusiata\u003c/em\u003e. Field trial results demonstrated that following application of a specific dose of SMS, \u003cem\u003eD. indusiata\u003c/em\u003e yield in the second year (S3-24) closely approached that of the first year and was significantly higher than the control group in the same period. Nutritional analysis of the fruiting body revealed that all major nutritional indicators in the treated group were superior to those in the control, indicating that bacterial granule application positively enhanced the nutritional quality of \u003cem\u003eD. indusiata\u003c/em\u003e. Furthermore, the treatment markedly improved soil quality, particularly in terms of physical and chemical properties and enzyme activities. It effectively mitigated soil acidification associated with continuous cropping, stabilized soil pH, and increased availability of key nutrients such as nitrogen and phosphorus. Microbial community analysis indicated a significantly increase in beneficial populations, reflecting an overall improvement in soil fertility and microbial balance, thereby confirming the microecological regulatory potential of this approach. Compared with conventional methods for managing continuous cropping obstacles, the SMS pretreatment method exhibited distinct advantages. Traditional high-temperature sterilization only partially reduces pathogens and is associated with high energy consumption and operational complexity; field rotation can alleviate soil degradation but compromises land use efficiency and production continuity; while chemical fumigation is effective against soil-borne diseases, it lacks selectivity, often harming beneficial microbes [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e], and poses risks of environmental pollution and residue accumulation. In contrast, pre-planting soil amendment with bacterial granules not only effectively alleviates yield decline due to continuous cropping but also directly enhances soil nutrients status and optimizes microbial community structure through a simple and practical operation. PLS-PM further confirmed that application of SMS distiller\u0026rsquo;s grains significantly increased the positive path coefficients between microorganisms and environmental factors and enhanced the model\u0026rsquo;s explanatory power, thereby improving the overall stability and coordination of the soil micro-ecological network. Moreover, this strategy enables high-value utilization of agricultural waste-derived bacterial granules, significantly reducing management costs and offering both ecological and economic benefits. In conclusion, this green, efficient, and cost-effective continuous cropping pathway, enhancing soil sustainability, and promoting high-yield, high-quality crop production.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOver two years of field experiments, this study evaluated fermented SMS for alleviating continuous cropping obstacles and improving soil microecology properties. Analysis of soil physicochemical properties, enzyme activities, microbial diversity, and agronomic and nutritional indicators showed that SMS significantly improved soil fertility. The 3-month treatment had the greatest effect: it stabilized soil pH, reduced acidification, and increased available phosphorus and total nitrogen, indicating enhanced soil nutrient availability. All SMS treatments suppressed excessive organic matter degradation, most effectively in 3-month group. These improvements were link to enrich functional microbes involved in phosphorus cycling, especially increased \u003cem\u003eBacillus\u003c/em\u003e abundance, which enhances phosphorus solubilization. Phenolic acid analysis revealed accumulation under continuous cropping, but SMS reduced coumaric acid levels. Notably, moderate phenolic acid promoted growth of \u003cem\u003eD. indusiata\u003c/em\u003e and some pathogens. SMS also enhanced bacterial-fungal network complexity and diversity by shaping microbial community structure and enriching beneficial bacteria associated with soil fertility and stability. This mechanism alleviated key soil dysfunctions from continuous cropping, such as microbial imbalance and reduced decomposition capacity. Crucially, SMS improved \u003cem\u003eD. indusiata\u003c/em\u003e yield and quality. Crude polysaccharides and protein also improved. Microscopy showed thicker, more robust hyphae in treated mycelia\u0026mdash;supporting high productivity. This study demonstrated successful SMS resource recovery, providing practical and theoretical solutions for \u003cem\u003eD. indusiate\u003c/em\u003e continuous cropping. Future work should optimize microbial strains and application methods, developed inoculants, and advance sustainable agriculture.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Guirong Lin from the Xinjundu Company for his technical guidance on \u003cem\u003eD. indusiata\u003c/em\u003e cultivation and Shaoqing Shu from the Xingwang family farm for managing the experimental site. Furthermore, we also thank the Shunchang Juncao Science and Technology Backyard for providing accommodation to our technicians and students.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJing Li and Xiongjie Lin: Conceptualization, Funding acquisition, Supervision, Writing\u0026ndash;original draft, Writing\u0026ndash;review \u0026amp; editing, Project administration. Xiaoyue Di: Conceptualization, Data curation, Formal analysis, Investigation, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing, Visualization. Yinghao Sun, Xianai Huang, Fengju Jiang, and Jiale Feng: Data curation, Formal analysis, Investigation. Dongmei Lin, and Zhanxi Lin: Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Key Research and Development Program of China \u0026ldquo;Research and Application Demonstration of Key Technologies for Juncao Medicinal and Edible Mushrooms and Orchids Cultivation\u0026rdquo; (2023YFD1000502); The Project for the Enhancement of the First-Class Discipline of Forestry (Juncao Science) at Fujian Agriculture and Forestry University (725025010A).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original dataset on microbial diversity for this study has been submitted to the National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/), along with the accession number PRJNA1234400 (this data will be made publicly available on December 31, 2026).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors agreed to the publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing financial interests or personal relationships that may have influenced the work reported in this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJeong SC, Jeong YT, Yang BK, Islam R, Koyyalamudi SR, Pang G, et al. 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Soil Ecol. 2022;174:104408. http:// doi.org/10.1016/j.apsoil.2022.104408.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"chemical-and-biological-technologies-in-agriculture","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Chemical and Biological Technologies in Agriculture](https://chembioagro.springeropen.com/)","snPcode":"40538","submissionUrl":"https://submission.nature.com/new-submission/40538/3","title":"Chemical and Biological Technologies in Agriculture","twitterHandle":"@SpringerPlants","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Dictyophora indusiata, Seafood mushroom spent substrate, Phenolic acid, Soil environment, Sustainable development","lastPublishedDoi":"10.21203/rs.3.rs-8761948/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8761948/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground \u003c/strong\u003eContinuous cropping poses a significant challenge to the sustainable cultivation of \u003cem\u003eDictyphora indusiata\u003c/em\u003e, often leading to soil degradation, microbial imbalance, and the accumulation of soil-borne pathogens, which severely compromise yield and quality. Conventional mitigation strategies, raise environmental and safety concerns. This two-year study investigated the efficacy of using fermented \u003cem\u003eHypsizygus marmoreus\u003c/em\u003e(seafood mushroom) spent substrate (SMS) as a sustainable, biocontrol-based approach to remediate soil, suppress pathogen communities, and alleviate the continuous cropping obstacles affecting \u003cem\u003eD. indusiata.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e By varying SMS application timing, we assessed it effects on yield, nutritional quality, soil physicochemical properties, enzyme activities, and microbial community diversity. Applying SMS three months before planting (S3-24) stabilized soil pH in the second year. Soil available phosphorus increased by 87.34%, total nitrogen by 14.14%, and organic matter declined less (14.14% decrease vs. 29.95% in control). Soil microbial diversity increased within 1-2 months, with phosphate-solubilizing bacteria reaching 7.52%. Soil phenolic acids increased, especially \u003cem\u003ep\u003c/em\u003e-coumaric acid (up 179.14% in S3-24). In \u003cem\u003evitro\u003c/em\u003e tests showed that at 250 mg/L phenolic acids, both \u003cem\u003eD. indusiata\u003c/em\u003e mycelium and pathogens grew faster, but pathogens had a stronger growth advantage. Fruiting body weight peaked at 19.61 g per fruit in S3-24, and fresh yield reached 12,758.4 kg/hm², representing a 745.07% higher than control. Crude polysaccharide and protein increased by 32.42% and 1.60%. Phenolic acid also increased mycelial diameter to 2.4-4.8 μm, improving mycelium structure and supporting higher yield. Moderate phenolic acid levels benefit both \u003cem\u003eD. indusiata\u003c/em\u003eand pathogens, but pathogens competitiveness may drive cropping obstacles. PLS-PM showed that S3-24 strengthened key pathways-from soil properties to enzyme activity (path coefficient: 16.29) and fungal communities (4.77)-and the enzyme-phenolic acid-microbe cascade effect (19.58), improving coordination and stability in the soil microecological network (GoF=0.78) and alleviating continuous cropping obstacles.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions \u003c/strong\u003eAppropriate SMS use mitigated these challenges, stabilizes yield, and improves quality. This approach recycled agricultural waste and offers practical solutions for future \u003cem\u003eD. indusiata\u003c/em\u003e continuous cultivation.\u003c/p\u003e","manuscriptTitle":"Effects of Hypsizygus marmoreus spent substrate on prevention and control potential of continuous cropping obstacle in Dictyophora indusiata cultivation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-17 10:25:31","doi":"10.21203/rs.3.rs-8761948/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-02T08:20:19+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-25T21:26:09+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-19T02:09:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"154882680248053272205910713483510753950","date":"2026-02-18T01:28:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"224097305148038663931654495536454526092","date":"2026-02-11T19:20:52+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-11T19:07:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-06T15:02:51+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-06T14:59:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"Chemical and Biological Technologies in Agriculture","date":"2026-02-02T07:26:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"chemical-and-biological-technologies-in-agriculture","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Chemical and Biological Technologies in Agriculture](https://chembioagro.springeropen.com/)","snPcode":"40538","submissionUrl":"https://submission.nature.com/new-submission/40538/3","title":"Chemical and Biological Technologies in Agriculture","twitterHandle":"@SpringerPlants","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4d437f49-91ad-47d4-84a2-84f5d0986a06","owner":[],"postedDate":"February 17th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-26T08:26:52+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-17 10:25:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8761948","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8761948","identity":"rs-8761948","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}