Cultivation of Morchella sextelata in saline-alkali soils of Ningxia: Multi-dimensional impacts on soil physico-chemical properties, enzyme activities, microbial ecology and functional genes

Cultivation of Morchella sextelata in saline-alkali soils of Ningxia: Multi-dimensional impacts on soil physico-chemical properties, enzyme activities, microbial ecology and functional genes | 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 Cultivation of Morchella sextelata in saline-alkali soils of Ningxia: Multi-dimensional impacts on soil physico-chemical properties, enzyme activities, microbial ecology and functional genes Yifan Wu, Xiaoli Yang, Yaya Zhang, Chunyan Xu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8232173/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Aims ( Morchella spp.), precious edible and medicinal fungi with important economic and scientific value, have always attracted the attention of scientific researchers at home and abroad, while successful large-scale cultivation in multiple provinces drives a northward expansion of production regions from the south. China’s northward expansion of morel mushrooms cultivation faces challenges from saline-alkali soils in arid regions. The purpose of this paper is to clarify the effects of Morchella cultivation on the saline-alkali soil of Ningxia, Northwest China. Methods In this study, A comparative analysis was performed on the physical and chemical properties of soil, microbial community structure and function, and the expression of enzymes related to nitrogen metabolism in saline-alkali soils under three treatments: (A) an unplanted control, (B) one year of Morchella cultivation, and (C) two years of Morchella cultivation. Results The results indicated that Morchella cultivation positively contributed to the amelioration of saline-alkali soil, evidenced by a significant reduction in pH, alongside remarkable enhancements in nutrient contents and enzyme activities. Results based on metagenomic sequencing also revealed that morel mushroom cultivation drives a functional remodeling of the soil microbial community. On the one hand, it enhances the overall metabolic activity of microorganisms by upregulating core metabolic pathways such as ABC transporters, and Morchella cultivation also positively promotes denitrification, nitrogen assimilation, and reutilization in soil nitrogen metabolism. On the other hand, it disrupts the microbial network responsible for phenolic acid degradation, manifested by an increased gene abundance of the synthesis key enzyme PAL, while the dominant microbial contributors to the degradation core enzyme P34O and its encoding genes pcaG / pcaH shift from the original state (dominated by genera such as Arthrobacter ) to a less efficient structure (dominated by Microvirga and Sphingomonas ), ultimately leading to phenolic acid accumulation and the risk of hindering sustainable continuous cropping. Conclusion This study highlights the extent of soil alterations induced by morel mushroom cultivation in saline-alkali environments and its underlying mechanisms. Morchella sextelata Saline-alkali soil Soil physicochemical properties Microbial community Functional genes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Introduction Morchella sextelata is a rare edible fungus belonging to the Pezizales order and Morchellaceae family. It is named after its lumpy cap, which resembles a sheep’s belly. It has a unique flavor and an exquisite aroma, and it is rich in over 20 amino acids and trace elements, making it highly sought after by consumers (Li et al. 2025 ). Accordingly, morel mushrooms are considered a rare type of edible fungi with both culinary value and medicinal benefits (Haider et al. 2025 ) (Wang et al. 2021b ) (Ramya et al. 2022 ). Therefore, the cultivation of morel mushrooms has long been a topic of concern to scientists. Currently, China has become the world’s largest producer and exporter of morel mushrooms ( Morchella sextelata ). In 2024, China’s exports of dried morel mushrooms reached 225.14 tons, valued at approximately USD 17.76 million (equivalent to roughly ¥126 million RMB) (Zhang et al. 2025a ). Concurrently, large-scale cultivation techniques for morel mushrooms have achieved success in provinces such as Sichuan, Yunnan, Hunan, Hubei, and Shaanxi. As the cultivation area gradually expands, the production regions for morel mushrooms are also extending northward from southern China (Xu et al. 2022 ). However, significant differences in soil quality exist between the northern and southern regions (Zhang et al. 2025b ). Unlike the high-humidity soils of southern China, arid and semi-arid northern regions exhibit soil salinisation. Soils with EC values exceeding 4 dS/m at 20 ℃ and with exchangeable Na + content above 15% are deemed saline (Sz´ekely and Barta 2025 ). Globally, saline-alkali land covers approximately 9.55×10 8 hm², of which China accounts for about 9.91×10 7 hm² (Arif et al. 2020 ). The saline-alkali land in the Hongsipu Yanghuang Irrigation District of Ningxia serves as a critical ecological restoration zone and features one of northwest China’s most technologically advanced lift irrigation systems (Wu et al. 2024 ). Morel-farming beds are very important for the growth of the morel mushroom. Different types of soil (forests, paddy fields, greenhouses and orchards) distinctly affect the growth of morel mushrooms and the ecological conditions of soil microorganisms (Yue et al. 2024 ). While the different types of morel-farming beds have an impact on the growth of morel mushrooms, their cultivation also improves the soil environment. Planting morel mushrooms can enhance soil health. During the fallow period, planting morel mushrooms can improve the soil characteristics of paddy fields and drylands and increase grain production (Duan et al. 2023 ). However, soil microorganisms, as key decomposers, are integral components of the soil ecosystem and play a key role in improving soil structure, regulating soil nutrient balance, and influencing crop yield (Wang et al. 2023 ). In addition, microorganisms are sensitive biological indicators of changes in the soil environment, and soil microbial diversity and community structure can serve as important indicators for assessing soil fertility (Jeffries et al. 2003 ) (Luo et al. 2016 ). Significant differences in soil microbial structure between morel-cultivated soils and non-cultivated soils (Zhang et al. 2023b ). Inoculation with morel mushrooms’ mycelia had a notable impact on soil bacterial diversity. In saline-alkali environments, where inherent constraints (high salinity, elevated pH, poor soil structure, and suppressed microbial activity) severely limit morel mushrooms (Dong et al. 2020 ). Understanding context-specific responses, particularly in saline-alkali soils, is therefore essential for sustainable cultivation. Since the introduction of morel mushroom species to Ningxia in 2018 and successful trials, the industry has developed rapidly. However, compared with reports in Yunnan and Sichuan, a gap exists regarding morel mushroom cultivation on saline-alkali soil. A comprehensive understanding of how Morchella sextelata cultivation itself, as a soil intervention, systematically affects key properties, including physicochemical characteristics, enzyme activities, microbial community composition/ diversity, and functional gene abundance, remains limited. Additionally, allelopathic autotoxicity (where plants release metabolites inhibiting their own growth) is a concern, as morel mushrooms cultivation typically accumulates phenolic acids in the soil. Therefore, this study aims to investigate differences in soil physicochemical properties, enzyme activities, microbial communities, and functional genes between morel-cultivated and non-cultivated soils in Ningxia’s saline-alkali land. Changes in soil microbial community structure and function were analyzed via metagenomics. Quantify phenolic acid content, key enzyme abundance, and related microbial communities across treatment groups. Materials and methods Soil sample collection As shown in Fig. 1 , the sampling sites were selected in the Ningxia morel mushroom planting base (37°20’N, 105°57’E, altitude 1240–1450 m) and 3 groups of samples were collected. They are soil that (A) unplanted control, (B) one year of Morchella cultivation, and (C) two years of Morchella cultivation. Set up two test greenhouses to collect series B and C. Soil samples were taken from the bare ground outside the shed. Each greenhouse is divided into three large quadrats, front, middle, and back, as three biological weights. In addition, a five-point sampling method was used to collect soil samples for each quadrate. Mix near the morel mushrooms and furrows with 0 to 20 cm of soil at each point. After the mixture is evenly mixed, 500 g of the mixed soil is weighed as the test soil. Part of the soil was preserved at -80 ℃ and sent to the biological company for metagenomic sequencing. The soil physical and chemical properties and enzyme activities of the remaining soil samples were tested after air drying. Determination of soil enzyme activities and physicochemical properties Soil physicochemical indexes and enzyme activities were determined according to soil science experiments and soil enzymes and their Research Method (Xie et al. 2025 ) (Liu et al. 2022 ), respectively, as detailed in Table 1 . Table 1 Methods for the determination of soil physicochemical properties and enzyme activities Index of measurement Method of determination pH value pH acidity meter method content of water Drying method organic matter Potassium dichromate oxidation method total nitrogen Semi-micro Kjeldahl method total phosphorous Mo-Sb colorimetric method total potassium Flame spectrometry olsen nitrogen alkali N-proliferation method olsen potassium Flame spectrometry olsen phosphorous Micro determination soil urease Phenol-hypochlorite colorimetry soil sucrase 3,5-dinitro salicylic acid colorimetry soil catalase Permanganate titration soil phosphatase Phenyl phosphate titration Extraction and determination of total phenolic acids Total phenolic acids were extracted using the modified method of alkali solution. 20 grams of dry soil samples were added to 100 mL NaOH solution (3 mol/L) and oscillated for 20 h at 150 r/min. The soil suspension was centrifuged at 5,000 g for 15 min, and the supernatant was filtered through filter paper. Then, the pH of the filtrate was adjusted to l.5 with HCl (5 mol/L), and extracted five times with ethyl acetate. The resultant extracts were pooled and evaporated to dryness using a rotary evaporator at 60 ℃. The residue was dissolved in 5 mL of distilled water and kept in the dark at 4 ℃. The content of phenolic acids was determined by the Folin-Ciocalteu Method using gallic acid as the standard. 0.5 mL soil extract and 1.5 mL deionized water were placed into a 10-ml centrifuge tube, then 5 mL of 1 mol/L Na 2 CO 3 and 1 mL Folin and Ciocalteu. Ciocalteu’s phenol reagent was added and incubated for 1 h in darkness at ambient temperature. Finally, the absorbance of the mixture was measured at 760 nm with a UV-Vis spectrophotometer. The total phenolic acid content in soil was expressed in micrograms of gallic acid equivalent per gram of soil. Analysis of microbial community structure and data processing Fungal and bacterial communities were analyzed after quality control and annotation of the sequencing data. The statistical analysis results of species abundance differences in the soil of different treatment groups were obtained, and the species with significant differences were analyzed. The relationship between functional genes and microbial communities was analyzed. Excel and SPSS 26 were used for data processing and Duncan’s significance test (significance level was p < 0.05), combined with information analysis cloud platform ( https://cloud.majorbio.com/page/project/overview.html ) analysis, and visualization mapping. The correlations between soil environmental factors and microbial communities were also analyzed by calculating the correlations between the environmental factors and the selected species (using Spearman and Pearson correlation coefficients, etc.). The obtained numerical matrix is visually presented through a Heatmap graph. The ggplot2 and ggtree in R were used to analyze the relationship between functional genes and microbial communities. Results Soil enzyme activities and physicochemical properties As shown in Fig. 2 and Fig. 3 , the pH value of cultivated morel mushrooms (B and C) was significantly lower than that of uncultivated morel mushrooms (A), suggesting that cultivation practices may induce soil acidification. However, there was no significant difference in pH value between morel mushrooms planted for one year (B) and two years (C). The contents of soil total nitrogen, total phosphorus, organic matter, soil urease, soil sucrase, and soil phosphatase in Groups B and C were significantly higher than those in Group A, indicating that cultivation enhances soil nutrient availability and biochemical activity. Again, no significant differences were detected between one-year and two-year cultivation. Notably, Olsen nitrogen and Olsen phosphorus levels showed significant differences across all groups, with Group C > Group B > Group A , implying that available nitrogen and phosphorus continue to accumulate with extended cultivation time. In contrast, total potassium was highest in Group B, significantly exceeding both Groups A and C, which did not differ from each other. Olsen potassium also varied significantly among all groups, following the order Group B > Group C > Group A, suggesting a more complex dynamics of potassium availability affected by cultivation duration. The total potassium content of Group B was significantly higher than that of Groups A and C, and there was no significant difference between A and C. Soil microbial community structure and diversity The composition of the soil microbial community differed significantly among the groups (Fig. 4 a- 4 f), a variation likely associated with changes in the soil microenvironment induced by different planting treatments. At the phylum level, Actinomycetota was the absolutely dominant phylum in Group A, while both Actinomycetota and Pseudomonadota were dominant in Groups B and C. A decrease in the relative abundance of Actinomycetota and an increase in Pseudomonadota were observed (Fig. 4 a), suggesting that planting activities may have promoted the proliferation of bacterial taxa involved in specific nutrient cycling processes. Regarding the fungal community, the abundance of Ascomycota decreased, while Mucoromycota and Basidiomycota increased (Fig. 4 b), indicating a possible enrichment of fungal groups related to decomposition or plant symbiosis following morel mushroom cultivation. At the genus level, a clear shift in bacterial dominance was observed, likely reflecting altered nutrient dynamics. Group A was dominated by Arthrobacter and Nocardioides , genera often associated with soil organic matter decomposition. In contrast, the rise of Sphingomonas (common in root-associated environments) and a significant increase in Nitrospira (a key nitrite-oxidizing bacterium) in Groups B and C suggest a shift toward bacterial communities involved in plant-microbe interactions and enhanced nitrification, respectively. A concomitant decline in Nocardia was also noted. Fungally, the community transitioned from being dominated by Alternaria in Group A to the dominance of Aspergillus , Rhizopus , and Fusarium in treated groups. The decline of Alternaria and the rise of Aspergillus, a potent decomposer, particularly point to a fungal community restructuring geared toward more efficient organic matter decomposition following morel mushroom cultivation (Fig. 4 c, 4 d). At the species level, there were 25,820 bacterial species in the three soil samples, accounting for 73.97% of the total in Group A, 82.2% in Group B, and 79.8% in Group C, respectively. There were 2,417 endemic species in Group A, 1,767 in Group B, and 1,156 in Group C (Fig. 4 e). There were 616 fungal species in the three soil samples, accounting for 60.4% of the total in Group A, 53.1% in Group B, and 42.7% in Group C. There were 210 endemic species in Group A, 126 in Group B, and 50 in Group C (Fig. 4 f). The number of endemic species exhibited significant trends with increasing cultivation duration. For bacteria, the number of endemic species significantly decreased with longer cultivation duration (Group A: 2417 species Group B: 1767 species Group C: 1156 species). Similarly, the number of fungal endemic species also decreased with increasing cultivation duration, but the decline was more pronounced and particularly sharp in Group C (Group A: 210 species > Group B: 126 species > Group C: 50 species). The endemic species in Group A represent the unique microbial community of pristine, undisturbed saline-alkaline soil. Following Morchella cultivation (Groups B and C), the number of these endemic species decreased significantly, which suggests that Morchella cultivation practices (including soil treatment, fungal inoculum introduction, cultivation management, etc.) resulted in the loss of a portion of indigenous microorganisms or the reduction of their abundance below detection thresholds. Notably, this phenomenon manifested more pronouncedly within the fungal community. From the perspective of fungi, the specific fungi unique to Group A are mostly Alternaria , Colletotrichum , Didymella , Fusarium , and Penicillium . The specific fungi unique to Group B are mostly Mortierella , Penicillium , Pseudogymnoascus and Coemansia , while the fungi unique to Group C are mostly Penicillium , Fusarium, Leptographium , Epichloe , and Pleurotus . From the perspective of bacteria, the specific bacteria unique to Group A are mostly Streptomyces , Arthrobacter , Methylobacterium , Bacillus , and Exiguobacterium . The specific bacteria unique to Group B are mostly Chryseobacterium , Pseudomonas , Flavobacterium , and Pedobacter . The specific bacteria unique to Group C are mostly Pseudomonas , Streptomyces , Burkholderia , Mesorhizobium , and Rhizobium . Alpha diversity analysis revealed a significant and consistent increase in bacterial diversity following morel mushroom cultivation. The rise in the Chao1, Shannon, and Simpson indices in Groups B and C compared to Group A indicates that planting morel mushrooms not only enriched bacterial species richness but also enhanced community evenness. This suggests that the cultivation practice created a more heterogeneous microenvironment or introduced diverse organic substrates, fostering a more complex and stable bacterial community. In stark contrast, the lack of significant change in fungal diversity across all indices underscores a fundamental difference in ecological strategy. The fungal community structure remained stable and resistant to the changes induced by short-term cultivation. This divergence highlights that morel mushroom cultivation selectively promotes bacterial diversity without disrupting the resident fungal network, potentially leading to a more multifaceted and resilient soil ecosystem (Fig. 5 c- 5 h). Correlations between soil microbial genera and environmental factors The correlation analysis at the genus level revealed distinct environmental preferences among key microbial taxa, linking community structure to soil enzyme activities and physicochemical properties. The significant negative correlation of Nocardioides and Arthrobacter with key fertility indicators (catalase, total nitrogen, potassium, and water content) suggests these taxa are better adapted to, or even thrive in, less fertile conditions, possibly acting as oligotrophic specialists. Conversely, the strong positive correlation of Nitrospira (a known nitrite-oxidising bacterium) with these same factors indicates its role in nutrient-rich environments, likely coupling its nitrification function with heightened potassium availability and microbial activity (catalase). Fungal genera also displayed clear niche partitioning: Alternaria and Fusarium were associated with higher pH, while the saprotrophic genera Mortierella and Rhizopus were positively linked with enhanced nutrient content (total nitrogen, potassium) and catalase activity, underscoring their importance in organic matter decomposition in fertile soils. KEGG functional enrichment analysis The differential pathway analysis underscores a microbial functional adaptation to the altered soil environment under morel mushroom cultivation. The upregulation of stress-associated pathways (lipopolysaccharide biosynthesis, two-component system) in the control group implies that cultivation alleviates certain environmental pressures on the microbial community. This is supported by the downregulation of core metabolic processes (amino acid biosynthesis, carbon metabolism) in the control, indicating a less metabolically active state. In contrast, the microbial community in the two-year cultivated soil demonstrated a pronounced shift toward heightened metabolic activity, evidenced by the upregulation of key pathways involved in cofactor synthesis, substrate transport (ABC transporters), and carbon utilization. The suppression of the thermogenic pathway further suggests an optimization of energy expenditure, favoring biosynthetic processes over heat dissipation in a more stable, cultivated environment (Fig. 7 a, 7 b). The marked increase in key enzymes involved in denitrification and dissimilatory nitrate reduction, including nitrous oxide reductase, nitric oxide reductase, and nitrate reductase, suggests a pronounced shift in the nitrogen cycle following morel cultivation. The elevated abundance of these enzymes implies an enhanced capacity for nitrate reduction to nitrite and subsequently to gaseous forms (NO and N 2 O). The upregulation of multiple enzymes in the denitrification pathway points to a systemic enrichment of microorganisms capable of anaerobic nitrogen respiration. This functional adaptation likely results from changes in soil structure, moisture, and organic matter input associated with morel mushroom farming, which may create microsites with localized low oxygen conditions. The consistency of this pattern across one and two years of cultivation indicates a stable restructuring of the microbial community’s metabolic network, orienting it toward nitrate and nitrite reduction as key energy-generating processes. Synthesis and degradation pathway analysis of phenolic acids Allelopathic autotoxicity refers to the phenomenon where plants release specific secondary metabolites into the environment, thereby causing harmful effects on their growth and development (R. Baziramakenga 1995). In the context of morel mushrooms cultivation, this phenomenon is of particular concern due to its implications for continuous cropping obstacles. It’s reported that the allelopathic substances of morel mushrooms include 4-coumaric acid, vanillin, ferulic acid, and vanillic acid (Qi et al. 2024 ). This is also the reason for the significant increase in total phenolic acid content in Group B compared with Group A. These phenolic acids not only contribute to the observed increase in total phenolic acid content in Group B compared with Group A but also play a crucial role in soil acidification and autotoxic feedback. This accumulation of phenolic acids reduces soil pH and, when excessively accumulated, exerts negative effects on both soil health and the crops themselves. Therefore, the rational management of phenolic acids has become a key focus. Through the determination of total phenolic acid content in soil samples collected under different treatments, it was found that the content of total phenolic acids in Group C was significantly higher than that in Groups B and A. Through the analysis of the synthetic and degradation pathways of phenolic acids (Fig. 9 ), PAL (phenylalanine ammonia-lyase) and P34O (protocatechuate 3,4-dioxygenase) were identified as the key enzymes in phenolic acid synthesis. The interplay between the abundance of key enzyme genes and the phenolic acid content reveals the core of the autotoxicity mechanism. The average difference in gene abundance of key enzymes between different treatments was analyzed (Fig. 10 ). The abundance of PAL, the key enzyme for phenolic acid synthesis, was significantly highest in Group C. Conversely, the abundance of P34O, the central enzyme for phenolic acid degradation, was significantly highest in Group A. This divergent profile creates a clear metabolic predisposition: Group C is genetically primed for high synthesis and low degradation, directly explaining the significant phenolic acid accumulation observed in this group. In contrast, Group A’s profile (low synthesis and high degradation capacity) aligns with its lower phenolic acid content, illustrating an effective metabolic balance. Figure 10 . Analysis of the gene abundance of key enzymes related to phenolic acids metabolism in different treatments The analysis of microbial functional contributions revealed a critical shift in the microbial decomposer community that underlies the observed differences in phenolic acid accumulation. Based on the correlation analysis of species contribution to the key enzyme P34O for phenolic acid decomposition, distinct patterns emerge: Group A (unplanted soil) exhibited a microbial community adapted to its native state. The genera Arthrobacter , Geodermatophilus , and Kocuria were identified as having high contributions to the P34O enzyme. Specifically for the functional gene pcaG , Rubrobacter was the dominant contributor, while for pcaH , it was Microvirga . Group B (one-year cultivated soil), a significant shift was observed, as there was no dominant contributing species for the pcaG . For the pcaH , Blastococcus emerged as the top contributor. This suggests that the first year of cropping disrupted the original stable community without yet establishing a new, dominant functional population. Group C (two-year cultivated soil following the “edible fungi-qilin melon” rotation ) established a distinctly restructured microbial community. For the functional gene pcaG , the bacteria belonging to Microvirga and Nocardioides were the dominant contributors. In contrast, for the pcaH gene, Sphingomonas was identified as the species with the highest contribution. In conclusion, the transition from native soil to cultivated systems not only restructures the microbial community but also appears to disrupt the efficacy of the phenolic acid degradation pathway. The “edible fungi-qilin melon” system, while promoting a distinct microbial consortium, is associated with a significant accumulation of phenolic acids, indicating a potential risk for soil sickness or allelopathy that could hinder sustainable cultivation unless managed appropriately. Discussion Dynamic changes in physico-chemical properties and enzyme activity of saline-alkali soil driven by morel mushroom cultivation An increasing number of studies have demonstrated that the cultivation of edible fungi can enhance soil environments. For instance, the undergrowth cultivation of P. glomus significantly improved soil porosity, soluble element content, biological enzyme activity, microbial diversity and metabolism, thereby reconstructing soil ecological functions and exhibiting considerable potential for soil remediation and sustainable crop production (Liu et al. 2021 ). Examined the effects on pH, total salt content, organic matter levels, available potassium, and ammonium nitrogen in soils following the planting of Stropharia rugosoannulata and investigated its efficacy in ameliorating saline-alkali soils (Yang et al. 2022 ). The ability of edible fungi to improve soil environments primarily stems from their residues’ application in soils, which leads to a series of changes in soil structure, material composition, and nutrient balance resulting in alterations to the composition of soil organic matter and subsequently affects microbial community composition, abundance, and activity. These changes influence both matter-energy conversion processes within the soil and nutrient availability from fertilizers (Li et al. 2015 ), which is in agreement with our experimental findings. Compared to Group A, significant improvements were observed in nutrient levels and pH values within Group B after one year of morel mushroom cultivation. However, total potassium and available potassium contents in Group C, where morel mushrooms were cultivated for two years, were markedly lower than those found in Group B after one year’s cultivation, suggesting that continuous morel mushroom cultivation may lead to potassium consumption within the soil (Zhang et al. 2023a ). In addition, compared with Groups B and A, Group C showed a rotation pattern of “edible fungi qilin melon”, which could effectively maintain the soil optimization brought by morel mushroom cultivation in Group B. Successional Dynamics of Soil Microbial Community Structure and Function Previous studies have shown that plant cultivation or agricultural management practices can reshape the soil microbial community through direct (such as root exudates) or indirect (such as changes in soil physical and chemical properties) mechanisms, and the impact of continuous cropping (long-term cultivation of the same crop) is more significant (Khmelevtsova et al. 2022 ).In addition, long-term plant encroachment enhanced microbial diversity and network stability in the special environment of bauxite residue (Deng et al. 2024 ). Analysis of the microbial community composition across treatments revealed a shift from a homogeneous to a more diversified state under morel mushroom cultivation. However, the rich microbial composition can make the soil more stable and versatile (Philippot et al. 2024 ). In group A, the Actinobacteria were the absolutely dominant phylum. However, in groups B and C, the abundance of the Actinobacteria decreased while that of the Deinococcus and Actinobacteria increased. Together, these two phyla became the dominant ones, but the Actinobacteria remained the core dominant phylum present in all three groups. The Actinobacteria usually dominate in natural, undisturbed soils. It has strong metabolic capabilities and can decompose complex organic substances (such as cellulose and lignin) (Asina et al. 2016 ). On the other hand, the Proteobacteria phylum is more sensitive to changes in environmental nutrients and its abundance tends to increase in cultivated soils or soils with increased nutrient input (Choudhary et al. 2020 ). Following the cultivation of morel mushrooms, a significant shift in soil fungal community composition was observed: the relative abundance of Ascomycota decreased, while increases were noted in the relative abundances of Mucoromycota and Basidiomycota. Given that morel mushrooms themselves belong to the Ascomycota phylum, it is hypothesized that their cultivation might lead to competitive suppression of other Ascomycota taxa, potentially through resource competition or niche occupation. However, other factors associated with cultivation practices could also contribute to these observed community shifts. In addition, the dominant bacteria in Groups B and C were Sphingomonas and Nitrospira . They play an important role in maintaining soil nitrogen cycling (Li et al. 2021 ) (Meng et al. 2023 ). Sphingomonas has been reported to play a role in the bioremediation and bacterial regulation of polycyclic aromatic hydrocarbons (PAH) contaminated saline soil (Song et al. 2021 ). For fungi, the abundance of Alternaria in soil decreased sharply after Morchella planting, with Aspergillus , Rhizopus , and Fusarium being the dominant genera in soil samples after one year and two years of planting. By analyzing the correlation between community structure and environmental factors, Alternaria , Fusarium , and Pyrenophora were found to be positively correlated with pH. The content of catalase, total nitrogen, total potassium, and available potassium was significantly positively correlated with the abundances of Mortierella and Rhizopus . According to previous literature, Alternaria exhibits a preference for alkaline environments, while increased levels of organic acids lead to a decline in its abundance (Lu et al. 2020 ). Furthermore, with the cultivation of Morchella , a decrease in soil pH and an increase in nutrient levels were observed, which promoted the accumulation of beneficial microorganisms such as Chryseobacterium (Majewska et al. 2022 ) and Chryseolinea . (Ortega et al. 2024 ) Furthermore, Venn analysis at the microbial species level revealed that the cultivation of morel mushrooms led to a loss of certain indigenous soil microorganisms. In particular, the loss of indigenous species in the fungal community has been more pronounced. This might be due to the fact that agricultural interference initially temporarily increases the total number of species by introducing new microorganisms, but long-term consecutive cultivation leads to the loss of native endemic species due to the homogenization of the habitat. It is speculated that since morel mushrooms occupy ecological niches by rapidly expanding their mycelial networks and competing for space and nutrients (such as potassium), some native fungi with similar nutritional requirements or slow growth rates have died due to the lack of resources. Response of microbial functional pathways to morel mushroom cultivation and soil microenvironment Compared with Group A, The Two-component system, Lipopolysaccharide biosynthesis, and Methane metabolism pathways in Group B were up-regulated, among which the two-component system was related to signal transduction in the category of environmental information processing. The two-component system plays an important role in maintaining the stability of microbial communities. During composting, penicillin residues may regulate community stability by affecting TCS gene expression, thereby improving community cohesion (Guo et al. 2024 ). Alternatively, the upregulation of the Lipopolysaccharide biosynthesis pathway may indirectly affect soil physical properties, such as soil aggregate stability. The stability of soil aggregates is related to soil organic carbon content, microbial activity, and soil structure. Lipopolysaccharide biosynthesis and the synthesis of other polysaccharides may improve soil structure by increasing the stability of soil aggregates (Wang et al. 2021a ). In addition, upregulation of methane metabolism Therefore, upregulation of methane metabolism not only affects methane oxidation, but may also affect nitrogen cycling in the soil by affecting denitrification, and consequently soil health (Chen et al. 2024 ). Compared with Group A, ABC transporters, Glycine, serine and threonine metabolism, and Steroid degradation in Group B were down-regulated, among which ABC transporter down-regulation had positive effects on the stability of soil structure and plant growth. With the development of biological soil crusts, the demand for ABC transporters for microbial osmotic potential-enhancing substances (glycine betaine/proline, osmotic protectants, erythritol, Na + , putrescine, 1-phosphatidylinositol) generally decreased (Zhang et al. 2020 ). Rhizosphere soil has a vital influence on the biogeochemical cycles and metabolic processes of basic elements such as carbon, nitrogen, phosphorus, sulfur, and iron, which is governed by the interactions among soil components, plants, and microorganisms (Liu et al. 2023 ). Indeed, the cycling of various elements in soil is essentially driven by complex microbial networks. Hence, this study focuses specifically on the nitrogen cycle as one of its key processes to gain deeper insights into how microbial activities regulate soil functionality. To assess the abundance of the nitrogen metabolic pathway in soil, differential tests and visual analyses of key enzymes were performed. The significance of differences in the abundance of enzymes involved in this pathway was evaluated across the three sample groups. These analyses revealed that enzymes catalyzing prokaryotic denitrification, including nitrous oxide reductase (EC 1.7.2.4), nitric oxide reductase (cytochrome c) (EC 1.7.2.5), and nitrite reductase (NO – forming) (EC 1.7.2.1), were more abundant in soils after one year and two-year of morel mushroom cultivation compared to the control group. It indicates that the soil denitrification capacity has significantly increased, which is conducive to converting active nitrogen (NO 3 – /NO 2 – ) into inert nitrogen gas (N 2 ) for release. They mainly enhance denitrification and nitrogen assimilation and reuse. At the same time, it could increase bacterial abundance and recruit beneficial soil microorganisms. Soil phenolic acid accumulation driven by morel mushroom cultivation Combined with our determination of total phenolic acid content in soil and metagenomic analysis, Group C exhibited the highest phenolic acid content. Notably, the level of PAL, a key enzyme involved in phenolic acid synthesis, was significantly greater than that observed in the other two groups. Conversely, Group A displayed the lowest phenolic acid content; however, it had a markedly higher concentration of P34O, an essential enzyme for decomposing phenolic acids compared to the other groups. The two results confirm each other. Since the gene abundance of PAL phenol acid synthase is very low in the metagenome, it is speculated that the main driving force of phenol acid synthesis in soil is not the microorganisms in the soil environment, but the edible fungi themselves. So the investigation focused on the phenol-acid-degrading enzyme P34O. Arthrobacter , Geodermatophilus , and Kocuria were also significant contributors to P34O enzyme activity. Arthrobacter demonstrates a strong capacity for degrading phenolic acids; indeed, Li Ming isolated an efficient strain capable of degrading these compounds from soil samples collected over many years from the Selenomargua growing area in Ningxia and identified it as Arthrobacter (Li et al. 2019 ). Bacteria belonging to the genus Geodermatophilus are typically found in extreme environments characterized by high temperatures and arid soils; they may possess unique metabolic pathways and stress resistance mechanisms. However, their specific role in phenolic acid degradation requires further investigation. During Kocuria flava degradation processes, catechol 2,3-dioxygenase (C23O) gene expression levels were 6.02 log-fold higher than those observed for Rhodococcus pyridinivoran s. This finding suggests that Kocuria flava may play a crucial role in polycyclic aromatic hydrocarbons (PAHs) degradation (Sakshi et al. 2023). Conclusion Morchella sextelata cultivation is a beneficial practice for ameliorating saline-alkali soils, effectively reducing pH, enhancing nutrient content, and stimulating nitrogen metabolism. However, the transition to a cultivated ecosystem induces a complex restructuring of the microbial community. While beneficial for overall soil quality, the concurrent issues of phenolic acid accumulation and potassium depletion pose a risk for soil sickness. Future management strategies should therefore integrate targeted approaches to mitigate phenolic acid accumulation and counteract potassium depletion, thereby ensuring the sustainable cultivation of this valuable fungus. Declarations Conflict of interest The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Funding This research was funded by an enterprise-sponsored project: University Local Cooperation Project: Research and Demonstration of Key Technologies for Morchella Cultivation in Hongsipu District (222136). Enterprise-Commissioned Project: Analysis of Physical and Chemical Properties and Microbial Community Structure of Saline-Alkali Soil After Morchella Cultivation (2025640101000053). Authors’ contributions Wu Yifan: investigation, methodology, software, visualization, writing-original draft. Yang Xiaoli: methodology, software, writing review and editing. Zhang Yaya: writing review and editing. Xu Chunyan: All authors read and approved the final manuscript. References Arif Y, Singh P, Siddiqui H et al (2020) Salinity induced physiological and biochemical changes in plants: An omic approach towards salt stress tolerance. Plant Physiol Biochem 156(Suppl. 1) Asina F, Brzonova I, Voeller K et al (2016) Biodegradation of lignin by fungi, bacteria and laccases. 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07:10:47","extension":"png","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":35145,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8232173/v1/76389e3dd0c2a8aa92150b43.png"},{"id":100951961,"identity":"4977d333-8d13-4dec-a237-3d769b89f772","added_by":"auto","created_at":"2026-01-23 07:11:36","extension":"png","order_by":29,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":40111,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8232173/v1/9787bee267a227d9479887a2.png"},{"id":100922661,"identity":"c2607c2f-8dbe-44cf-9c1b-81d9e918c2ca","added_by":"auto","created_at":"2026-01-22 20:41:21","extension":"xml","order_by":30,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":115470,"visible":true,"origin":"","legend":"","description":"","filename":"PLSOD25046720structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8232173/v1/0623ae0804cd988d8ec44a97.xml"},{"id":100922662,"identity":"ac584fc3-912e-4a06-b504-9a9d91a37da3","added_by":"auto","created_at":"2026-01-22 20:41:21","extension":"html","order_by":31,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":128345,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8232173/v1/1a8ae3688ab4b2a151a61d7d.html"},{"id":100922633,"identity":"d1d95f98-cca3-42e8-8e51-a873364b6c22","added_by":"auto","created_at":"2026-01-22 20:41:20","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":116356,"visible":true,"origin":"","legend":"\u003cp\u003eMap showing sampling site locations.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8232173/v1/0f04d39abff3c3c7da0b78a6.jpg"},{"id":100951989,"identity":"39e9c853-9be8-483f-a335-aae01e040429","added_by":"auto","created_at":"2026-01-23 07:11:39","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":120732,"visible":true,"origin":"","legend":"\u003cp\u003eSoil physicochemical properties under different treatments. (a) pH value, (b) total phenolic acid, (c) organic matter, (d) total nitrogen, (e) total phosphorous, (f) total potassium, (g) Olsen nitrogen, (h) Olsen potassium, (i) Olsen phosphorus. Bar charts and error bars represent the mean and standard deviation of three biological replicates.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8232173/v1/0f9ebd2963b52a66270aeff9.jpg"},{"id":100922630,"identity":"020feaec-967a-44b1-b14e-534d96da98f0","added_by":"auto","created_at":"2026-01-22 20:41:20","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":81382,"visible":true,"origin":"","legend":"\u003cp\u003eEnzyme activities under different treatments. (a) soil phosphatase, (b)soil catalase, (c) soil urease, and (d) soil sucrase. Bar charts and error bars represent the mean and standard deviation of three biological replicates.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8232173/v1/8316b1340c29b100f021cda3.jpg"},{"id":100952126,"identity":"5f9aeec3-d844-480e-9a25-6be6c1323f18","added_by":"auto","created_at":"2026-01-23 07:11:59","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":183308,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of soil microbial community structure Community composition of bacteria (a) and fungi (b) at the phylum level, community composition of bacteria (c) and fungi (d) at the genus level, Venn diagram of species composition at the bacterial (e) and fungal(f) species level.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8232173/v1/368f6decc4b2de4753d9eeda.jpg"},{"id":100922642,"identity":"f7f43197-989d-4db2-b8bb-9f919861222b","added_by":"auto","created_at":"2026-01-22 20:41:20","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":155398,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of soil microbial diversity PCA analysis at the genus level of fungi (a) and bacteria (b), Analysis of group differences in chao1(c), Shannon (d) and Simpson (e) indices in alpha diversity of soil bacteria, Analysis of group differences in chao1(f), Shannon (g) and Simpson (h) indices in alpha diversity of soil fungi.\u003c/p\u003e\n\u003cp\u003eCorrelations between soil microbial genera and environmental factors\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8232173/v1/6309c16ac68cd0bffb2be087.jpg"},{"id":100922634,"identity":"462213a8-2117-46c5-96df-9930c0f6203f","added_by":"auto","created_at":"2026-01-22 20:41:20","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":201692,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation analysis of environmental factors (Heat map of the correlation between environmental factors and different fungal genera (a), and heat map of the correlation between environmental factors and different bacterial genera (b).\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8232173/v1/55abc633037a7d2e7e55b93c.jpg"},{"id":101751373,"identity":"3eecf8a5-db2b-44e5-ae7b-41df694c1193","added_by":"auto","created_at":"2026-02-03 10:19:49","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":253861,"visible":true,"origin":"","legend":"\u003cp\u003eKEGG pathway enrichment bubble plots of (a) Group A vs. Group B and (b) Group B vs Group C\u003c/p\u003e","description":"","filename":"Picture7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8232173/v1/d9db64faab8fac58ffedb158.jpg"},{"id":100951403,"identity":"42fa1170-a8f2-44c4-b1ca-af07fe3feac1","added_by":"auto","created_at":"2026-01-23 07:10:35","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":103280,"visible":true,"origin":"","legend":"\u003cp\u003eThe pathway map of N-metabolism and comparison in abundance of key enzymes\u003c/p\u003e\n\u003cp\u003eSynthesis and degradation pathway analysis of phenolic acids\u003c/p\u003e","description":"","filename":"Picture8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8232173/v1/942b4c50d8eedb4472f56271.jpg"},{"id":100951562,"identity":"5316a765-5e62-4e00-8a11-144c0877821d","added_by":"auto","created_at":"2026-01-23 07:10:52","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":146089,"visible":true,"origin":"","legend":"\u003cp\u003eSynthesis and degradation analysis of phenolic acids.\u003c/p\u003e","description":"","filename":"Picture9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8232173/v1/5118f1f4b33c24bfdce0c336.jpg"},{"id":100951465,"identity":"ad3c9dc4-5e45-40a3-972e-76d2f1e916b7","added_by":"auto","created_at":"2026-01-23 07:10:38","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":54716,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of the gene abundance of key enzymes related to phenolic acids metabolism in different treatments\u003c/p\u003e","description":"","filename":"Picture10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8232173/v1/9baa948422e6fb4842c62f60.jpg"},{"id":100951665,"identity":"4eafd37d-fb31-4645-9258-737e74b8ae7a","added_by":"auto","created_at":"2026-01-23 07:11:04","extension":"jpg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":121253,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation analysis of contribution degree between species and functional enzymes.\u003c/p\u003e","description":"","filename":"Picture11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8232173/v1/872f038489be9cafca21da11.jpg"},{"id":100922659,"identity":"b6144245-ff1c-45a2-bec1-991887c24670","added_by":"auto","created_at":"2026-01-22 20:41:21","extension":"jpg","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":57368,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation analysis of the contribution degree between species and functional gene.\u003c/p\u003e","description":"","filename":"Picture12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8232173/v1/63c451a99890a235602a8309.jpg"},{"id":103451864,"identity":"8795ba9f-45f1-4344-ae1b-6269bdd7cb86","added_by":"auto","created_at":"2026-02-25 21:39:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2161931,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8232173/v1/6bc1135f-ec87-47d3-afeb-80e22429bd25.pdf"}],"financialInterests":"","formattedTitle":"Cultivation of Morchella sextelata in saline-alkali soils of Ningxia: Multi-dimensional impacts on soil physico-chemical properties, enzyme activities, microbial ecology and functional genes","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eMorchella sextelata\u003c/em\u003e is a rare edible fungus belonging to the Pezizales order and Morchellaceae family. It is named after its lumpy cap, which resembles a sheep\u0026rsquo;s belly. It has a unique flavor and an exquisite aroma, and it is rich in over 20 amino acids and trace elements, making it highly sought after by consumers (Li et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Accordingly, morel mushrooms are considered a rare type of edible fungi with both culinary value and medicinal benefits (Haider et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) (Wang et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e) (Ramya et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Therefore, the cultivation of morel mushrooms has long been a topic of concern to scientists. Currently, China has become the world\u0026rsquo;s largest producer and exporter of morel mushrooms (\u003cem\u003eMorchella sextelata\u003c/em\u003e). In 2024, China\u0026rsquo;s exports of dried morel mushrooms reached 225.14 tons, valued at approximately USD 17.76\u0026nbsp;million (equivalent to roughly \u0026yen;126\u0026nbsp;million RMB) (Zhang et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2025a\u003c/span\u003e). Concurrently, large-scale cultivation techniques for morel mushrooms have achieved success in provinces such as Sichuan, Yunnan, Hunan, Hubei, and Shaanxi. As the cultivation area gradually expands, the production regions for morel mushrooms are also extending northward from southern China (Xu et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHowever, significant differences in soil quality exist between the northern and southern regions (Zhang et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2025b\u003c/span\u003e). Unlike the high-humidity soils of southern China, arid and semi-arid northern regions exhibit soil salinisation. Soils with EC values exceeding 4 dS/m at 20 ℃ and with exchangeable Na\u003csup\u003e+\u003c/sup\u003e content above 15% are deemed saline (Sz\u0026acute;ekely and Barta \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Globally, saline-alkali land covers approximately 9.55\u0026times;10\u003csup\u003e8\u003c/sup\u003e hm\u0026sup2;, of which China accounts for about 9.91\u0026times;10\u003csup\u003e7\u003c/sup\u003e hm\u0026sup2; (Arif et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The saline-alkali land in the Hongsipu Yanghuang Irrigation District of Ningxia serves as a critical ecological restoration zone and features one of northwest China\u0026rsquo;s most technologically advanced lift irrigation systems (Wu et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Morel-farming beds are very important for the growth of the morel mushroom. Different types of soil (forests, paddy fields, greenhouses and orchards) distinctly affect the growth of morel mushrooms and the ecological conditions of soil microorganisms (Yue et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). While the different types of morel-farming beds have an impact on the growth of morel mushrooms, their cultivation also improves the soil environment. Planting morel mushrooms can enhance soil health. During the fallow period, planting morel mushrooms can improve the soil characteristics of paddy fields and drylands and increase grain production (Duan et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, soil microorganisms, as key decomposers, are integral components of the soil ecosystem and play a key role in improving soil structure, regulating soil nutrient balance, and influencing crop yield (Wang et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In addition, microorganisms are sensitive biological indicators of changes in the soil environment, and soil microbial diversity and community structure can serve as important indicators for assessing soil fertility (Jeffries et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) (Luo et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Significant differences in soil microbial structure between morel-cultivated soils and non-cultivated soils (Zhang et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e). Inoculation with morel mushrooms\u0026rsquo; mycelia had a notable impact on soil bacterial diversity.\u003c/p\u003e \u003cp\u003eIn saline-alkali environments, where inherent constraints (high salinity, elevated pH, poor soil structure, and suppressed microbial activity) severely limit morel mushrooms (Dong et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Understanding context-specific responses, particularly in saline-alkali soils, is therefore essential for sustainable cultivation. Since the introduction of morel mushroom species to Ningxia in 2018 and successful trials, the industry has developed rapidly. However, compared with reports in Yunnan and Sichuan, a gap exists regarding morel mushroom cultivation on saline-alkali soil. A comprehensive understanding of how \u003cem\u003eMorchella sextelata\u003c/em\u003e cultivation itself, as a soil intervention, systematically affects key properties, including physicochemical characteristics, enzyme activities, microbial community composition/ diversity, and functional gene abundance, remains limited. Additionally, allelopathic autotoxicity (where plants release metabolites inhibiting their own growth) is a concern, as morel mushrooms cultivation typically accumulates phenolic acids in the soil.\u003c/p\u003e \u003cp\u003eTherefore, this study aims to investigate differences in soil physicochemical properties, enzyme activities, microbial communities, and functional genes between morel-cultivated and non-cultivated soils in Ningxia\u0026rsquo;s saline-alkali land. Changes in soil microbial community structure and function were analyzed via metagenomics. Quantify phenolic acid content, key enzyme abundance, and related microbial communities across treatment groups.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eSoil sample collection\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the sampling sites were selected in the Ningxia morel mushroom planting base (37\u0026deg;20\u0026rsquo;N, 105\u0026deg;57\u0026rsquo;E, altitude 1240\u0026ndash;1450 m) and 3 groups of samples were collected. They are soil that (A) unplanted control, (B) one year of \u003cem\u003eMorchella\u003c/em\u003e cultivation, and (C) two years of \u003cem\u003eMorchella\u003c/em\u003e cultivation. Set up two test greenhouses to collect series B and C. Soil samples were taken from the bare ground outside the shed. Each greenhouse is divided into three large quadrats, front, middle, and back, as three biological weights. In addition, a five-point sampling method was used to collect soil samples for each quadrate. Mix near the morel mushrooms and furrows with 0 to 20 cm of soil at each point. After the mixture is evenly mixed, 500 g of the mixed soil is weighed as the test soil. Part of the soil was preserved at -80 ℃ and sent to the biological company for metagenomic sequencing. The soil physical and chemical properties and enzyme activities of the remaining soil samples were tested after air drying.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDetermination of soil enzyme activities and physicochemical properties\u003c/p\u003e \u003cp\u003eSoil physicochemical indexes and enzyme activities were determined according to soil science experiments and soil enzymes and their Research Method (Xie et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) (Liu et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), respectively, as detailed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMethods for the determination of soil physicochemical properties and enzyme activities\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIndex of measurement\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMethod of determination\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epH value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epH acidity meter method\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003econtent of water\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDrying method\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eorganic matter\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePotassium dichromate oxidation method\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003etotal nitrogen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSemi-micro Kjeldahl method\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003etotal phosphorous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMo-Sb colorimetric method\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003etotal potassium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFlame spectrometry\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eolsen nitrogen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ealkali N-proliferation method\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eolsen potassium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFlame spectrometry\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eolsen phosphorous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMicro determination\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esoil urease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePhenol-hypochlorite colorimetry\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esoil sucrase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3,5-dinitro salicylic acid colorimetry\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esoil catalase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePermanganate titration\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esoil phosphatase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePhenyl phosphate titration\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"2\"\u003eExtraction and determination of total phenolic acids\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTotal phenolic acids were extracted using the modified method of alkali solution. 20 grams of dry soil samples were added to 100 mL NaOH solution (3 mol/L) and oscillated for 20 h at 150 r/min. The soil suspension was centrifuged at 5,000 g for 15 min, and the supernatant was filtered through filter paper. Then, the pH of the filtrate was adjusted to l.5 with HCl (5 mol/L), and extracted five times with ethyl acetate. The resultant extracts were pooled and evaporated to dryness using a rotary evaporator at 60 ℃. The residue was dissolved in 5 mL of distilled water and kept in the dark at 4 ℃. The content of phenolic acids was determined by the Folin-Ciocalteu Method using gallic acid as the standard. 0.5 mL soil extract and 1.5 mL deionized water were placed into a 10-ml centrifuge tube, then 5 mL of 1 mol/L Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e and 1 mL Folin and Ciocalteu. Ciocalteu\u0026rsquo;s phenol reagent was added and incubated for 1 h in darkness at ambient temperature. Finally, the absorbance of the mixture was measured at 760 nm with a UV-Vis spectrophotometer. The total phenolic acid content in soil was expressed in micrograms of gallic acid equivalent per gram of soil.\u003c/p\u003e \u003cp\u003eAnalysis of microbial community structure and data processing\u003c/p\u003e \u003cp\u003eFungal and bacterial communities were analyzed after quality control and annotation of the sequencing data. The statistical analysis results of species abundance differences in the soil of different treatment groups were obtained, and the species with significant differences were analyzed. The relationship between functional genes and microbial communities was analyzed. Excel and SPSS 26 were used for data processing and Duncan\u0026rsquo;s significance test (significance level was \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), combined with information analysis cloud platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cloud.majorbio.com/page/project/overview.html\u003c/span\u003e\u003cspan address=\"https://cloud.majorbio.com/page/project/overview.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) analysis, and visualization mapping. The correlations between soil environmental factors and microbial communities were also analyzed by calculating the correlations between the environmental factors and the selected species (using Spearman and Pearson correlation coefficients, etc.). The obtained numerical matrix is visually presented through a Heatmap graph. The ggplot2 and ggtree in R were used to analyze the relationship between functional genes and microbial communities.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eSoil enzyme activities and physicochemical properties\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the pH value of cultivated morel mushrooms (B and C) was significantly lower than that of uncultivated morel mushrooms (A), suggesting that cultivation practices may induce soil acidification. However, there was no significant difference in pH value between morel mushrooms planted for one year (B) and two years (C). The contents of soil total nitrogen, total phosphorus, organic matter, soil urease, soil sucrase, and soil phosphatase in Groups B and C were significantly higher than those in Group A, indicating that cultivation enhances soil nutrient availability and biochemical activity. Again, no significant differences were detected between one-year and two-year cultivation. Notably, Olsen nitrogen and Olsen phosphorus levels showed significant differences across all groups, with \u003cem\u003eGroup C\u0026thinsp;\u0026gt;\u0026thinsp;Group B\u0026thinsp;\u0026gt;\u0026thinsp;Group A\u003c/em\u003e, implying that available nitrogen and phosphorus continue to accumulate with extended cultivation time. In contrast, total potassium was highest in Group B, significantly exceeding both Groups A and C, which did not differ from each other. Olsen potassium also varied significantly among all groups, following the order Group B\u0026thinsp;\u0026gt;\u0026thinsp;Group C\u0026thinsp;\u0026gt;\u0026thinsp;Group A, suggesting a more complex dynamics of potassium availability affected by cultivation duration. The total potassium content of Group B was significantly higher than that of Groups A and C, and there was no significant difference between A and C.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSoil microbial community structure and diversity\u003c/p\u003e \u003cp\u003eThe composition of the soil microbial community differed significantly among the groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef), a variation likely associated with changes in the soil microenvironment induced by different planting treatments. At the phylum level, Actinomycetota was the absolutely dominant phylum in Group A, while both Actinomycetota and Pseudomonadota were dominant in Groups B and C. A decrease in the relative abundance of Actinomycetota and an increase in Pseudomonadota were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), suggesting that planting activities may have promoted the proliferation of bacterial taxa involved in specific nutrient cycling processes. Regarding the fungal community, the abundance of Ascomycota decreased, while Mucoromycota and Basidiomycota increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb), indicating a possible enrichment of fungal groups related to decomposition or plant symbiosis following morel mushroom cultivation. At the genus level, a clear shift in bacterial dominance was observed, likely reflecting altered nutrient dynamics. Group A was dominated by \u003cem\u003eArthrobacter\u003c/em\u003e and \u003cem\u003eNocardioides\u003c/em\u003e, genera often associated with soil organic matter decomposition. In contrast, the rise of \u003cem\u003eSphingomonas\u003c/em\u003e (common in root-associated environments) and a significant increase in \u003cem\u003eNitrospira\u003c/em\u003e (a key nitrite-oxidizing bacterium) in Groups B and C suggest a shift toward bacterial communities involved in plant-microbe interactions and enhanced nitrification, respectively. A concomitant decline in \u003cem\u003eNocardia\u003c/em\u003e was also noted. Fungally, the community transitioned from being dominated by \u003cem\u003eAlternaria\u003c/em\u003e in Group A to the dominance of \u003cem\u003eAspergillus\u003c/em\u003e, \u003cem\u003eRhizopus\u003c/em\u003e, and \u003cem\u003eFusarium\u003c/em\u003e in treated groups. The decline of \u003cem\u003eAlternaria\u003c/em\u003e and the rise of Aspergillus, a potent decomposer, particularly point to a fungal community restructuring geared toward more efficient organic matter decomposition following morel mushroom cultivation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eAt the species level, there were 25,820 bacterial species in the three soil samples, accounting for 73.97% of the total in Group A, 82.2% in Group B, and 79.8% in Group C, respectively. There were 2,417 endemic species in Group A, 1,767 in Group B, and 1,156 in Group C (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). There were 616 fungal species in the three soil samples, accounting for 60.4% of the total in Group A, 53.1% in Group B, and 42.7% in Group C. There were 210 endemic species in Group A, 126 in Group B, and 50 in Group C (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). The number of endemic species exhibited significant trends with increasing cultivation duration. For bacteria, the number of endemic species significantly decreased with longer cultivation duration (Group A: 2417 species Group B: 1767 species Group C: 1156 species). Similarly, the number of fungal endemic species also decreased with increasing cultivation duration, but the decline was more pronounced and particularly sharp in Group C (Group A: 210 species\u0026thinsp;\u003cem\u003e\u0026gt;\u003c/em\u003e\u0026thinsp;Group B: 126 species\u0026thinsp;\u003cem\u003e\u0026gt;\u003c/em\u003e\u0026thinsp;Group C: 50 species). The endemic species in Group A represent the unique microbial community of pristine, undisturbed saline-alkaline soil. Following \u003cem\u003eMorchella\u003c/em\u003e cultivation (Groups B and C), the number of these endemic species decreased significantly, which suggests that \u003cem\u003eMorchella\u003c/em\u003e cultivation practices (including soil treatment, fungal inoculum introduction, cultivation management, etc.) resulted in the loss of a portion of indigenous microorganisms or the reduction of their abundance below detection thresholds. Notably, this phenomenon manifested more pronouncedly within the fungal community. From the perspective of fungi, the specific fungi unique to Group A are mostly \u003cem\u003eAlternaria\u003c/em\u003e, \u003cem\u003eColletotrichum\u003c/em\u003e, \u003cem\u003eDidymella\u003c/em\u003e, \u003cem\u003eFusarium\u003c/em\u003e, and \u003cem\u003ePenicillium\u003c/em\u003e. The specific fungi unique to Group B are mostly \u003cem\u003eMortierella\u003c/em\u003e, \u003cem\u003ePenicillium\u003c/em\u003e, \u003cem\u003ePseudogymnoascus\u003c/em\u003e and \u003cem\u003eCoemansia\u003c/em\u003e, while the fungi unique to Group C are mostly \u003cem\u003ePenicillium\u003c/em\u003e, Fusarium, \u003cem\u003eLeptographium\u003c/em\u003e, \u003cem\u003eEpichloe\u003c/em\u003e, and \u003cem\u003ePleurotus\u003c/em\u003e. From the perspective of bacteria, the specific bacteria unique to Group A are mostly \u003cem\u003eStreptomyces\u003c/em\u003e, \u003cem\u003eArthrobacter\u003c/em\u003e, \u003cem\u003eMethylobacterium\u003c/em\u003e, \u003cem\u003eBacillus\u003c/em\u003e, and \u003cem\u003eExiguobacterium\u003c/em\u003e. The specific bacteria unique to Group B are mostly \u003cem\u003eChryseobacterium\u003c/em\u003e, \u003cem\u003ePseudomonas\u003c/em\u003e, \u003cem\u003eFlavobacterium\u003c/em\u003e, and \u003cem\u003ePedobacter\u003c/em\u003e. The specific bacteria unique to Group C are mostly \u003cem\u003ePseudomonas\u003c/em\u003e, \u003cem\u003eStreptomyces\u003c/em\u003e, \u003cem\u003eBurkholderia\u003c/em\u003e, \u003cem\u003eMesorhizobium\u003c/em\u003e, and \u003cem\u003eRhizobium\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAlpha diversity analysis revealed a significant and consistent increase in bacterial diversity following morel mushroom cultivation. The rise in the Chao1, Shannon, and Simpson indices in Groups B and C compared to Group A indicates that planting morel mushrooms not only enriched bacterial species richness but also enhanced community evenness. This suggests that the cultivation practice created a more heterogeneous microenvironment or introduced diverse organic substrates, fostering a more complex and stable bacterial community. In stark contrast, the lack of significant change in fungal diversity across all indices underscores a fundamental difference in ecological strategy. The fungal community structure remained stable and resistant to the changes induced by short-term cultivation. This divergence highlights that morel mushroom cultivation selectively promotes bacterial diversity without disrupting the resident fungal network, potentially leading to a more multifaceted and resilient soil ecosystem (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCorrelations between soil microbial genera and environmental factors\u003c/p\u003e \u003cp\u003eThe correlation analysis at the genus level revealed distinct environmental preferences among key microbial taxa, linking community structure to soil enzyme activities and physicochemical properties. The significant negative correlation of \u003cem\u003eNocardioides\u003c/em\u003e and \u003cem\u003eArthrobacter\u003c/em\u003e with key fertility indicators (catalase, total nitrogen, potassium, and water content) suggests these taxa are better adapted to, or even thrive in, less fertile conditions, possibly acting as oligotrophic specialists. Conversely, the strong positive correlation of \u003cem\u003eNitrospira\u003c/em\u003e (a known nitrite-oxidising bacterium) with these same factors indicates its role in nutrient-rich environments, likely coupling its nitrification function with heightened potassium availability and microbial activity (catalase). Fungal genera also displayed clear niche partitioning: \u003cem\u003eAlternaria\u003c/em\u003e and \u003cem\u003eFusarium\u003c/em\u003e were associated with higher pH, while the saprotrophic genera \u003cem\u003eMortierella\u003c/em\u003e and \u003cem\u003eRhizopus\u003c/em\u003e were positively linked with enhanced nutrient content (total nitrogen, potassium) and catalase activity, underscoring their importance in organic matter decomposition in fertile soils.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eKEGG functional enrichment analysis\u003c/p\u003e \u003cp\u003eThe differential pathway analysis underscores a microbial functional adaptation to the altered soil environment under morel mushroom cultivation. The upregulation of stress-associated pathways (lipopolysaccharide biosynthesis, two-component system) in the control group implies that cultivation alleviates certain environmental pressures on the microbial community. This is supported by the downregulation of core metabolic processes (amino acid biosynthesis, carbon metabolism) in the control, indicating a less metabolically active state. In contrast, the microbial community in the two-year cultivated soil demonstrated a pronounced shift toward heightened metabolic activity, evidenced by the upregulation of key pathways involved in cofactor synthesis, substrate transport (ABC transporters), and carbon utilization. The suppression of the thermogenic pathway further suggests an optimization of energy expenditure, favoring biosynthetic processes over heat dissipation in a more stable, cultivated environment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe marked increase in key enzymes involved in denitrification and dissimilatory nitrate reduction, including nitrous oxide reductase, nitric oxide reductase, and nitrate reductase, suggests a pronounced shift in the nitrogen cycle following morel cultivation. The elevated abundance of these enzymes implies an enhanced capacity for nitrate reduction to nitrite and subsequently to gaseous forms (NO and N\u003csub\u003e2\u003c/sub\u003eO). The upregulation of multiple enzymes in the denitrification pathway points to a systemic enrichment of microorganisms capable of anaerobic nitrogen respiration. This functional adaptation likely results from changes in soil structure, moisture, and organic matter input associated with morel mushroom farming, which may create microsites with localized low oxygen conditions. The consistency of this pattern across one and two years of cultivation indicates a stable restructuring of the microbial community\u0026rsquo;s metabolic network, orienting it toward nitrate and nitrite reduction as key energy-generating processes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSynthesis and degradation pathway analysis of phenolic acids\u003c/p\u003e \u003cp\u003eAllelopathic autotoxicity refers to the phenomenon where plants release specific secondary metabolites into the environment, thereby causing harmful effects on their growth and development (R. Baziramakenga 1995). In the context of morel mushrooms cultivation, this phenomenon is of particular concern due to its implications for continuous cropping obstacles. It\u0026rsquo;s reported that the allelopathic substances of morel mushrooms include 4-coumaric acid, vanillin, ferulic acid, and vanillic acid (Qi et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This is also the reason for the significant increase in total phenolic acid content in Group B compared with Group A. These phenolic acids not only contribute to the observed increase in total phenolic acid content in Group B compared with Group A but also play a crucial role in soil acidification and autotoxic feedback. This accumulation of phenolic acids reduces soil pH and, when excessively accumulated, exerts negative effects on both soil health and the crops themselves. Therefore, the rational management of phenolic acids has become a key focus. Through the determination of total phenolic acid content in soil samples collected under different treatments, it was found that the content of total phenolic acids in Group C was significantly higher than that in Groups B and A. Through the analysis of the synthetic and degradation pathways of phenolic acids (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e), PAL (phenylalanine ammonia-lyase) and P34O (protocatechuate 3,4-dioxygenase) were identified as the key enzymes in phenolic acid synthesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe interplay between the abundance of key enzyme genes and the phenolic acid content reveals the core of the autotoxicity mechanism. The average difference in gene abundance of key enzymes between different treatments was analyzed (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e10\u003c/span\u003e). The abundance of PAL, the key enzyme for phenolic acid synthesis, was significantly highest in Group C. Conversely, the abundance of P34O, the central enzyme for phenolic acid degradation, was significantly highest in Group A. This divergent profile creates a clear metabolic predisposition: Group C is genetically primed for high synthesis and low degradation, directly explaining the significant phenolic acid accumulation observed in this group. In contrast, Group A\u0026rsquo;s profile (low synthesis and high degradation capacity) aligns with its lower phenolic acid content, illustrating an effective metabolic balance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e10\u003c/span\u003e. Analysis of the gene abundance of key enzymes related to phenolic acids metabolism in different treatments\u003c/p\u003e \u003cp\u003eThe analysis of microbial functional contributions revealed a critical shift in the microbial decomposer community that underlies the observed differences in phenolic acid accumulation. Based on the correlation analysis of species contribution to the key enzyme P34O for phenolic acid decomposition, distinct patterns emerge: Group A (unplanted soil) exhibited a microbial community adapted to its native state. The genera \u003cem\u003eArthrobacter\u003c/em\u003e, \u003cem\u003eGeodermatophilus\u003c/em\u003e, and \u003cem\u003eKocuria\u003c/em\u003e were identified as having high contributions to the P34O enzyme. Specifically for the functional gene \u003cem\u003epcaG\u003c/em\u003e, \u003cem\u003eRubrobacter\u003c/em\u003e was the dominant contributor, while for \u003cem\u003epcaH\u003c/em\u003e, it was \u003cem\u003eMicrovirga\u003c/em\u003e. Group B (one-year cultivated soil), a significant shift was observed, as there was no dominant contributing species for the \u003cem\u003epcaG\u003c/em\u003e. For the \u003cem\u003epcaH\u003c/em\u003e, \u003cem\u003eBlastococcus\u003c/em\u003e emerged as the top contributor. This suggests that the first year of cropping disrupted the original stable community without yet establishing a new, dominant functional population. Group C (two-year cultivated soil following the \u0026ldquo;edible fungi-qilin melon\u0026rdquo; rotation ) established a distinctly restructured microbial community. For the functional gene \u003cem\u003epcaG\u003c/em\u003e, the bacteria belonging to \u003cem\u003eMicrovirga\u003c/em\u003e and \u003cem\u003eNocardioides\u003c/em\u003e were the dominant contributors. In contrast, for the \u003cem\u003epcaH\u003c/em\u003e gene, \u003cem\u003eSphingomonas\u003c/em\u003e was identified as the species with the highest contribution.\u003c/p\u003e \u003cp\u003eIn conclusion, the transition from native soil to cultivated systems not only restructures the microbial community but also appears to disrupt the efficacy of the phenolic acid degradation pathway. The \u0026ldquo;edible fungi-qilin melon\u0026rdquo; system, while promoting a distinct microbial consortium, is associated with a significant accumulation of phenolic acids, indicating a potential risk for soil sickness or allelopathy that could hinder sustainable cultivation unless managed appropriately.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eDynamic changes in physico-chemical properties and enzyme activity of saline-alkali soil driven by morel mushroom cultivation\u003c/p\u003e \u003cp\u003eAn increasing number of studies have demonstrated that the cultivation of edible fungi can enhance soil environments. For instance, the undergrowth cultivation of \u003cem\u003eP. glomus\u003c/em\u003e significantly improved soil porosity, soluble element content, biological enzyme activity, microbial diversity and metabolism, thereby reconstructing soil ecological functions and exhibiting considerable potential for soil remediation and sustainable crop production (Liu et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Examined the effects on pH, total salt content, organic matter levels, available potassium, and ammonium nitrogen in soils following the planting of Stropharia rugosoannulata and investigated its efficacy in ameliorating saline-alkali soils (Yang et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The ability of edible fungi to improve soil environments primarily stems from their residues\u0026rsquo; application in soils, which leads to a series of changes in soil structure, material composition, and nutrient balance resulting in alterations to the composition of soil organic matter and subsequently affects microbial community composition, abundance, and activity. These changes influence both matter-energy conversion processes within the soil and nutrient availability from fertilizers (Li et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), which is in agreement with our experimental findings. Compared to Group A, significant improvements were observed in nutrient levels and pH values within Group B after one year of morel mushroom cultivation. However, total potassium and available potassium contents in Group C, where morel mushrooms were cultivated for two years, were markedly lower than those found in Group B after one year\u0026rsquo;s cultivation, suggesting that continuous morel mushroom cultivation may lead to potassium consumption within the soil (Zhang et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e). In addition, compared with Groups B and A, Group C showed a rotation pattern of \u0026ldquo;edible fungi qilin melon\u0026rdquo;, which could effectively maintain the soil optimization brought by morel mushroom cultivation in Group B.\u003c/p\u003e \u003cp\u003eSuccessional Dynamics of Soil Microbial Community Structure and Function\u003c/p\u003e \u003cp\u003ePrevious studies have shown that plant cultivation or agricultural management practices can reshape the soil microbial community through direct (such as root exudates) or indirect (such as changes in soil physical and chemical properties) mechanisms, and the impact of continuous cropping (long-term cultivation of the same crop) is more significant (Khmelevtsova et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).In addition, long-term plant encroachment enhanced microbial diversity and network stability in the special environment of bauxite residue (Deng et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Analysis of the microbial community composition across treatments revealed a shift from a homogeneous to a more diversified state under morel mushroom cultivation. However, the rich microbial composition can make the soil more stable and versatile (Philippot et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In group A, the Actinobacteria were the absolutely dominant phylum. However, in groups B and C, the abundance of the Actinobacteria decreased while that of the Deinococcus and Actinobacteria increased. Together, these two phyla became the dominant ones, but the Actinobacteria remained the core dominant phylum present in all three groups. The Actinobacteria usually dominate in natural, undisturbed soils. It has strong metabolic capabilities and can decompose complex organic substances (such as cellulose and lignin) (Asina et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). On the other hand, the Proteobacteria phylum is more sensitive to changes in environmental nutrients and its abundance tends to increase in cultivated soils or soils with increased nutrient input (Choudhary et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Following the cultivation of morel mushrooms, a significant shift in soil fungal community composition was observed: the relative abundance of Ascomycota decreased, while increases were noted in the relative abundances of Mucoromycota and Basidiomycota. Given that morel mushrooms themselves belong to the Ascomycota phylum, it is hypothesized that their cultivation might lead to competitive suppression of other Ascomycota taxa, potentially through resource competition or niche occupation. However, other factors associated with cultivation practices could also contribute to these observed community shifts.\u003c/p\u003e \u003cp\u003eIn addition, the dominant bacteria in Groups B and C were \u003cem\u003eSphingomonas\u003c/em\u003e and \u003cem\u003eNitrospira\u003c/em\u003e. They play an important role in maintaining soil nitrogen cycling (Li et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) (Meng et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). \u003cem\u003eSphingomonas\u003c/em\u003e has been reported to play a role in the bioremediation and bacterial regulation of polycyclic aromatic hydrocarbons (PAH) contaminated saline soil (Song et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). For fungi, the abundance of \u003cem\u003eAlternaria\u003c/em\u003e in soil decreased sharply after \u003cem\u003eMorchella\u003c/em\u003e planting, with \u003cem\u003eAspergillus\u003c/em\u003e, \u003cem\u003eRhizopus\u003c/em\u003e, and \u003cem\u003eFusarium\u003c/em\u003e being the dominant genera in soil samples after one year and two years of planting. By analyzing the correlation between community structure and environmental factors, \u003cem\u003eAlternaria\u003c/em\u003e, \u003cem\u003eFusarium\u003c/em\u003e, and \u003cem\u003ePyrenophora\u003c/em\u003e were found to be positively correlated with pH. The content of catalase, total nitrogen, total potassium, and available potassium was significantly positively correlated with the abundances of \u003cem\u003eMortierella\u003c/em\u003e and \u003cem\u003eRhizopus\u003c/em\u003e. According to previous literature, \u003cem\u003eAlternaria\u003c/em\u003e exhibits a preference for alkaline environments, while increased levels of organic acids lead to a decline in its abundance (Lu et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Furthermore, with the cultivation of \u003cem\u003eMorchella\u003c/em\u003e, a decrease in soil pH and an increase in nutrient levels were observed, which promoted the accumulation of beneficial microorganisms such as \u003cem\u003eChryseobacterium\u003c/em\u003e (Majewska et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and \u003cem\u003eChryseolinea\u003c/em\u003e. (Ortega et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eFurthermore, Venn analysis at the microbial species level revealed that the cultivation of morel mushrooms led to a loss of certain indigenous soil microorganisms. In particular, the loss of indigenous species in the fungal community has been more pronounced. This might be due to the fact that agricultural interference initially temporarily increases the total number of species by introducing new microorganisms, but long-term consecutive cultivation leads to the loss of native endemic species due to the homogenization of the habitat. It is speculated that since morel mushrooms occupy ecological niches by rapidly expanding their mycelial networks and competing for space and nutrients (such as potassium), some native fungi with similar nutritional requirements or slow growth rates have died due to the lack of resources.\u003c/p\u003e \u003cp\u003eResponse of microbial functional pathways to morel mushroom cultivation and soil microenvironment\u003c/p\u003e \u003cp\u003eCompared with Group A, The Two-component system, Lipopolysaccharide biosynthesis, and Methane metabolism pathways in Group B were up-regulated, among which the two-component system was related to signal transduction in the category of environmental information processing. The two-component system plays an important role in maintaining the stability of microbial communities. During composting, penicillin residues may regulate community stability by affecting TCS gene expression, thereby improving community cohesion (Guo et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Alternatively, the upregulation of the Lipopolysaccharide biosynthesis pathway may indirectly affect soil physical properties, such as soil aggregate stability. The stability of soil aggregates is related to soil organic carbon content, microbial activity, and soil structure. Lipopolysaccharide biosynthesis and the synthesis of other polysaccharides may improve soil structure by increasing the stability of soil aggregates (Wang et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e). In addition, upregulation of methane metabolism Therefore, upregulation of methane metabolism not only affects methane oxidation, but may also affect nitrogen cycling in the soil by affecting denitrification, and consequently soil health (Chen et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Compared with Group A, ABC transporters, Glycine, serine and threonine metabolism, and Steroid degradation in Group B were down-regulated, among which ABC transporter down-regulation had positive effects on the stability of soil structure and plant growth. With the development of biological soil crusts, the demand for ABC transporters for microbial osmotic potential-enhancing substances (glycine betaine/proline, osmotic protectants, erythritol, Na\u003csup\u003e+\u003c/sup\u003e, putrescine, 1-phosphatidylinositol) generally decreased (Zhang et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRhizosphere soil has a vital influence on the biogeochemical cycles and metabolic processes of basic elements such as carbon, nitrogen, phosphorus, sulfur, and iron, which is governed by the interactions among soil components, plants, and microorganisms (Liu et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Indeed, the cycling of various elements in soil is essentially driven by complex microbial networks. Hence, this study focuses specifically on the nitrogen cycle as one of its key processes to gain deeper insights into how microbial activities regulate soil functionality. To assess the abundance of the nitrogen metabolic pathway in soil, differential tests and visual analyses of key enzymes were performed. The significance of differences in the abundance of enzymes involved in this pathway was evaluated across the three sample groups. These analyses revealed that enzymes catalyzing prokaryotic denitrification, including nitrous oxide reductase (EC 1.7.2.4), nitric oxide reductase (cytochrome c) (EC 1.7.2.5), and nitrite reductase (NO\u003csup\u003e\u0026ndash;\u003c/sup\u003eforming) (EC 1.7.2.1), were more abundant in soils after one year and two-year of morel mushroom cultivation compared to the control group. It indicates that the soil denitrification capacity has significantly increased, which is conducive to converting active nitrogen (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e/NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e) into inert nitrogen gas (N\u003csub\u003e2\u003c/sub\u003e) for release. They mainly enhance denitrification and nitrogen assimilation and reuse. At the same time, it could increase bacterial abundance and recruit beneficial soil microorganisms.\u003c/p\u003e \u003cp\u003eSoil phenolic acid accumulation driven by morel mushroom cultivation\u003c/p\u003e \u003cp\u003eCombined with our determination of total phenolic acid content in soil and metagenomic analysis, Group C exhibited the highest phenolic acid content. Notably, the level of PAL, a key enzyme involved in phenolic acid synthesis, was significantly greater than that observed in the other two groups. Conversely, Group A displayed the lowest phenolic acid content; however, it had a markedly higher concentration of P34O, an essential enzyme for decomposing phenolic acids compared to the other groups. The two results confirm each other. Since the gene abundance of PAL phenol acid synthase is very low in the metagenome, it is speculated that the main driving force of phenol acid synthesis in soil is not the microorganisms in the soil environment, but the edible fungi themselves. So the investigation focused on the phenol-acid-degrading enzyme P34O. \u003cem\u003eArthrobacter\u003c/em\u003e, \u003cem\u003eGeodermatophilus\u003c/em\u003e, and \u003cem\u003eKocuria\u003c/em\u003e were also significant contributors to P34O enzyme activity. \u003cem\u003eArthrobacter\u003c/em\u003e demonstrates a strong capacity for degrading phenolic acids; indeed, Li Ming isolated an efficient strain capable of degrading these compounds from soil samples collected over many years from the \u003cem\u003eSelenomargua\u003c/em\u003e growing area in Ningxia and identified it as \u003cem\u003eArthrobacter\u003c/em\u003e (Li et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Bacteria belonging to the genus \u003cem\u003eGeodermatophilus\u003c/em\u003e are typically found in extreme environments characterized by high temperatures and arid soils; they may possess unique metabolic pathways and stress resistance mechanisms. However, their specific role in phenolic acid degradation requires further investigation. During \u003cem\u003eKocuria flava\u003c/em\u003e degradation processes, catechol 2,3-dioxygenase (C23O) gene expression levels were 6.02 log-fold higher than those observed for \u003cem\u003eRhodococcus pyridinivoran\u003c/em\u003es. This finding suggests that \u003cem\u003eKocuria flava\u003c/em\u003e may play a crucial role in polycyclic aromatic hydrocarbons (PAHs) degradation (Sakshi et al. 2023).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003e \u003cem\u003eMorchella sextelata\u003c/em\u003e cultivation is a beneficial practice for ameliorating saline-alkali soils, effectively reducing pH, enhancing nutrient content, and stimulating nitrogen metabolism. However, the transition to a cultivated ecosystem induces a complex restructuring of the microbial community. While beneficial for overall soil quality, the concurrent issues of phenolic acid accumulation and potassium depletion pose a risk for soil sickness. Future management strategies should therefore integrate targeted approaches to mitigate phenolic acid accumulation and counteract potassium depletion, thereby ensuring the sustainable cultivation of this valuable fungus.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eThe authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was funded by an enterprise-sponsored project: University Local Cooperation Project: Research and Demonstration of Key Technologies for \u003cem\u003eMorchella\u003c/em\u003e Cultivation in Hongsipu District (222136).\u003c/p\u003e \u003cp\u003eEnterprise-Commissioned Project: Analysis of Physical and Chemical Properties and Microbial Community Structure of Saline-Alkali Soil After \u003cem\u003eMorchella\u003c/em\u003e Cultivation (2025640101000053).\u003c/p\u003e\n\u003ch3\u003eAuthors’ contributions\u003c/h3\u003e\n\u003cp\u003eWu Yifan: investigation, methodology, software, visualization, writing-original draft. Yang Xiaoli: methodology, software, writing review and editing. Zhang Yaya: writing review and editing. Xu Chunyan: All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eArif Y, Singh P, Siddiqui H et al (2020) Salinity induced physiological and biochemical changes in plants: An omic approach towards salt stress tolerance. Plant Physiol Biochem 156(Suppl. 1)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAsina F, Brzonova I, Voeller K et al (2016) Biodegradation of lignin by fungi, bacteria and laccases. Bioresour Technol pp 414\u0026ndash;424\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen KH, Feng J, Bodelier PLE et al (2024) Metabolic coupling between soil aerobic methanotrophs and denitrifiers in rice paddy fields. 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Ecol Evol 15(5):e71289\u0026ndash;e71289\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, Zhao Q, Uroz S et al (2023b) The cultivation regimes of morchella sextelata trigger shifts in the community assemblage and ecological traits of soil bacteria\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFrontiers in Microbiology 14(1):10\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Morchella sextelata, Saline-alkali soil, Soil physicochemical properties, Microbial community, Functional genes","lastPublishedDoi":"10.21203/rs.3.rs-8232173/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8232173/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eAims\u003c/h2\u003e \u003cp\u003e(\u003cem\u003eMorchella\u003c/em\u003e spp.), precious edible and medicinal fungi with important economic and scientific value, have always attracted the attention of scientific researchers at home and abroad, while successful large-scale cultivation in multiple provinces drives a northward expansion of production regions from the south. China\u0026rsquo;s northward expansion of morel mushrooms cultivation faces challenges from saline-alkali soils in arid regions. The purpose of this paper is to clarify the effects of \u003cem\u003eMorchella\u003c/em\u003e cultivation on the saline-alkali soil of Ningxia, Northwest China.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eIn this study, A comparative analysis was performed on the physical and chemical properties of soil, microbial community structure and function, and the expression of enzymes related to nitrogen metabolism in saline-alkali soils under three treatments: (A) an unplanted control, (B) one year of \u003cem\u003eMorchella\u003c/em\u003e cultivation, and (C) two years of \u003cem\u003eMorchella\u003c/em\u003e cultivation.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe results indicated that \u003cem\u003eMorchella\u003c/em\u003e cultivation positively contributed to the amelioration of saline-alkali soil, evidenced by a significant reduction in pH, alongside remarkable enhancements in nutrient contents and enzyme activities. Results based on metagenomic sequencing also revealed that morel mushroom cultivation drives a functional remodeling of the soil microbial community. On the one hand, it enhances the overall metabolic activity of microorganisms by upregulating core metabolic pathways such as ABC transporters, and \u003cem\u003eMorchella\u003c/em\u003e cultivation also positively promotes denitrification, nitrogen assimilation, and reutilization in soil nitrogen metabolism. On the other hand, it disrupts the microbial network responsible for phenolic acid degradation, manifested by an increased gene abundance of the synthesis key enzyme PAL, while the dominant microbial contributors to the degradation core enzyme P34O and its encoding genes \u003cem\u003epcaG\u003c/em\u003e/\u003cem\u003epcaH\u003c/em\u003e shift from the original state (dominated by genera such as \u003cem\u003eArthrobacter\u003c/em\u003e) to a less efficient structure (dominated by \u003cem\u003eMicrovirga\u003c/em\u003e and \u003cem\u003eSphingomonas\u003c/em\u003e), ultimately leading to phenolic acid accumulation and the risk of hindering sustainable continuous cropping.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis study highlights the extent of soil alterations induced by morel mushroom cultivation in saline-alkali environments and its underlying mechanisms.\u003c/p\u003e","manuscriptTitle":"Cultivation of Morchella sextelata in saline-alkali soils of Ningxia: Multi-dimensional impacts on soil physico-chemical properties, enzyme activities, microbial ecology and functional genes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-22 20:41:15","doi":"10.21203/rs.3.rs-8232173/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4691579c-7614-421b-8d7b-8232fc74bbfb","owner":[],"postedDate":"January 22nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-25T21:39:17+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-22 20:41:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8232173","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8232173","identity":"rs-8232173","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}