Cascading effects of soil salinization on agricultural soil biodiversity-multifunctionality relationship

Cascading effects of soil salinization on agricultural soil biodiversity-multifunctionality relationship | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Cascading effects of soil salinization on agricultural soil biodiversity-multifunctionality relationship Shuwen Hu, Tairan Zhou, Yun Zhang, Xu yang, Jiaxin Hu, Lingyu Kong, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5835602/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Soil salinization poses a threat to global food security by deteriorating soil biodiversity and especially various soil functions. However, how soil salinization affects the soil biodiversity-multifunctionality relationship remains largely unknown in agro-ecosystems. Here, we conducted a standardized survey of agro-ecosystems across a typical soil salinization gradient at 115 representative agricultural sites, aiming to investigate the relationships between soil biodiversity (encompassing the diversity of bacteria, fungi, archaea, protists, and metazoans) and nine soil functions as well as multifunctionality. We found that soil multitrophic biodiversity and multifunctionality declined with increasing soil salinization, and the relationships between multiple functions and the degree of salinization exhibited extensive nonlinear changes and threshold effects. We further demonstrated that soil salinization reduced the general strength of the biodiversity-multifunctionality relationship, by significantly altering soil physicochemical properties and the interplay between microbial communities and soil properties. The inconsistent changes in biodiversity–ecosystem functioning (BEF) relationships were attributed to the multi-threshold relationships between different soil functions and multitrophic biodiversity, as well as the differential responses of various biological communities to the multifunctionality of the soil. This study highlights the cascading hazards of soil salinization in agro-ecosystems, emphasizing the importance of integrating salinization prevention and control into sustainable agricultural management strategies to maintain soil biodiversity and ecosystem functioning. Earth and environmental sciences/Ecology/Agroecology Earth and environmental sciences/Ecology/Biodiversity Saline soil Threshold effect Soil health Soil function Agricultural ecosystem Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Soil constitutes a core component of the earth's critical zone, providing numerous vital functions including nutrient cycling, water retention, biodiversity, and habitat 1 , 2 . Maintaining soil health is also pivotal for ensuring human survival and sustainable development, particularly for agricultural soils, which supply humans with the most direct provisions such as food and fiber 3 . In recent years, intensifying land-use conversion and intensification due to factors like population growth and economic development have exacerbated global soil degradation and increased the complexity and uncertainty of global changes 4 , 5 , 6 . This situation poses greater challenges to the sustainable management of agricultural soil, as it necessitates maintaining soil multifunctionality (EMF) while sustaining high productivity 5 , 7 . Consequently, there is an urgent need to enhance our understanding of the functions of agricultural soil and the mechanisms underpinning their maintenance, in order to better secure global food security 8 , 9 . Soil salinization, a major global change factor, refers to the excessive accumulation of soluble salts in soil 10 . Salt-affected soils now cover 20% of the total global cultivated land area and are expanding at a rate of 10% per year 11 . Besides primary salinization caused by rainfall, eolian processes, and physical or chemical weathering of parent rock materials 12 , escalating anthropogenic activities, such as irrigation with saline water and excessive fertilization, have also made salt stress in soils prominent 13 . Soil salinization has become a crucial issue threatening global agricultural productivity 10 and led to multiple levels of biodiversity loss 14 . Early global synthetic analyses identified soil salinity as a major environmental determinant of microbial community composition 15 , whereas subsequent local studies suggested that the influence of soil salinity might not be significant 16 . This controversial understanding may arise from the challenges posed by differences in soil background characteristics and vegetation types in field experiments, which act as barriers to controlling variables and hinder efforts to isolate the effects of soil salinity on microbial communities 13 . Overall, there are fundamental differences in the responses of different soil microbial taxa to salt stress 14 . For instance, some studies have suggested that archaeal communities are more susceptible to changes in soil salinity than bacterial communities 16 , while fungi are generally considered to have stronger abilities to cope with osmotic pressure changes compared to bacteria 14 . However, the response mechanisms of these communities across a broader range of salinity changes remain unclear 11 , and few studies have explored the diversity and functions of archaea, protists, and metazoans, despite their crucial roles in soil microbial food webs and microbially mediated biochemistry in saline soils 14 . Moreover, changes in soil salinity have been proven to significantly impact various soil functions 12 . For example, agricultural productivity and soil organic matter decomposition capacity are significantly inhibited by soil salinity, and the influence of salt stress on soil nitrification has also been shown to be closely related to the degree of soil salinization 13 , 14 . However, most of these studies have focused on single soil functions and are based on simple linear relationships or binary hypotheses. Increasing evidence indicates that ecosystems do not always respond progressively to environmental changes and can sometimes undergo abrupt changes, highlighting the unpredictability of ecological consequences 17 , 18 . The lack of research on the effects of soil salinization makes it difficult to fully understand how soil salinization affects soil multifunctionality and the overall stability of ecosystems 13 , 14 . Therefore, we need to conduct more in-depth and comprehensive research to reveal the true effects of soil salinization and provide a scientific basis for formulating effective soil management and ecological protection strategies. Soil salinization not only affects a variety of soil functions and biodiversity but may also significantly alter the relationships between them, shedding light on the intricate interplay between Earth's biodiversity and the ecosystem services it provides to humanity 19 , 20 . The manner in which global change factors, such as soil salinization, reshape BEF relationships has emerged as a pivotal topic within ecological and environmental sciences, as well as in emerging interdisciplinary fields 19 . Past research has demonstrated that biodiversity loss can diminish the functioning of most ecosystems 21 . Furthermore, soil biodiversity, encompassing a diverse array of taxa such as bacteria, fungi, archaea, protists, and metazoans, has been proven to have a significant driving effect on multiple ecosystem functions 22 . Furthermore, soil multitrophic biodiversity can also effectively sustain multiple ecosystem functions and crop productivity in agriculture 23 . However, global change factors alter diversity and functions in different directions, potentially due to the direct and indirect influences of climate, soil, and numerous other external factors on various aspects of BEF 19 , 24 . For instance, drought can drive shifts in biodiversity-soil multifunctionality relationships 17 . Therefore, exploring the mechanisms by which global change factors influence soil multi-dimensional BEF relationships is one of the key issues in ensuring the sustainable production of resources for humanity in agriculture 23 . Currently, research on BEF is rapidly evolving towards EMF, but it has primarily focused on natural ecosystems such as forests and grasslands 7 , 25 . The changes in BEF relationships in salt-affected agricultural soils remain understudied and poorly understood 5 , 9 , 19 . Here, we specifically investigate the responses of biodiverse soil organisms and key soil functions. We hypothesize that (1) the impacts of soil salinization on diversity and function may be complex, potentially exhibiting distinct nonlinear relationships and threshold effects; and (2) the effect of soil salinization will further alter the soil BEF relationship, a change that may be jointly mediated by other soil factors. This is due to the intensification of resource-dependent competition driven by environmental stress, which impacts the survival of biocommunities associated with ecosystem functions 14 , 26 . Consequently, this leads to a cascade effect of decreased abundance and diversity in soil biotic communities, ultimately resulting in the loss of multiple soil functions 27 . This may represent an unrevealed pivotal mechanism by which soil salinization currently jeopardizes global soil health and food security. To test our hypothesis, we evaluated how the BEF varies along a broad salinity gradient in Northeast China. This region is one of the three major saline soil distribution areas in the world, encompassing an area of approximately 3.73 million hectares, which is roughly 1.3 times the size of Belgium 28 . We focus on soil multifunctionality because of the importance of multiple soil functions in regulating global biogeochemical cycles 9 , 19 . To achieve this, we measured typical soil functions and soil biodiversity, which collectively represent good proxies for nutrient cycling, climate regulation, and soil fertility. A total of 115 field sites were selected along a 100-kilometer salinization gradient, encompassing typical multiple farmland cultivation types and cropping histories (Fig. S1 and Table S1 ). To exclude the influence of other environmental gradients, these sites exhibited similar farmland management practices, water availability, and drought intensity. Results and discussion 1.Cascading effect of salinization on soil biocommunities and soil functions Soil salinization, as indicated by soil electrical conductivity (EC), governs biocommunities across trophic levels, influencing biodiversity, total abundance, species richness, and key community abundances (Figs. S2A-C, S3; Supplementary Text 3), and significantly impacts community biodiversity aspects in random forest models (Fig. S4A-F). We use the enumeration method to obtain a significant causal path and construct a structural equation model (SEM) to reveal the significant and complex cascading effects of different soil biotic communities on salinity changes (Fig. 1 A). The direct negative effect of soil salinization on the fungus community was detected, while the archaea community was found to be positively affected by soil salinization (Fig. 1 B). This is consistent with previous research results, where the fungus community is considered to have a more sensitive response to the increase in soil salinity, and the eukaryotic community shifts towards the prokaryotic community 29 , 30 . Certain groups of archaea have been shown to exhibit higher salinity tolerance, for instance, ammonia oxidizing archaea can dominate nitrification and exhibit higher abundance at intermediate salinity 31 . Previous studies have mainly focused on bacterial and fungal communities, and our results provide important reference for exploring the soil food web relationships including archaea, protists, and metazoans under the context of soil salinization 14 . Our results emphasize the key central role of the fungus community in driving multi-community changes under the context of soil salinization, affecting the changes in soil biological community abundance through multiple direct and indirect pathways (Fig. 1 A). We found that the fungus community has the relatively highest individual contribution percentage to multitrophic biodiversity, soil structure, fertility, plant productivity, and multifunctionality (Fig. 1 C). On the one hand, arbuscular mycorrhizal fungi can regulate the mineral nutrient metabolism and physiological ecological responses of host plants to enhance their salt tolerance 32 . For example, inoculation with arbuscular mycorrhizal fungi can increase the activity of various antioxidant enzymes, which can scavenge reactive oxygen species and alleviate salt stress 14 , 32 . On the other hand, certain fungal groups exhibit stronger adaptability to salt stress, thereby playing a more significant role in maintaining soil functions 29 . Our results also highlight the important role of Metazoans community in soil functions such as carbon sequestration, symbiosis, pathogen control, and ARG control (Fig. 1 C). Metazoans, by feeding on bacteria and fungi, affect symbiotic relationships and can promote the accumulation of microbial residues, thus playing an important role in the stabilization of soil carbon pools 33 , 34 . The activity of Metazoans may influence the selection pressure and transmission pathways of pathogens and ARGs in the soil, thereby controlling related environmental risks to some extent 7 , 35 . However, it is also necessary to emphasize that this multi-community relationship may have regional and climatic environmental limitations due to the high variability of biotic communities 14 . Therefore, future global-scale research across soil background characteristics and vegetation types is still needed to bridge the knowledge gap 15 , 22 . The random forest model indicates that soil salinity significantly affects multiple soil functions and multifunctionality (Fig. S4G-P). Biodiversity across multiple trophic levels is interconnected through food webs, generating internal interactions through predation or resource-dependent competition 36 , 37 . Consequently, they may differ in the manner and sequence of their responses to soil salinity changes 14 , 15 . For instance, the biodiversity indices of bacteria and fungi increase with the increase in soil EC but decrease after crossing a threshold value as EC continues to rise (Fig. 2 A, B). In contrast, the biodiversity of archaea and protists exhibits a nearly linear negative correlation with increasing EC (Fig. 2 C, D). Conversely, the biodiversity of metazoans shows a slight increasing trend with the increase in soil EC (Fig. 2 E). Multitrophic biodiversity indices consider interactions among multiple trophic levels, which can lead to complex responses to soil EC. Initially, increasing soil EC may impose moderate environmental stress, prompting the soil biotic community to release additional ecological niches. However, beyond a certain threshold, high EC can become toxic or stressful for some organisms, leading to a decline in diversity. The linear relationship observed with individual soil diversity indices is likely due to the direct influence of soil EC on specific groups (Fig. S5), while the hump-shaped pattern in multitrophic indices reflects the complex interplay of nutrient availability and environmental stress across multiple trophic levels (Fig. 2 F). Our study also reveals that surpassing specific thresholds of soil EC induces abrupt shifts in individual soil functions and multifunctionality, with the exception of carbon sequestration, mutualism, and pathogen control (Fig. 2 L, M, N) ( P < 0.05). Notably, the responses of various soil functions to salinization exhibit significant divergence. Beyond a certain salinity threshold (2.2), the soil may reach a relatively stable state, slowing further structural degradation and potentially decelerating the decline in water regulation (Fig. 2 G). As soil structure surpasses the salinity threshold (1.95), its trend reverses from decline to increase (Fig. 2 H), possibly due to the accumulation of soil minerals and mineral particle formation associated with increased salinity 38 . In general, elevated soil salinity may displace calcium and magnesium ions with sodium, disrupting the binding between soil particles and preventing the formation of stable aggregate structures 10 , 13 . Additionally, the increase in soluble salts in saline soils weakens the cohesion between particles, leading to the degradation of soil structure, which adversely affects water regulation and soil structure 12 , 39 . The model of organic-mineral interactions further explains the bonding mechanisms between soil minerals and organic matter, accounting for the slight improvement in soil structure post-salinity threshold 39 . The carbon storage function of soil continues to decline with the increase of soil salinity (Fig. 2 L) ( P < 0.05). Soil salinity may diminish carbon storage by affecting microbial carbon pumps and physical protection mechanisms of organic matter 40 . High salinity environments decrease biotic metabolic and extracellular enzyme activities, directly reducing soil fertility and biogeochemical cycling processes, explaining the changes in fertility and organic matter decomposition 41 . This may explain why soil fertility function accelerates its decline after soil salinity crosses the threshold (2.01) (Fig. 2 I). Reduced soil porosity not only directly affects aggregation but also restricts the movement of oxygen and water, which may restrict microorganisms from decomposing organic matter under hypoxic or dehydrated conditions 42 . Therefore, the decomposition of soil organic matter decreases continuously with the increase of soil salinity (Fig. 2 J) ( P < 0.05). Moreover, as salinity crosses the threshold (2.87), the rate of decline in organic matter decomposition slows down, which may be due to the adaptive adjustment of biotic communities to environmental changes 41 , 43 . With increasing soil salinity, declines in plant productivity and pathogen control may create ecological niches, potentially filled by other microbes or organisms, leading to new symbiotic relationships 26 . Under reduced competition for resources due to decreased plant productivity, other organisms may gain more living space and resources, potentially enhancing symbiotic stability through resource-sharing 43 . This explains the increase in mutualism function with rising soil salinity (Fig. 2 M) ( P < 0.05). Moreover, the control function of the pathogen decreased with the increase of salinity (Fig. 2 N) ( P < 0.05). This further confirms that biotic interactions undergo significant changes that may sacrifice the control functions of some pathogens due to the need to adjust their physiological and metabolic mechanisms to accommodate high osmotic pressures 14 , 16 . High salinity environments may indirectly affect the spread and dissemination of antibiotic resistance genes (ARGs) by altering soil biotic community structures and influencing the degradation and transformation of antibiotics 35 . The results indicate that the control of ARGs increases with increasing salinity (Fig. 2 O). This is an interesting phenomenon, and we believe it may be due to a decrease in human activity intensity on high salt soils, which requires further research. Collectively, high salinity environments exert direct and indirect effects on multiple individual soil functions 5 , with the cumulative total effects leading to a reduction in soil multifunctionality and the formation of thresholds (2.32) ( P < 0.05) (Fig. 2 P). We used cluster analysis to better explore the hidden patterns in soil function and thereby construct 5 cluster centers of soil functions through the elbow diagram method (Fig. S6, Supplementary Text 5). A SEM was developed to elucidate the cascading effects of salinization on soil multitrophic biodiversity and multidimensional functions (Fig. 3 A, Supplementary Text 4). Soil multitrophic biodiversity is significantly negatively affected by soil salinization, directly impacting Dimension 3 (Carbon storage, Fertility, Organic matter decomposition) and dimension 5 (ARG control) of soil functions. The decline in soil biodiversity may lead to a reduction in the biological groups involved in key soil functions, thereby constraining critical processes such as soil organic matter decomposition, phosphorus mineralization, and nitrogen transformation 9 , 21 . In contrast, the decrease in biotic diversity could imply the presence of fewer biotic groups, potentially suppressing the spread and enrichment of ARGs 35 . Dimension 3 and dimension 4 (Soil structure) of soil functions are directly and indirectly affected by soil salinization (Fig. 3 B), exhibiting significant direct effects on all other dimensions of soil functions. Moreover, Dimensions 3 and 4 have a higher unique, average share, and individual contribution to soil multifunctionality (Fig. 3 C), with individual contribution ratios as high as 46.01% and 29.96%, respectively (Fig. 3 D). Therefore, we consider Dimensions 3 and 4 of soil functions to be key dimensions that influence soil multifunctionality through cascading effects. The results of this study are consistent with previous hypotheses regarding the impact of soil salinization on soil functions and provide direct evidence that the effects of soil salinization on soil structure are multifaceted, and these impacts can have cascading effects on other soil functions 28 , 39 . This cascading effect is primarily driven by the disruption of soil structure. The accumulation of salts in the soil alters the interactions between soil particles, leading to the collapse of aggregates and their pore network 5 , 38 . The reduced infiltration and distribution of water and oxygen ultimately limit soil biodiversity and activity, thereby weakening their support for soil functions 39 . Ultimately, the collapse of structure affects the multifunctionality of soil, including multiple dimensions such as dimension 1 (Water regulation, Pathogen control) and dimension 2 (Plant productivity, Mutualism) (Fig. 3 A). Therefore, the protection and restoration of soil structure are essential for maintaining soil health and ecosystem services 14 , 28 . 2.Inconsistent changes in BEF relationships along with soil salinization gradient Global observational and experimental microcosm studies collectively provide evidence that soil biodiversity is significantly and positively correlated with multiple ecosystem functions 7 , 37 . Moreover, in agricultural ecosystems, BEF relationships are typically weaker than in natural ecosystems (e.g., grasslands and forests) 19 . Our findings also indicate a pervasive threshold effect in the BEF relationship as EC varies in agricultural ecosystems (Fig. 4 A, B, C, D, G, I, J) ( P < 0.05). Prior to the threshold (3.55), the strength of the biodiversity-ecosystem functioning (BEF) relationship diminishes gradually with increasing soil salinization, eventually transitioning from a positive to a negative correlation (Fig. 4 J) ( P < 0.05). This might be due to N deposition or fertilization, which increase functions but decrease soil multitrophic diversity 44 , 45 . This also suggests that during the initial stages of salinization, competitive interactions among soil organisms intensify due to increased soil salinity 26 , 27 , and the significant reduction in mutualism before the threshold supports this view (Fig. 4 G). In other words, non-productive species may gradually gain a competitive advantage at this stage, thereby reducing the overall resource turnover efficiency of the biological community 17 , 18 . As salinization continues to increase, the BEF relationship becomes dominated by a negative correlation. The Stress Gradient Hypothesis (SGH) may explain this shift, as the decline in abundance of dominant species releases more niches 11 , 14 . Consequently, the competitive pressure on non-dominant species may decrease, leading to potential increases in biodiversity within certain biological communities 19 , 26 . Additionally, positive interactions among communities decrease at this time, ultimately affecting the strength and direction of BEF 27 . Post-threshold, as soil salinization increases, the negative BEF correlation gradually weakens. The decoupled relationship here is largely due to the change in both diversity and functions or because niche differentiation caused by salinization reduces competition among species 11 . In general, we observe threshold effects in the BEF responses of various functions, with soil EMF exhibiting the highest EC threshold (Fig. 4 J). These results suggest that changes in the BEF relationship of soil EMF may exhibit a certain lag, where the supportive role of biodiversity for some functions may be suppressed, but its relationships with other functions may be enhanced to compensate for this loss 17 , 27 . Our findings indicate that soil structure, organic matter decomposition, and mutualism functions all exhibit similar response patterns in the BEF relationship of soil EMF, with completely different change patterns before and after the threshold (Fig. 4 C, D, G). However, the BEF relationship changes for fertility, and ARG control functions increase with increasing soil EC (Fig. 4 E, I). This suggests that under soil salinization stress, biological communities related to generating the above two functions may gradually dominate 9 , 14 . The BEF relationships for other functions generally decrease with increasing soil EC, indicating that the contribution of soil biodiversity to these functions may be less significant 19 . We acknowledge that different functional selections may ultimately affect the evaluation results of the BEF relationship. Therefore, we recommend incorporating more ecosystem functions into the multifunctionality framework to achieve more robust outcomes. Considering a range of independent multifunctionality indexes with multiple thresholds (multi-threshold multifunctionality, including > 10%, > 25%, > 50%, > 75%, and > 90% thresholds; Fig. 4 K) ( P < 0.05), similar conclusions were also found 46 . Specifically, the most significant decline was observed at thresholds of 10% and 25%, indicating that ecosystems with lower levels of function are more susceptible to the effects of soil salinization. This multi-threshold approach, which accounts for correlations between functions, effectively reveals BEF changes at different functional levels while limiting the number of functions considered 17 , 22 . At higher threshold levels (90%), BEF tends to remain unchanged with increasing salinization. This suggests that selective pressures intensify under these conditions 46 . The biological community's survival strategies shift more actively to adapt to saline environments, thereby offsetting changes in the BEF relationship 14 , 16 . This attenuated BEF relationships is evident across various soil functions and multiple community factors (Fig. S7 A, B). This is primarily attributed to the monoculture practices which diminish the interactions and interdependencies among species within the system 20 , 27 . Additionally, the frequent application of fertilizers, irrigation, weed control, and pest management in agricultural ecosystems may directly influence their functionality 9 , 43 . To further elucidate the mechanisms underlying the changes in soil biodiversity and BEF relationships caused by soil salinization, we analyzed the variations in BEF relationships associated with key soil biota groups (Fig. 4 L - P). At the mean level, we found that BEF relationships for all biological groups, except for fungi and protists, significantly decline with increasing soil salinization. This suggests that the impact of soil salinization on function-supporting biological communities may be a comprehensive effect across food webs 24 , 37 . Bacteria and fungi primarily act as decomposers in ecosystems, and due to the diversity of their physiological characteristics and metabolic pathways, they can often survive and function in adverse environments such as salinization 14 . Thus, they are more important in BEF relationships at high salinization levels (i.e., lower functional levels) (Fig. 4 L, M). Moreover, the BEF relationship for fungi tends to increase with soil salinization, particularly at high threshold levels (T90), implying that symbiotic relationships among species may be promoted (Fig. 4 M). This could partially offset the negative impacts of soil salinization and further emphasize the more critical role of fungal communities in maintaining ecosystem function in saline soils 29 , 30 . Additionally, protists exhibit different patterns in BEF relationships across threshold levels (Fig. 4 O), reflecting the complexity and variability of biodiversity and ecological functions under different environmental conditions 7 , 18 . Interestingly, the BEF relationships for bacteria and archaea exhibit a higher rate of decline at low threshold levels (T10) with increasing soil salinization, while metazoans show the opposite pattern (T90) (Fig. 4 L, N, P) ( P < 0.05). This indicates that the diversity of metazoans is more sensitive to soil salinization in ecosystems supporting high-level functions 7 , 18 . Metazoans, typically multicellular organisms with complex body structures and physiological functions, can prey on other organisms or utilize more complex organic materials as food sources 37 , 43 . Therefore, metazoans generally possess more complex physiological systems and higher energy demands, making them more important in BEF relationships at higher functional levels 33 . In order to reveal the reasons why the BEF relationship changes with the degree of soil salinization, we conducted further analysis on the soil biotic community and its relationship with soil factors. We have unveiled a reduction in the significant association pathways between biological communities and environmental factors in salinized soils compared to non-salinized soils, a pattern that is universally present and consistent across multiple communities (Fig. 5 A, C). The BEF research has delineated a comprehensive array of mechanisms, including but not exhaustive of the expansion of biotope space indicating an augmented diversity of ecological niches, improved resource utilization efficiency, multitrophic interactions, and facilitation processes, which collectively facilitate the enhancement of ecosystem functioning in response to increased biodiversity 19 . Interestingly, we observed that in salinized soils, the co-occurrence networks of biological communities are more complex and tightly connected (Fig. 5 B, D). Upon soil salinization, there is a noticeable increase in the number of nodes and the quantity of positive correlations within the total network and each sub-community network (Table S2). This indicates that positive interactions among biological communities are enhanced in salinized soils, reflecting the adaptive strategies of these communities to the adversity of saline soil conditions 14 , 31 . This indicates that as soil salinization increases, the decoupling of the BEF relationship may be attributed to changes in soil food web complexity and interactions among different trophic levels 20 .We also believe these changes are due to the increased limitation of biologically available resources caused by salinization, which in turn stimulates the intensification of various biological interactions 29 , 47 . The SGH helps to explain how soil biological communities cope with the stress of soil salinization by enhancing positive interactions and complementary effects 48 . On the other hand, the associated soil environmental changes also serve as a powerful filtering factor for existing soil biological species 11 , 30 . We found that in salinized soils, the bacteria-metazoans sub-networks exhibit higher network complexity and connectivity, whereas in non-salinized soils, the bacteria-fungus communities play a more significant role (Table S2). This suggests that in salinized soils, top-down effects may be somewhat enhanced, meaning that species at higher trophic levels may play a more important role in controlling species and BEF relationships 49 . Overall, our understanding of BEF relationships across biological communities remains limited 19 . Our results also provide a reference for future studies on the BEF relationships of specific biological communities with ecosystem functions and for revealing the complex internal connections among species 19 , 20 . The random forest model demonstrates that soil EC is a key environmental factor affecting multi-trophic BEF relationships, encompassing biodiversity indices across multiple biological groups and soil functions (Fig. S8). Among the various environmental variables assessed, EC significantly impacts BEF relationships, and we also found that the impact of soil EC on the BEF relationship is significantly regulated by other soil factors (Fig. S9). The increase in soil pH, bulk density, clay content, and catalase enzyme activity may exacerbate the loss of BEF relationships induced by soil salinization, whereas other soil factors exhibit opposite effects. This is because the augmentation of factors such as soil pH, among others, indicates stress on plants and soil organisms from various perspectives, thereby hindering the ability of soil organisms to maintain soil functions 46 . For instance, an increase in pH may lead to reduced availability of essential nutrients such as phosphorus, iron, manganese, and zinc. An elevation in bulk density and clay content typically signifies increased soil compaction, which reduces soil aeration and permeability 13 , 47 . Heightened catalase activity may indicate the accumulation of hydrogen peroxide in the soil, potentially damaging the cellular structures of soil organisms and impairing their normal functions 32 . These results validate the notion that direct interactions among various soil physicochemical factors can influence soil functions, and that the degree of salinization has the potential to interact with other soil factors as a concentration amplifier 6 . We further elucidate the interactive effects of soil EC and other critical environmental factors on BEF relationships of EMF using generalized linear models (Table S3). Soil EC exhibits a significantly negative effect in all models except for alkaline available phosphorus and microbial biomass phosphorus, with its estimates substantially stronger than other soil factors, indicating that soil salinization is a primary driver of BEF relationships ( P < 0.05). Moreover, soil salinization significantly interacts with Total nitrogen, Alkaline hydrolyzed nitrogen, Clay, and S gene abundance to affect soil BEF relationships negatively (Table S3). Soil salinization can impair soil organisms' ability to transform and utilize nitrogen, leading to reduced availability of total nitrogen and alkaline hydrolyzed nitrogen 40 . Thus, we posit that nitrogen management in saline soils should be a focal point. On the other hand, in saline soils, clay particles adsorb significant amounts of salts, leading to reduced soil permeability and affecting water and nutrient cycling 39 . The interaction between salinization and clay may disrupt soil biotic communities and impact S gene expression, consequently affecting soil biodiversity and functions 38 , 39 . The imbalance in biotic communities caused by salinization can disrupt key ecosystem processes, thereby exerting a greater influence on BEF relationships (Fig. 6 ). These impacts collectively lead to reduced stability and productivity of soil ecosystems, adversely affecting agricultural production and the ecological environment 12 , 13 . Therefore, effective measures are needed to prevent soil salinization and protect health and sustainable development of soil ecosystems. Our results have significant implications for better promoting soil health restoration and sustainable agricultural productivity development in the context of global soil salinization. Conclusion Our findings integrated threshold analysis to separately reveal the patterns of changes in soil multitrophic biodiversity and multiple-level soil functions as soil salinity increases. Furthermore, we elucidated the changes and decoupling patterns of soil biodiversity-ecosystem function relationships as soil salinization progresses. Moreover, we have unveiled the cascading effects among biotic communities and the interactive effect of soil environmental factors, which can amplify the impacts of soil salinization. These conclusions underscore the urgency and importance of soil salinization management; without robust measures, soil salinization may lead to greater losses in soil functions and agricultural productivity than previously anticipated. In summary, this study comprehensively elucidates the mechanisms underlying the changes in soil BEF relationships in the context of soil salinization, contributing innovative interdisciplinary insights to safeguarding global food security. We call for future research to connect our findings with ecosystem service assessments and socio-economic data, to comprehensively advance the sustainable remediation of soil ecosystems in the Anthropocene era, marked by rapid environmental changes and the reduction of natural resources. Overall, we have demonstrated that the impact of soil salinization results in biodiversity changes at one trophic level cascading to other trophic levels, potentially leading to greater losses in soil EMF than previously anticipated. Methods Site information and sampling protocol Our study area is located in the Songnen Plain of northeastern China, a major region for salt-affected soil distribution globally 5 , 12 . Soil salinization in this area is primarily due to the low-lying terrain, with an annual rainfall of 423.57 mm and high evaporation rates exceeding 1200 mm, leading to the formation of extensive saline soils. The salinity composition of the soils is predominantly sodium carbonate (Na 2 CO 3 ) and sodium bicarbonate (NaHCO 3 ), with Na + ions accounting for more than 70% of the total soil cation content. The region's soils are mainly clay-based and fall under a temperate monsoon climate, with an average annual temperature of approximately 6.10°C and significant diurnal temperature variation. Between 2023 and 2024, soil sampling was conducted across 115 typical agricultural sites within an area of over 10,000 square kilometers, representing a typical salinization gradient with soil EC ranging from 0.37 dS/m to 6.03 dS/m. These agricultural fields were historically cultivated at different time intervals (Table S1 ) and were previously salt-affected grasslands with low vegetation coverage. The dominant plant species in these grasslands were Leymus chinensis and Suaeda glauca . The agricultural fields are managed by local farming corporations under similar cultivation practices regulated by the government. Specific agricultural management practices for paddy fields (rice) and upland fields (corn), including fertilization, tillage, and planting density, are described in the reference 5 , 41 . Soil sampling was carried out following standard procedures, with a 50m×50m plot selected at each site 22 . Ten soil samples were collected using an 'S' pattern and combined into a single composite sample. Additionally, an undisturbed soil sample was collected from each site to analyze soil structure and aggregate composition. Furthermore, average yield records for the past five years for each site were obtained from local farming corporations. After the field investigation, the composite soil samples were sieved (< 2 mm) and divided into two subsamples. One soil subsample was air-dried to analyze its physical and chemical properties, while the other subsample was immediately frozen at -20°C for molecular biological analysis. Methods for soil physical and biochemical analysis are detailed in the Supplementary Text 1. Methods of soil molecular analysis Total DNA is extracted from soil samples using the E.Z.N.A.® Soil DNA Kit (OmegaBio-Tek, USA) following the manufacturer's guidelines. The diversity of soil bacteria, fungi, archaea, protists, and metazoans is assessed via amplicon sequencing on the Illumina MiSeq platform 22 . Bacterial identification employs 16S V4 region primers (515F and 806R), fungal identification uses ITS1-5F/ITS2 region primers, archaeal 16S rRNA gene V3-V5 regions are amplified with primers Arch344F and Arch915R, and eukaryotic organisms are identified using 18S V4 region primers (528F and 706R) 17 , 22 , 36 . Operational taxonomic units (OTUs) are defined at 100% sequence identity 17 , 36 , 37 . Taxonomic classification is conducted using RDPclassifier (version 2.2) against the Silva16S rRNA database (v138) with an 80% similarity threshold 50 . Community composition at taxonomic levels including domain, phylum, class, order, family, genus, and species is analyzed using the uclust algorithm. Metagenomic shotgun sequencing libraries were constructed and sequenced at Shanghai Biozeron Biological Technology Co. Ltd. In briefly, for each sample, TruSeq DNA Library Preparation kit (catalog no: FC-121-2001, Illumina, USA) were used to construct sequencing libraries and the concentration of all libraries were measured by High Sensitivity Double Stranded DNA kit on a Qubit Fluorometer (Thermo Fisher Scientific) 51 . All samples were sequenced in the NGS instrument with pair-end 150bp (PE150) mode. Following sequencing, detailed steps of a bioinformatics pipeline for processing and analyzing metagenomic sequencing data, encompassing quality control, read assembly, contig filtering, gene prediction, clustering, and functional analysis, are elaborated in the Supplementary Text 2. KEGG ortholog annotation was conducted utilizing the KofamScan with the HMMSEARCH package 52 . A total of 239 carbon metabolism-related functional genes were identified, encompassing processes such as methane metabolism, carbon fixation, and aerobic respiration 53 . Additionally, 33 nitrogen cycle-related functional genes were detected, including denitrification, nitrite reduction, and ammonia oxidation 54 . The study also identified 41 phosphorus cycle-related functional genes, which are involved in organic P mineralization, P transportation, and P regulation 55 . Furthermore, 73 sulfur cycle-related functional genes were detected, including the synthesis of sulfite reductase, sulfur transferase, and sulfur relay protein 56 . Soil biodiversity index calculation The Shannon diversity index of soil bacteria, fungi, archaea, protists, and metazoans is ascertained from rarefied ASV abundance datasets. The top 10 most abundant soil organisms from bacteria, fungus, archaea, protist, and metazoans communities were selected to compare their relative abundance responses to soil salinization Supplementary Text 3. To derive a quantitative metric of soil biodiversity per sample, the biodiversity attributes of our soil organism groups are integrated by averaging the biodiversity index across all groups 17 , 21 . This composite index is termed the multitrophic diversity index, reflecting the comprehensive biodiversity across trophic levels 37 . This methodology is frequently applied to calculate multiple biodiversity indices for both soil and plant communities 19 , 36 . Co-occurrence network traits of soil organism Biotic communities were categorized into salinized and non-salinized based on the current definition of soil EC (> 2 dS/m) 11, 26 to construct co-occurrence networks. Subsequently, co-occurrence networks for these communities, including sub-networks between them and within individual community, were established to assess the impact of soil salinization on the overall structure and potential biological interactions of dominant soil organisms. These networks were constructed based on Spearman's correlation algorithm of different ASV proportions. Specifically, the "WGCNA" and "igraph" R packages were used to construct and analyze the networks. Nodes in the network represent taxonomic ASVs of soil organisms, and edges correspond to significant connections between nodes 46 . A series of topological parameters were calculated to describe the biological connections in the network, including the number of nodes and edges, positive and negative correlation numbers, average degree, graph density, degree centralization, and modularity 20 . Definition of individual soil functions and assessing soil multifunctionality In each plot, multiple soil indicators reflecting ecosystem functions, processes, or attributes were used to calculate nine typical soil functions, which are regulated by soil biology and considered to comprehensively reflect the functionality of agricultural ecosystems 21 , 22 , 46 : water regulation (water retention capacity, calculated from soil porosity), soil structure (including bulk density, water-stable aggregate content), carbon storage (calculated from soil organic carbon content and bulk density), fertility (including available potassium, calcium, sodium, and magnesium ion content), organic matter decomposition (including the activity of nine enzymes associated with soil redox processes, carbon-nitrogen-phosphorus metabolism, and key substrate decomposition), plant productivity (based on the average yield per hectare over the past five years, from 2018 to 2023), mutualism (the proportion of ectomycorrhizal fungi and arbuscular mycorrhizal fungi), pathogen control (the relative abundance reduction of plant pathogens in the soil), and ARG control (the reduction of ARG abundance in the soil). We first calculated the standardized index for each function according to the methods in the literature to represent each function, ensuring that data with different characteristics are on the same scale. To obtain quantitative indicators of multiple ecosystem functions, multifunctionality was calculated using two of the most common methods: "averaging" and "multiple thresholds" 22 , 37 . The averaging method assesses by calculating the mean of all standardized (0–1 normalized) ecosystem function indicators. The threshold-based method evaluates the total number of functions that exceed or are equal to a predefined percentage of the maximum observed value for each individual function. The specific calculation methods have been widely described in the literature. We used a set of thresholds: 10%, 25%, 50%, 75%, and 90% to represent the multifunctionality at low, medium, and high thresholds of the observed maximum functionality 46 . Statistical analyses The local soil BEF relationships were analyzed from multiple perspectives: the correlation between individual soil functions and soil EC, the relationship between biodiversity and soil functions, biodiversity and multifunctionality using both averaging and multiple threshold approaches, and the biodiversity of different organism groups (including bacteria, fungi, archaea, protists, and metazoans) versus individual ecosystem functions. Spearman's correlation coefficients were calculated to represent local soil BEF relationships, with stronger positive correlations indicating a higher capacity of soil biodiversity to enhance ecosystem functions 20 , 46 . A moving window approach was applied to explore the determinants of boundary function relationships, aiding in the analysis of multidimensional data along ecological gradients 17 . To ensure robust data analysis, a sliding window averaging algorithm was applied, wherein two window sizes consisting of 60 consecutive samples were selected at each site, yielding 56 data points (e.g., 1–60, 2–61...56–115) 20 . After reordering along the soil salinity gradient, the window was advanced across sampling points, creating adjacent subsets to better estimate the complexity of BEF relationships. Acknowledging that data points from the moving window are not independent and may exhibit correlations with the window, a bootstrapping procedure was conducted, involving random sampling of 55 data points without replacement from the original dataset 1000 times 17 . Bootstrapping helps address these dependencies, aiding in the estimation of confidence intervals for these correlation coefficients and providing more information about the strength of relationships and more accurate statistical estimates 17 , 20 . Segmented regression analysis was performed using the "segmented" package, initially setting the threshold at the mean EC value. A grid search function was defined to systematically evaluate different threshold values by calculating the Akaike Information Criterion (AIC) for each potential threshold 17 . The grid search was conducted across a range of EC values, and the threshold with the minimum AIC was identified as the optimal threshold. The data were split into two segments based on the identified threshold, and linear regression models were fitted to each segment to assess changes in slope before and after the threshold 18 . The Mann–Whitney U-test was implicitly applied through the comparison of slopes and predicted values before and after each threshold to validate the statistical significance of the identified threshold. Linear models before and after the threshold were fitted using the lm function, and their summaries were examined. If no threshold was detected within the selected EC range, linear regression analysis was conducted on the entire dataset. Additionally, Generalized Additive Models (GAMs) were fitted using the mgcv package, modeling each soil function as a smooth function of EC 17 . The "ggplot2" package was utilized to create visualizations, including boxplots and fitted lines from the GAM model, segmented regression, and linear models. Additionally, correlation analyses were performed using the "corrplo" R package. The obtained P values were adjusted for multiple comparisons using the "fdr" method to control the likelihood of false positives. A Random Forest analysis was conducted using the "randomForest" package to identify key soil environmental factors affecting individual soil functions, multifunctionality, and their BEF relationships 18 , 22 . The "lavaan" package in R was utilized for SEM. The logic behind model construction and references are described in Fig S10, S11 and Supplementary Text 4. This package facilitates the specification of latent variables and estimation of path coefficients, aiding in the comprehension of direct and indirect effects within our model. The k-means function in R was used for clustering analysis, the specific details of cluster analysis are detailed in Supplementary Text 5. Mantel analysis, a statistical method for testing correlations between two distance matrices, was employed to assess if there is a significant relationship between dissimilarities in species composition and environmental variables 5 . The "vegan" package was used to execute Mantel tests, with the mantel function calculating the Mantel statistic and evaluating its significance through permutation tests. The generalized linear model is constructed using the "stats" package in R, and the specific details are explained in detail in the Supplementary Text 6. 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J Soil Sediment 17 , 70-84 (2017). Mondaca P , et al. Effects of sustainable agricultural practices on soil microbial diversity, composition, and functions. Agric Ecosyst Environ 370 , 109053 (2024). Chen X, Chen HYH, Searle EB, Chen C, Reich PB. Negative to positive shifts in diversity effects on soil nitrogen over time. Nat Sustain 4 , 225-232 (2021). Zhang Ta, Chen HYH, Ruan H. Global negative effects of nitrogen deposition on soil microbes. ISME J 12 , 1817-1825 (2018). Feng J, Liu Y-R, Eldridge D, Huang Q, Tan W, Delgado-Baquerizo M. Geologically younger ecosystems are more dependent on soil biodiversity for supporting function. Nat Commun 15 , 4141 (2024). Rath KM, Fierer N, Murphy DV, Rousk J. Linking bacterial community composition to soil salinity along environmental gradients. ISME J 13 , 836-846 (2019). Gao C , et al. Co-occurrence networks reveal more complexity than community composition in resistance and resilience of microbial communities. Nat Commun 13 , 3867 (2022). Conti E, Di Mauro LS, Pluchino A, Mulder C. Testing for top-down cascading effects in a biomass-driven ecological network of soil invertebrates. Ecol Evol 10 , 7062-7072 (2020). Wang Q, Garrity GM, Tiedje JM, Cole JR. Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Applied and Environmental Microbiology 73 , 5261-5267 (2007). Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30 , 2114-2120 (2014). Lombard V, Golaconda Ramulu H, Drula E, Coutinho PM, Henrissat B. The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res 42 , D490-495 (2014). Llorens-Marès T , et al. Connecting biodiversity and potential functional role in modern euxinic environments by microbial metagenomics. ISME J 9 , 1648-1661 (2015). Mosley OE, Gios E, Close M, Weaver L, Daughney C, Handley KM. Nitrogen cycling and microbial cooperation in the terrestrial subsurface. ISME J 16 , 2561-2573 (2022). Liang J-L , et al. Novel phosphate-solubilizing bacteria enhance soil phosphorus cycling following ecological restoration of land degraded by mining. ISME J 14 , 1600-1613 (2020). Anantharaman K , et al. Expanded diversity of microbial groups that shape the dissimilatory sulfur cycle. ISME J 12 , 1715-1728 (2018). Additional Declarations There is NO Competing Interest. Supplementary Files P6SupplementaryInformation.docx Cascading effects of soil salinization on agricultural soil biodiversity-multifunctionality relationship Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5835602","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":412107567,"identity":"3fafe2fe-5000-4126-94a3-57d05d9b4387","order_by":0,"name":"Shuwen Hu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzElEQVRIiWNgGAWjYDACCTB5gIEfwmUmQYtkA8laDA4Qq0V+do+ZxMcddxI3Hz/8TIKhwjqxgf3sAbxaDO6cMZOceeZZ4rYzaWYSDGfSExt48hLwa5HIMbvN23Y4cdsNBjMJRiCjQYLHAL/DZgC1/AWq3DyD/ZsE4z8itDDcAGoBGb5BggdoSwMRWgxupJX/7G07bDzjTE6xRcKxdOM2nhxCDkvebPCz7bBsf/vxjTc+1FjL9rOfIeAwFJAAxGwkqB8Fo2AUjIJRgAMAAJoxSHDIQrXaAAAAAElFTkSuQmCC","orcid":"","institution":"College of Resources and Environment Sciences, China Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Shuwen","middleName":"","lastName":"Hu","suffix":""},{"id":412107568,"identity":"1f4957f9-dc14-409b-a7f2-c13bdeadd389","order_by":1,"name":"Tairan Zhou","email":"","orcid":"","institution":"College of Resources and Environment Sciences, China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Tairan","middleName":"","lastName":"Zhou","suffix":""},{"id":412107569,"identity":"f286aa8b-438a-4cb5-8e76-49fc4aea051c","order_by":2,"name":"Yun Zhang","email":"","orcid":"","institution":"College of Resources and Environment Sciences, China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yun","middleName":"","lastName":"Zhang","suffix":""},{"id":412107570,"identity":"180398a1-788f-42eb-8cf5-bf2f54a299db","order_by":3,"name":"Xu yang","email":"","orcid":"","institution":"College of Resources and Environment Sciences, China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xu","middleName":"","lastName":"yang","suffix":""},{"id":412107571,"identity":"5d3c0975-3410-4198-810c-41844540bf33","order_by":4,"name":"Jiaxin Hu","email":"","orcid":"","institution":"College of Resources and Environment Sciences, China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jiaxin","middleName":"","lastName":"Hu","suffix":""},{"id":412107572,"identity":"ef6570b5-0aa2-4401-a82a-8c0c9dd36e73","order_by":5,"name":"Lingyu Kong","email":"","orcid":"","institution":"College of Resources and Environment Sciences, China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Lingyu","middleName":"","lastName":"Kong","suffix":""},{"id":412107573,"identity":"aaf8dfe6-b222-44ab-b72d-c2fc8ce4d6a4","order_by":6,"name":"Qilin Lv","email":"","orcid":"","institution":"College of Resources and Environment Sciences, China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Qilin","middleName":"","lastName":"Lv","suffix":""},{"id":412107574,"identity":"b04e2d9a-ae5c-4f68-b6a7-a044ced14a1d","order_by":7,"name":"Jie Wang","email":"","orcid":"https://orcid.org/0000-0001-5657-8109","institution":"China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Wang","suffix":""},{"id":412107575,"identity":"b166914c-09df-4d85-88e8-ccd6da2baa5e","order_by":8,"name":"Xueqin Ren","email":"","orcid":"","institution":"College of Resources and Environment Sciences, China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xueqin","middleName":"","lastName":"Ren","suffix":""},{"id":412107576,"identity":"c75e31c5-1e68-4c34-8845-1a93382b671d","order_by":9,"name":"Chen Ning","email":"","orcid":"https://orcid.org/0000-0002-1779-915X","institution":"Lanzhou University","correspondingAuthor":false,"prefix":"","firstName":"Chen","middleName":"","lastName":"Ning","suffix":""}],"badges":[],"createdAt":"2025-01-15 15:16:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5835602/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5835602/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":75694722,"identity":"4e45dbfb-43c0-45e5-95eb-78a4b14baebf","added_by":"auto","created_at":"2025-02-07 07:58:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":173300,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe response of various composition and biodiversity of soil biological communities to soil salinization and their contribution to soil function.\u003c/strong\u003e (A) A structural equation model (SEM) is fitted to dissect the influences of soil salinization on various biological communities. The blue and red arrows indicate negative and positive effects, respectively. (B) The total effects, encompassing both direct and indirect influences, were extracted from the SEM depicted previously. (C) A mixed-effects model was established to assess the individual contribution of different biological communities on the variance of multitrophic biodiversity, soil function, and soil multifunctionality.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5835602/v1/1718e1e29918940b28f5cbb3.png"},{"id":75694701,"identity":"060f28c8-e846-4470-b2ec-fd1eb7dba8ab","added_by":"auto","created_at":"2025-02-07 07:58:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":363468,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLinear and nonlinear responses of biodiversity index, multifunctionality and individual soil functions to increasing salinization, and their respective salinization thresholds\u003c/strong\u003e. Box plots demonstrate the distribution of bootstrapped standardized coefficients corresponding to those in for each subset window (N = 1000 independent simulations), showing the median (centerline), 25th and 75th percentiles of each distribution. The orange solid lines indicate the nonlinear trends fitted by generalized additive models (GAMs). The red dashed lines represent the detected salinization thresholds, while the green and blue solid lines before and after these thresholds denote the linear regression relationships on either side of each threshold. For those indicators where no threshold is detected, the purple solid line signifies the overall linear regression relationship. The significance levels are denoted as *** for \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001, ** for \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, and * for \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, respectively, and the same convention applies hereinafter.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5835602/v1/aafa1ed51fb73e5aac71f85e.png"},{"id":75694725,"identity":"a0b28e89-1026-44f2-9393-cdd4b95330cd","added_by":"auto","created_at":"2025-02-07 07:58:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":274298,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe response of soil biodiversity and function to soil salinization and their contribution to soil multifunctionality. \u003c/strong\u003eThe interconnections between soil multitrophic biodiversity and soil function across various dimensions were delineated utilizing a fitted SEM, where blue arrows signify negative effects and red arrows denote positive effects (A). The total effects, encompassing both direct and indirect impacts, were extrapolated from the aforementioned SEM (B). The mixed-effects model was constructed to evaluate the influence of soil salinization on soil multitrophic biodiversity and soil function across different dimensions, with the inclusive of unique, average share, and individual contributions of various effects to the variance of soil multifunctionality (C), and the individual contribution percentages to soil multifunctionality (D).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5835602/v1/7905f97f0b514aac3024a707.png"},{"id":75694944,"identity":"9b01e7b3-d0f0-4426-8980-c9f99a045e28","added_by":"auto","created_at":"2025-02-07 08:06:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":372345,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLinear and nonlinear responses of BEF relationships to increasing salinization, and their respective salinization thresholds\u003c/strong\u003e. The BEF relationships between soil biodiversity and function were represented by average and multi-threshold method respectively (A-J). The Averaged, T10, T25, T50, T75 and T90, represents BEF relationship between diversity and multifunctionality quantified using averaged method and at threshold of 10%, 25%, 75% and 90% (K-P).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5835602/v1/c8286cab0f4175f60ba9dc7c.png"},{"id":75694706,"identity":"d46cc33d-80b0-42b8-98c9-f0fe8943fa3b","added_by":"auto","created_at":"2025-02-07 07:58:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":655542,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eResponse of environmental factors and intercommunity relationship to soil salinization in different soil biomes\u003c/strong\u003e. The relationship between key biological communities and various soil properties in non-saline (EC≤2 dS/M) (A) and saline (EC\u0026gt;2 dS/M) (C) soils was detected by the Mantel test. Edge width corresponds to Mantel's r value, and edge color denotes statistical significance. Shaded areas show 95% confidence intervals of the fit in the line regression analysis. Biotic co-occurrence networks in non-saline (B) and saline (D) soils.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5835602/v1/1fb54a6cf5ed7cf1a1dce3cd.png"},{"id":75694946,"identity":"e5366fa0-e199-45c7-8384-0bb0c254e017","added_by":"auto","created_at":"2025-02-07 08:06:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":325898,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA conceptual model illustrating soil salinization changes soil diversity and multifunction through a cascade effect.\u003c/strong\u003e The lines of different colors represent four typical patterns of soil function as soil salinity increases: continuous decrease, increase followed by decrease, continuous increase, and decrease followed by increase. As soil salinity increases, the associations within the soil biocenosis intensify, yet their supporting role in soil function and overall biodiversity decrease significantly.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5835602/v1/0df762f3a619875df5663e73.png"},{"id":83936579,"identity":"99881a7d-32ae-4bc1-b6e2-f45899df6b3d","added_by":"auto","created_at":"2025-06-04 16:40:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2938805,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5835602/v1/14e450ea-fad4-466b-8dc5-08b064356ead.pdf"},{"id":75695681,"identity":"20d72d48-4eed-48df-9ec1-acfc032f5812","added_by":"auto","created_at":"2025-02-07 08:14:49","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":33679787,"visible":true,"origin":"","legend":"Cascading effects of soil salinization on agricultural soil biodiversity-multifunctionality relationship","description":"","filename":"P6SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-5835602/v1/c9858edc91a5775345fad1d5.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Cascading effects of soil salinization on agricultural soil biodiversity-multifunctionality relationship","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSoil constitutes a core component of the earth's critical zone, providing numerous vital functions including nutrient cycling, water retention, biodiversity, and habitat\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Maintaining soil health is also pivotal for ensuring human survival and sustainable development, particularly for agricultural soils, which supply humans with the most direct provisions such as food and fiber\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. In recent years, intensifying land-use conversion and intensification due to factors like population growth and economic development have exacerbated global soil degradation and increased the complexity and uncertainty of global changes\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. This situation poses greater challenges to the sustainable management of agricultural soil, as it necessitates maintaining soil multifunctionality (EMF) while sustaining high productivity\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Consequently, there is an urgent need to enhance our understanding of the functions of agricultural soil and the mechanisms underpinning their maintenance, in order to better secure global food security\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSoil salinization, a major global change factor, refers to the excessive accumulation of soluble salts in soil\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Salt-affected soils now cover 20% of the total global cultivated land area and are expanding at a rate of 10% per year\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Besides primary salinization caused by rainfall, eolian processes, and physical or chemical weathering of parent rock materials\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, escalating anthropogenic activities, such as irrigation with saline water and excessive fertilization, have also made salt stress in soils prominent\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Soil salinization has become a crucial issue threatening global agricultural productivity\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e and led to multiple levels of biodiversity loss\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Early global synthetic analyses identified soil salinity as a major environmental determinant of microbial community composition\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, whereas subsequent local studies suggested that the influence of soil salinity might not be significant\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. This controversial understanding may arise from the challenges posed by differences in soil background characteristics and vegetation types in field experiments, which act as barriers to controlling variables and hinder efforts to isolate the effects of soil salinity on microbial communities\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Overall, there are fundamental differences in the responses of different soil microbial taxa to salt stress\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. For instance, some studies have suggested that archaeal communities are more susceptible to changes in soil salinity than bacterial communities\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, while fungi are generally considered to have stronger abilities to cope with osmotic pressure changes compared to bacteria\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. However, the response mechanisms of these communities across a broader range of salinity changes remain unclear\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, and few studies have explored the diversity and functions of archaea, protists, and metazoans, despite their crucial roles in soil microbial food webs and microbially mediated biochemistry in saline soils\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMoreover, changes in soil salinity have been proven to significantly impact various soil functions\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. For example, agricultural productivity and soil organic matter decomposition capacity are significantly inhibited by soil salinity, and the influence of salt stress on soil nitrification has also been shown to be closely related to the degree of soil salinization\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. However, most of these studies have focused on single soil functions and are based on simple linear relationships or binary hypotheses. Increasing evidence indicates that ecosystems do not always respond progressively to environmental changes and can sometimes undergo abrupt changes, highlighting the unpredictability of ecological consequences\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. The lack of research on the effects of soil salinization makes it difficult to fully understand how soil salinization affects soil multifunctionality and the overall stability of ecosystems\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Therefore, we need to conduct more in-depth and comprehensive research to reveal the true effects of soil salinization and provide a scientific basis for formulating effective soil management and ecological protection strategies.\u003c/p\u003e \u003cp\u003eSoil salinization not only affects a variety of soil functions and biodiversity but may also significantly alter the relationships between them, shedding light on the intricate interplay between Earth's biodiversity and the ecosystem services it provides to humanity\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. The manner in which global change factors, such as soil salinization, reshape BEF relationships has emerged as a pivotal topic within ecological and environmental sciences, as well as in emerging interdisciplinary fields\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Past research has demonstrated that biodiversity loss can diminish the functioning of most ecosystems\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Furthermore, soil biodiversity, encompassing a diverse array of taxa such as bacteria, fungi, archaea, protists, and metazoans, has been proven to have a significant driving effect on multiple ecosystem functions\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Furthermore, soil multitrophic biodiversity can also effectively sustain multiple ecosystem functions and crop productivity in agriculture\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. However, global change factors alter diversity and functions in different directions, potentially due to the direct and indirect influences of climate, soil, and numerous other external factors on various aspects of BEF\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. For instance, drought can drive shifts in biodiversity-soil multifunctionality relationships\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Therefore, exploring the mechanisms by which global change factors influence soil multi-dimensional BEF relationships is one of the key issues in ensuring the sustainable production of resources for humanity in agriculture\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Currently, research on BEF is rapidly evolving towards EMF, but it has primarily focused on natural ecosystems such as forests and grasslands\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. The changes in BEF relationships in salt-affected agricultural soils remain understudied and poorly understood\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHere, we specifically investigate the responses of biodiverse soil organisms and key soil functions. We hypothesize that (1) the impacts of soil salinization on diversity and function may be complex, potentially exhibiting distinct nonlinear relationships and threshold effects; and (2) the effect of soil salinization will further alter the soil BEF relationship, a change that may be jointly mediated by other soil factors. This is due to the intensification of resource-dependent competition driven by environmental stress, which impacts the survival of biocommunities associated with ecosystem functions\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Consequently, this leads to a cascade effect of decreased abundance and diversity in soil biotic communities, ultimately resulting in the loss of multiple soil functions\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. This may represent an unrevealed pivotal mechanism by which soil salinization currently jeopardizes global soil health and food security. To test our hypothesis, we evaluated how the BEF varies along a broad salinity gradient in Northeast China. This region is one of the three major saline soil distribution areas in the world, encompassing an area of approximately 3.73\u0026nbsp;million hectares, which is roughly 1.3 times the size of Belgium\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. We focus on soil multifunctionality because of the importance of multiple soil functions in regulating global biogeochemical cycles\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. To achieve this, we measured typical soil functions and soil biodiversity, which collectively represent good proxies for nutrient cycling, climate regulation, and soil fertility. A total of 115 field sites were selected along a 100-kilometer salinization gradient, encompassing typical multiple farmland cultivation types and cropping histories (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). To exclude the influence of other environmental gradients, these sites exhibited similar farmland management practices, water availability, and drought intensity.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e1.Cascading effect of salinization on soil biocommunities and soil functions\u003c/h2\u003e \u003cp\u003eSoil salinization, as indicated by soil electrical conductivity (EC), governs biocommunities across trophic levels, influencing biodiversity, total abundance, species richness, and key community abundances (Figs. S2A-C, S3; Supplementary Text 3), and significantly impacts community biodiversity aspects in random forest models (Fig. S4A-F). We use the enumeration method to obtain a significant causal path and construct a structural equation model (SEM) to reveal the significant and complex cascading effects of different soil biotic communities on salinity changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The direct negative effect of soil salinization on the fungus community was detected, while the archaea community was found to be positively affected by soil salinization (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). This is consistent with previous research results, where the fungus community is considered to have a more sensitive response to the increase in soil salinity, and the eukaryotic community shifts towards the prokaryotic community\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Certain groups of archaea have been shown to exhibit higher salinity tolerance, for instance, ammonia oxidizing archaea can dominate nitrification and exhibit higher abundance at intermediate salinity\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Previous studies have mainly focused on bacterial and fungal communities, and our results provide important reference for exploring the soil food web relationships including archaea, protists, and metazoans under the context of soil salinization\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Our results emphasize the key central role of the fungus community in driving multi-community changes under the context of soil salinization, affecting the changes in soil biological community abundance through multiple direct and indirect pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). We found that the fungus community has the relatively highest individual contribution percentage to multitrophic biodiversity, soil structure, fertility, plant productivity, and multifunctionality (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eOn the one hand, arbuscular mycorrhizal fungi can regulate the mineral nutrient metabolism and physiological ecological responses of host plants to enhance their salt tolerance\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. For example, inoculation with arbuscular mycorrhizal fungi can increase the activity of various antioxidant enzymes, which can scavenge reactive oxygen species and alleviate salt stress\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. On the other hand, certain fungal groups exhibit stronger adaptability to salt stress, thereby playing a more significant role in maintaining soil functions\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Our results also highlight the important role of Metazoans community in soil functions such as carbon sequestration, symbiosis, pathogen control, and ARG control (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Metazoans, by feeding on bacteria and fungi, affect symbiotic relationships and can promote the accumulation of microbial residues, thus playing an important role in the stabilization of soil carbon pools\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. The activity of Metazoans may influence the selection pressure and transmission pathways of pathogens and ARGs in the soil, thereby controlling related environmental risks to some extent\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. However, it is also necessary to emphasize that this multi-community relationship may have regional and climatic environmental limitations due to the high variability of biotic communities\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Therefore, future global-scale research across soil background characteristics and vegetation types is still needed to bridge the knowledge gap\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe random forest model indicates that soil salinity significantly affects multiple soil functions and multifunctionality (Fig. S4G-P). Biodiversity across multiple trophic levels is interconnected through food webs, generating internal interactions through predation or resource-dependent competition\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Consequently, they may differ in the manner and sequence of their responses to soil salinity changes\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. For instance, the biodiversity indices of bacteria and fungi increase with the increase in soil EC but decrease after crossing a threshold value as EC continues to rise (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). In contrast, the biodiversity of archaea and protists exhibits a nearly linear negative correlation with increasing EC (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D). Conversely, the biodiversity of metazoans shows a slight increasing trend with the increase in soil EC (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Multitrophic biodiversity indices consider interactions among multiple trophic levels, which can lead to complex responses to soil EC. Initially, increasing soil EC may impose moderate environmental stress, prompting the soil biotic community to release additional ecological niches. However, beyond a certain threshold, high EC can become toxic or stressful for some organisms, leading to a decline in diversity. The linear relationship observed with individual soil diversity indices is likely due to the direct influence of soil EC on specific groups (Fig. S5), while the hump-shaped pattern in multitrophic indices reflects the complex interplay of nutrient availability and environmental stress across multiple trophic levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eOur study also reveals that surpassing specific thresholds of soil EC induces abrupt shifts in individual soil functions and multifunctionality, with the exception of carbon sequestration, mutualism, and pathogen control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL, M, N) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Notably, the responses of various soil functions to salinization exhibit significant divergence. Beyond a certain salinity threshold (2.2), the soil may reach a relatively stable state, slowing further structural degradation and potentially decelerating the decline in water regulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). As soil structure surpasses the salinity threshold (1.95), its trend reverses from decline to increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH), possibly due to the accumulation of soil minerals and mineral particle formation associated with increased salinity\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. In general, elevated soil salinity may displace calcium and magnesium ions with sodium, disrupting the binding between soil particles and preventing the formation of stable aggregate structures\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Additionally, the increase in soluble salts in saline soils weakens the cohesion between particles, leading to the degradation of soil structure, which adversely affects water regulation and soil structure\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. The model of organic-mineral interactions further explains the bonding mechanisms between soil minerals and organic matter, accounting for the slight improvement in soil structure post-salinity threshold\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe carbon storage function of soil continues to decline with the increase of soil salinity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Soil salinity may diminish carbon storage by affecting microbial carbon pumps and physical protection mechanisms of organic matter\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. High salinity environments decrease biotic metabolic and extracellular enzyme activities, directly reducing soil fertility and biogeochemical cycling processes, explaining the changes in fertility and organic matter decomposition\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. This may explain why soil fertility function accelerates its decline after soil salinity crosses the threshold (2.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). Reduced soil porosity not only directly affects aggregation but also restricts the movement of oxygen and water, which may restrict microorganisms from decomposing organic matter under hypoxic or dehydrated conditions\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Therefore, the decomposition of soil organic matter decreases continuously with the increase of soil salinity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Moreover, as salinity crosses the threshold (2.87), the rate of decline in organic matter decomposition slows down, which may be due to the adaptive adjustment of biotic communities to environmental changes\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWith increasing soil salinity, declines in plant productivity and pathogen control may create ecological niches, potentially filled by other microbes or organisms, leading to new symbiotic relationships\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Under reduced competition for resources due to decreased plant productivity, other organisms may gain more living space and resources, potentially enhancing symbiotic stability through resource-sharing\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. This explains the increase in mutualism function with rising soil salinity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eM) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Moreover, the control function of the pathogen decreased with the increase of salinity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eN) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This further confirms that biotic interactions undergo significant changes that may sacrifice the control functions of some pathogens due to the need to adjust their physiological and metabolic mechanisms to accommodate high osmotic pressures\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. High salinity environments may indirectly affect the spread and dissemination of antibiotic resistance genes (ARGs) by altering soil biotic community structures and influencing the degradation and transformation of antibiotics\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. The results indicate that the control of ARGs increases with increasing salinity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eO). This is an interesting phenomenon, and we believe it may be due to a decrease in human activity intensity on high salt soils, which requires further research. Collectively, high salinity environments exert direct and indirect effects on multiple individual soil functions\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, with the cumulative total effects leading to a reduction in soil multifunctionality and the formation of thresholds (2.32) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eP).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe used cluster analysis to better explore the hidden patterns in soil function and thereby construct 5 cluster centers of soil functions through the elbow diagram method (Fig. S6, Supplementary Text 5). A SEM was developed to elucidate the cascading effects of salinization on soil multitrophic biodiversity and multidimensional functions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, Supplementary Text 4). Soil multitrophic biodiversity is significantly negatively affected by soil salinization, directly impacting Dimension 3 (Carbon storage, Fertility, Organic matter decomposition) and dimension 5 (ARG control) of soil functions. The decline in soil biodiversity may lead to a reduction in the biological groups involved in key soil functions, thereby constraining critical processes such as soil organic matter decomposition, phosphorus mineralization, and nitrogen transformation\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. In contrast, the decrease in biotic diversity could imply the presence of fewer biotic groups, potentially suppressing the spread and enrichment of ARGs\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Dimension 3 and dimension 4 (Soil structure) of soil functions are directly and indirectly affected by soil salinization (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), exhibiting significant direct effects on all other dimensions of soil functions. Moreover, Dimensions 3 and 4 have a higher unique, average share, and individual contribution to soil multifunctionality (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), with individual contribution ratios as high as 46.01% and 29.96%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Therefore, we consider Dimensions 3 and 4 of soil functions to be key dimensions that influence soil multifunctionality through cascading effects. The results of this study are consistent with previous hypotheses regarding the impact of soil salinization on soil functions and provide direct evidence that the effects of soil salinization on soil structure are multifaceted, and these impacts can have cascading effects on other soil functions\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. This cascading effect is primarily driven by the disruption of soil structure. The accumulation of salts in the soil alters the interactions between soil particles, leading to the collapse of aggregates and their pore network\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. The reduced infiltration and distribution of water and oxygen ultimately limit soil biodiversity and activity, thereby weakening their support for soil functions\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Ultimately, the collapse of structure affects the multifunctionality of soil, including multiple dimensions such as dimension 1 (Water regulation, Pathogen control) and dimension 2 (Plant productivity, Mutualism) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Therefore, the protection and restoration of soil structure are essential for maintaining soil health and ecosystem services\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e2.Inconsistent changes in BEF relationships along with soil salinization gradient\u003c/h3\u003e\n\u003cp\u003eGlobal observational and experimental microcosm studies collectively provide evidence that soil biodiversity is significantly and positively correlated with multiple ecosystem functions\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Moreover, in agricultural ecosystems, BEF relationships are typically weaker than in natural ecosystems (e.g., grasslands and forests)\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Our findings also indicate a pervasive threshold effect in the BEF relationship as EC varies in agricultural ecosystems (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B, C, D, G, I, J) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Prior to the threshold (3.55), the strength of the biodiversity-ecosystem functioning (BEF) relationship diminishes gradually with increasing soil salinization, eventually transitioning from a positive to a negative correlation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This might be due to N deposition or fertilization, which increase functions but decrease soil multitrophic diversity\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. This also suggests that during the initial stages of salinization, competitive interactions among soil organisms intensify due to increased soil salinity\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, and the significant reduction in mutualism before the threshold supports this view (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). In other words, non-productive species may gradually gain a competitive advantage at this stage, thereby reducing the overall resource turnover efficiency of the biological community\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. As salinization continues to increase, the BEF relationship becomes dominated by a negative correlation. The Stress Gradient Hypothesis (SGH) may explain this shift, as the decline in abundance of dominant species releases more niches\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Consequently, the competitive pressure on non-dominant species may decrease, leading to potential increases in biodiversity within certain biological communities\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Additionally, positive interactions among communities decrease at this time, ultimately affecting the strength and direction of BEF\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Post-threshold, as soil salinization increases, the negative BEF correlation gradually weakens. The decoupled relationship here is largely due to the change in both diversity and functions or because niche differentiation caused by salinization reduces competition among species\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn general, we observe threshold effects in the BEF responses of various functions, with soil EMF exhibiting the highest EC threshold (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ). These results suggest that changes in the BEF relationship of soil EMF may exhibit a certain lag, where the supportive role of biodiversity for some functions may be suppressed, but its relationships with other functions may be enhanced to compensate for this loss\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Our findings indicate that soil structure, organic matter decomposition, and mutualism functions all exhibit similar response patterns in the BEF relationship of soil EMF, with completely different change patterns before and after the threshold (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D, G). However, the BEF relationship changes for fertility, and ARG control functions increase with increasing soil EC (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, I). This suggests that under soil salinization stress, biological communities related to generating the above two functions may gradually dominate\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. The BEF relationships for other functions generally decrease with increasing soil EC, indicating that the contribution of soil biodiversity to these functions may be less significant\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. We acknowledge that different functional selections may ultimately affect the evaluation results of the BEF relationship. Therefore, we recommend incorporating more ecosystem functions into the multifunctionality framework to achieve more robust outcomes.\u003c/p\u003e \u003cp\u003eConsidering a range of independent multifunctionality indexes with multiple thresholds (multi-threshold multifunctionality, including\u0026thinsp;\u0026gt;\u0026thinsp;10%, \u0026gt;\u0026thinsp;25%, \u0026gt;\u0026thinsp;50%, \u0026gt;\u0026thinsp;75%, and \u0026gt;\u0026thinsp;90% thresholds; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), similar conclusions were also found\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Specifically, the most significant decline was observed at thresholds of 10% and 25%, indicating that ecosystems with lower levels of function are more susceptible to the effects of soil salinization. This multi-threshold approach, which accounts for correlations between functions, effectively reveals BEF changes at different functional levels while limiting the number of functions considered\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. At higher threshold levels (90%), BEF tends to remain unchanged with increasing salinization. This suggests that selective pressures intensify under these conditions\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. The biological community's survival strategies shift more actively to adapt to saline environments, thereby offsetting changes in the BEF relationship\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThis attenuated BEF relationships is evident across various soil functions and multiple community factors (Fig. S7 A, B). This is primarily attributed to the monoculture practices which diminish the interactions and interdependencies among species within the system\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Additionally, the frequent application of fertilizers, irrigation, weed control, and pest management in agricultural ecosystems may directly influence their functionality\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. To further elucidate the mechanisms underlying the changes in soil biodiversity and BEF relationships caused by soil salinization, we analyzed the variations in BEF relationships associated with key soil biota groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL - P). At the mean level, we found that BEF relationships for all biological groups, except for fungi and protists, significantly decline with increasing soil salinization. This suggests that the impact of soil salinization on function-supporting biological communities may be a comprehensive effect across food webs\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBacteria and fungi primarily act as decomposers in ecosystems, and due to the diversity of their physiological characteristics and metabolic pathways, they can often survive and function in adverse environments such as salinization\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Thus, they are more important in BEF relationships at high salinization levels (i.e., lower functional levels) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL, M). Moreover, the BEF relationship for fungi tends to increase with soil salinization, particularly at high threshold levels (T90), implying that symbiotic relationships among species may be promoted (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eM). This could partially offset the negative impacts of soil salinization and further emphasize the more critical role of fungal communities in maintaining ecosystem function in saline soils\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Additionally, protists exhibit different patterns in BEF relationships across threshold levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eO), reflecting the complexity and variability of biodiversity and ecological functions under different environmental conditions\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eInterestingly, the BEF relationships for bacteria and archaea exhibit a higher rate of decline at low threshold levels (T10) with increasing soil salinization, while metazoans show the opposite pattern (T90) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL, N, P) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This indicates that the diversity of metazoans is more sensitive to soil salinization in ecosystems supporting high-level functions\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Metazoans, typically multicellular organisms with complex body structures and physiological functions, can prey on other organisms or utilize more complex organic materials as food sources\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Therefore, metazoans generally possess more complex physiological systems and higher energy demands, making them more important in BEF relationships at higher functional levels\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn order to reveal the reasons why the BEF relationship changes with the degree of soil salinization, we conducted further analysis on the soil biotic community and its relationship with soil factors. We have unveiled a reduction in the significant association pathways between biological communities and environmental factors in salinized soils compared to non-salinized soils, a pattern that is universally present and consistent across multiple communities (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, C). The BEF research has delineated a comprehensive array of mechanisms, including but not exhaustive of the expansion of biotope space indicating an augmented diversity of ecological niches, improved resource utilization efficiency, multitrophic interactions, and facilitation processes, which collectively facilitate the enhancement of ecosystem functioning in response to increased biodiversity\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Interestingly, we observed that in salinized soils, the co-occurrence networks of biological communities are more complex and tightly connected (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, D). Upon soil salinization, there is a noticeable increase in the number of nodes and the quantity of positive correlations within the total network and each sub-community network (Table S2). This indicates that positive interactions among biological communities are enhanced in salinized soils, reflecting the adaptive strategies of these communities to the adversity of saline soil conditions\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. This indicates that as soil salinization increases, the decoupling of the BEF relationship may be attributed to changes in soil food web complexity and interactions among different trophic levels\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.We also believe these changes are due to the increased limitation of biologically available resources caused by salinization, which in turn stimulates the intensification of various biological interactions\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. The SGH helps to explain how soil biological communities cope with the stress of soil salinization by enhancing positive interactions and complementary effects\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. On the other hand, the associated soil environmental changes also serve as a powerful filtering factor for existing soil biological species\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. We found that in salinized soils, the bacteria-metazoans sub-networks exhibit higher network complexity and connectivity, whereas in non-salinized soils, the bacteria-fungus communities play a more significant role (Table S2). This suggests that in salinized soils, top-down effects may be somewhat enhanced, meaning that species at higher trophic levels may play a more important role in controlling species and BEF relationships\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Overall, our understanding of BEF relationships across biological communities remains limited\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Our results also provide a reference for future studies on the BEF relationships of specific biological communities with ecosystem functions and for revealing the complex internal connections among species\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe random forest model demonstrates that soil EC is a key environmental factor affecting multi-trophic BEF relationships, encompassing biodiversity indices across multiple biological groups and soil functions (Fig. S8). Among the various environmental variables assessed, EC significantly impacts BEF relationships, and we also found that the impact of soil EC on the BEF relationship is significantly regulated by other soil factors (Fig. S9). The increase in soil pH, bulk density, clay content, and catalase enzyme activity may exacerbate the loss of BEF relationships induced by soil salinization, whereas other soil factors exhibit opposite effects. This is because the augmentation of factors such as soil pH, among others, indicates stress on plants and soil organisms from various perspectives, thereby hindering the ability of soil organisms to maintain soil functions\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. For instance, an increase in pH may lead to reduced availability of essential nutrients such as phosphorus, iron, manganese, and zinc. An elevation in bulk density and clay content typically signifies increased soil compaction, which reduces soil aeration and permeability\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Heightened catalase activity may indicate the accumulation of hydrogen peroxide in the soil, potentially damaging the cellular structures of soil organisms and impairing their normal functions\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. These results validate the notion that direct interactions among various soil physicochemical factors can influence soil functions, and that the degree of salinization has the potential to interact with other soil factors as a concentration amplifier\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe further elucidate the interactive effects of soil EC and other critical environmental factors on BEF relationships of EMF using generalized linear models (Table S3). Soil EC exhibits a significantly negative effect in all models except for alkaline available phosphorus and microbial biomass phosphorus, with its estimates substantially stronger than other soil factors, indicating that soil salinization is a primary driver of BEF relationships (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Moreover, soil salinization significantly interacts with Total nitrogen, Alkaline hydrolyzed nitrogen, Clay, and S gene abundance to affect soil BEF relationships negatively (Table S3). Soil salinization can impair soil organisms' ability to transform and utilize nitrogen, leading to reduced availability of total nitrogen and alkaline hydrolyzed nitrogen\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Thus, we posit that nitrogen management in saline soils should be a focal point. On the other hand, in saline soils, clay particles adsorb significant amounts of salts, leading to reduced soil permeability and affecting water and nutrient cycling\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. The interaction between salinization and clay may disrupt soil biotic communities and impact S gene expression, consequently affecting soil biodiversity and functions\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe imbalance in biotic communities caused by salinization can disrupt key ecosystem processes, thereby exerting a greater influence on BEF relationships (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). These impacts collectively lead to reduced stability and productivity of soil ecosystems, adversely affecting agricultural production and the ecological environment\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Therefore, effective measures are needed to prevent soil salinization and protect health and sustainable development of soil ecosystems. Our results have significant implications for better promoting soil health restoration and sustainable agricultural productivity development in the context of global soil salinization.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur findings integrated threshold analysis to separately reveal the patterns of changes in soil multitrophic biodiversity and multiple-level soil functions as soil salinity increases. Furthermore, we elucidated the changes and decoupling patterns of soil biodiversity-ecosystem function relationships as soil salinization progresses. Moreover, we have unveiled the cascading effects among biotic communities and the interactive effect of soil environmental factors, which can amplify the impacts of soil salinization. These conclusions underscore the urgency and importance of soil salinization management; without robust measures, soil salinization may lead to greater losses in soil functions and agricultural productivity than previously anticipated. In summary, this study comprehensively elucidates the mechanisms underlying the changes in soil BEF relationships in the context of soil salinization, contributing innovative interdisciplinary insights to safeguarding global food security. We call for future research to connect our findings with ecosystem service assessments and socio-economic data, to comprehensively advance the sustainable remediation of soil ecosystems in the Anthropocene era, marked by rapid environmental changes and the reduction of natural resources. Overall, we have demonstrated that the impact of soil salinization results in biodiversity changes at one trophic level cascading to other trophic levels, potentially leading to greater losses in soil EMF than previously anticipated.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eSite information and sampling protocol\u003c/h2\u003e \u003cp\u003eOur study area is located in the Songnen Plain of northeastern China, a major region for salt-affected soil distribution globally\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Soil salinization in this area is primarily due to the low-lying terrain, with an annual rainfall of 423.57 mm and high evaporation rates exceeding 1200 mm, leading to the formation of extensive saline soils. The salinity composition of the soils is predominantly sodium carbonate (Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e) and sodium bicarbonate (NaHCO\u003csub\u003e3\u003c/sub\u003e), with Na\u003csup\u003e+\u003c/sup\u003e ions accounting for more than 70% of the total soil cation content. The region's soils are mainly clay-based and fall under a temperate monsoon climate, with an average annual temperature of approximately 6.10\u0026deg;C and significant diurnal temperature variation. Between 2023 and 2024, soil sampling was conducted across 115 typical agricultural sites within an area of over 10,000 square kilometers, representing a typical salinization gradient with soil EC ranging from 0.37 dS/m to 6.03 dS/m. These agricultural fields were historically cultivated at different time intervals (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) and were previously salt-affected grasslands with low vegetation coverage. The dominant plant species in these grasslands were \u003cem\u003eLeymus chinensis\u003c/em\u003e and \u003cem\u003eSuaeda glauca\u003c/em\u003e. The agricultural fields are managed by local farming corporations under similar cultivation practices regulated by the government. Specific agricultural management practices for paddy fields (rice) and upland fields (corn), including fertilization, tillage, and planting density, are described in the reference\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSoil sampling was carried out following standard procedures, with a 50m\u0026times;50m plot selected at each site\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Ten soil samples were collected using an 'S' pattern and combined into a single composite sample. Additionally, an undisturbed soil sample was collected from each site to analyze soil structure and aggregate composition. Furthermore, average yield records for the past five years for each site were obtained from local farming corporations. After the field investigation, the composite soil samples were sieved (\u0026lt;\u0026thinsp;2 mm) and divided into two subsamples. One soil subsample was air-dried to analyze its physical and chemical properties, while the other subsample was immediately frozen at -20\u0026deg;C for molecular biological analysis. Methods for soil physical and biochemical analysis are detailed in the Supplementary Text 1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMethods of soil molecular analysis\u003c/h2\u003e \u003cp\u003eTotal DNA is extracted from soil samples using the E.Z.N.A.\u0026reg; Soil DNA Kit (OmegaBio-Tek, USA) following the manufacturer's guidelines. The diversity of soil bacteria, fungi, archaea, protists, and metazoans is assessed via amplicon sequencing on the Illumina MiSeq platform\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Bacterial identification employs 16S V4 region primers (515F and 806R), fungal identification uses ITS1-5F/ITS2 region primers, archaeal 16S rRNA gene V3-V5 regions are amplified with primers Arch344F and Arch915R, and eukaryotic organisms are identified using 18S V4 region primers (528F and 706R)\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Operational taxonomic units (OTUs) are defined at 100% sequence identity\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Taxonomic classification is conducted using RDPclassifier (version 2.2) against the Silva16S rRNA database (v138) with an 80% similarity threshold\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Community composition at taxonomic levels including domain, phylum, class, order, family, genus, and species is analyzed using the uclust algorithm.\u003c/p\u003e \u003cp\u003eMetagenomic shotgun sequencing libraries were constructed and sequenced at Shanghai Biozeron Biological Technology Co. Ltd. In briefly, for each sample, TruSeq DNA Library Preparation kit (catalog no: FC-121-2001, Illumina, USA) were used to construct sequencing libraries and the concentration of all libraries were measured by High Sensitivity Double Stranded DNA kit on a Qubit Fluorometer (Thermo Fisher Scientific)\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. All samples were sequenced in the NGS instrument with pair-end 150bp (PE150) mode. Following sequencing, detailed steps of a bioinformatics pipeline for processing and analyzing metagenomic sequencing data, encompassing quality control, read assembly, contig filtering, gene prediction, clustering, and functional analysis, are elaborated in the Supplementary Text 2.\u003c/p\u003e \u003cp\u003eKEGG ortholog annotation was conducted utilizing the KofamScan with the HMMSEARCH package\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. A total of 239 carbon metabolism-related functional genes were identified, encompassing processes such as methane metabolism, carbon fixation, and aerobic respiration\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Additionally, 33 nitrogen cycle-related functional genes were detected, including denitrification, nitrite reduction, and ammonia oxidation\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. The study also identified 41 phosphorus cycle-related functional genes, which are involved in organic P mineralization, P transportation, and P regulation\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Furthermore, 73 sulfur cycle-related functional genes were detected, including the synthesis of sulfite reductase, sulfur transferase, and sulfur relay protein\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSoil biodiversity index calculation\u003c/h3\u003e\n\u003cp\u003eThe Shannon diversity index of soil bacteria, fungi, archaea, protists, and metazoans is ascertained from rarefied ASV abundance datasets. The top 10 most abundant soil organisms from bacteria, fungus, archaea, protist, and metazoans communities were selected to compare their relative abundance responses to soil salinization Supplementary Text 3. To derive a quantitative metric of soil biodiversity per sample, the biodiversity attributes of our soil organism groups are integrated by averaging the biodiversity index across all groups\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. This composite index is termed the multitrophic diversity index, reflecting the comprehensive biodiversity across trophic levels\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. This methodology is frequently applied to calculate multiple biodiversity indices for both soil and plant communities\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eCo-occurrence network traits of soil organism\u003c/h3\u003e\n\u003cp\u003eBiotic communities were categorized into salinized and non-salinized based on the current definition of soil EC (\u0026gt;\u0026thinsp;2 dS/m)\u003csup\u003e11, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e to construct co-occurrence networks. Subsequently, co-occurrence networks for these communities, including sub-networks between them and within individual community, were established to assess the impact of soil salinization on the overall structure and potential biological interactions of dominant soil organisms. These networks were constructed based on Spearman's correlation algorithm of different ASV proportions. Specifically, the \"WGCNA\" and \"igraph\" R packages were used to construct and analyze the networks. Nodes in the network represent taxonomic ASVs of soil organisms, and edges correspond to significant connections between nodes\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. A series of topological parameters were calculated to describe the biological connections in the network, including the number of nodes and edges, positive and negative correlation numbers, average degree, graph density, degree centralization, and modularity\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDefinition of individual soil functions and assessing soil multifunctionality\u003c/h2\u003e \u003cp\u003eIn each plot, multiple soil indicators reflecting ecosystem functions, processes, or attributes were used to calculate nine typical soil functions, which are regulated by soil biology and considered to comprehensively reflect the functionality of agricultural ecosystems\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e: water regulation (water retention capacity, calculated from soil porosity), soil structure (including bulk density, water-stable aggregate content), carbon storage (calculated from soil organic carbon content and bulk density), fertility (including available potassium, calcium, sodium, and magnesium ion content), organic matter decomposition (including the activity of nine enzymes associated with soil redox processes, carbon-nitrogen-phosphorus metabolism, and key substrate decomposition), plant productivity (based on the average yield per hectare over the past five years, from 2018 to 2023), mutualism (the proportion of ectomycorrhizal fungi and arbuscular mycorrhizal fungi), pathogen control (the relative abundance reduction of plant pathogens in the soil), and ARG control (the reduction of ARG abundance in the soil). We first calculated the standardized index for each function according to the methods in the literature to represent each function, ensuring that data with different characteristics are on the same scale.\u003c/p\u003e \u003cp\u003eTo obtain quantitative indicators of multiple ecosystem functions, multifunctionality was calculated using two of the most common methods: \"averaging\" and \"multiple thresholds\"\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. The averaging method assesses by calculating the mean of all standardized (0\u0026ndash;1 normalized) ecosystem function indicators. The threshold-based method evaluates the total number of functions that exceed or are equal to a predefined percentage of the maximum observed value for each individual function. The specific calculation methods have been widely described in the literature. We used a set of thresholds: 10%, 25%, 50%, 75%, and 90% to represent the multifunctionality at low, medium, and high thresholds of the observed maximum functionality\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eThe local soil BEF relationships were analyzed from multiple perspectives: the correlation between individual soil functions and soil EC, the relationship between biodiversity and soil functions, biodiversity and multifunctionality using both averaging and multiple threshold approaches, and the biodiversity of different organism groups (including bacteria, fungi, archaea, protists, and metazoans) versus individual ecosystem functions. Spearman's correlation coefficients were calculated to represent local soil BEF relationships, with stronger positive correlations indicating a higher capacity of soil biodiversity to enhance ecosystem functions\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. A moving window approach was applied to explore the determinants of boundary function relationships, aiding in the analysis of multidimensional data along ecological gradients\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo ensure robust data analysis, a sliding window averaging algorithm was applied, wherein two window sizes consisting of 60 consecutive samples were selected at each site, yielding 56 data points (e.g., 1\u0026ndash;60, 2\u0026ndash;61...56\u0026ndash;115)\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. After reordering along the soil salinity gradient, the window was advanced across sampling points, creating adjacent subsets to better estimate the complexity of BEF relationships. Acknowledging that data points from the moving window are not independent and may exhibit correlations with the window, a bootstrapping procedure was conducted, involving random sampling of 55 data points without replacement from the original dataset 1000 times\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Bootstrapping helps address these dependencies, aiding in the estimation of confidence intervals for these correlation coefficients and providing more information about the strength of relationships and more accurate statistical estimates\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSegmented regression analysis was performed using the \"segmented\" package, initially setting the threshold at the mean EC value. A grid search function was defined to systematically evaluate different threshold values by calculating the Akaike Information Criterion (AIC) for each potential threshold\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The grid search was conducted across a range of EC values, and the threshold with the minimum AIC was identified as the optimal threshold. The data were split into two segments based on the identified threshold, and linear regression models were fitted to each segment to assess changes in slope before and after the threshold\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. The Mann\u0026ndash;Whitney U-test was implicitly applied through the comparison of slopes and predicted values before and after each threshold to validate the statistical significance of the identified threshold. Linear models before and after the threshold were fitted using the lm function, and their summaries were examined. If no threshold was detected within the selected EC range, linear regression analysis was conducted on the entire dataset. Additionally, Generalized Additive Models (GAMs) were fitted using the mgcv package, modeling each soil function as a smooth function of EC\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The \"ggplot2\" package was utilized to create visualizations, including boxplots and fitted lines from the GAM model, segmented regression, and linear models.\u003c/p\u003e \u003cp\u003eAdditionally, correlation analyses were performed using the \"corrplo\" R package. The obtained P values were adjusted for multiple comparisons using the \"fdr\" method to control the likelihood of false positives. A Random Forest analysis was conducted using the \"randomForest\" package to identify key soil environmental factors affecting individual soil functions, multifunctionality, and their BEF relationships\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. The \"lavaan\" package in R was utilized for SEM. The logic behind model construction and references are described in Fig S10, S11 and Supplementary Text 4. This package facilitates the specification of latent variables and estimation of path coefficients, aiding in the comprehension of direct and indirect effects within our model. The k-means function in R was used for clustering analysis, the specific details of cluster analysis are detailed in Supplementary Text 5. Mantel analysis, a statistical method for testing correlations between two distance matrices, was employed to assess if there is a significant relationship between dissimilarities in species composition and environmental variables\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The \"vegan\" package was used to execute Mantel tests, with the mantel function calculating the Mantel statistic and evaluating its significance through permutation tests. The generalized linear model is constructed using the \"stats\" package in R, and the specific details are explained in detail in the Supplementary Text 6.\u003c/p\u003e \u003c/div\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBanwart SA, Nikolaidis NP, Zhu Y-G, Peacock CL, Sparks DL. 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However, how soil salinization affects the soil biodiversity-multifunctionality relationship remains largely unknown in agro-ecosystems. Here, we conducted a standardized survey of agro-ecosystems across a typical soil salinization gradient at 115 representative agricultural sites, aiming to investigate the relationships between soil biodiversity (encompassing the diversity of bacteria, fungi, archaea, protists, and metazoans) and nine soil functions as well as multifunctionality. We found that soil multitrophic biodiversity and multifunctionality declined with increasing soil salinization, and the relationships between multiple functions and the degree of salinization exhibited extensive nonlinear changes and threshold effects. We further demonstrated that soil salinization reduced the general strength of the biodiversity-multifunctionality relationship, by significantly altering soil physicochemical properties and the interplay between microbial communities and soil properties. The inconsistent changes in biodiversity\u0026ndash;ecosystem functioning (BEF) relationships were attributed to the multi-threshold relationships between different soil functions and multitrophic biodiversity, as well as the differential responses of various biological communities to the multifunctionality of the soil. This study highlights the cascading hazards of soil salinization in agro-ecosystems, emphasizing the importance of integrating salinization prevention and control into sustainable agricultural management strategies to maintain soil biodiversity and ecosystem functioning.\u003c/p\u003e","manuscriptTitle":"Cascading effects of soil salinization on agricultural soil biodiversity-multifunctionality relationship","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-07 07:58:43","doi":"10.21203/rs.3.rs-5835602/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-food","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"natfood","sideBox":"Learn more about [Nature Food](http://www.nature.com/natfood/)","snPcode":"","submissionUrl":"","title":"Nature Food","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Research","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ae38000d-8939-4826-bed6-7a64bfba7926","owner":[],"postedDate":"February 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":43945471,"name":"Earth and environmental sciences/Ecology/Agroecology"},{"id":43945472,"name":"Earth and environmental sciences/Ecology/Biodiversity"}],"tags":[],"updatedAt":"2026-05-06T09:58:21+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-07 07:58:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5835602","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5835602","identity":"rs-5835602","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}