Both biotic and abiotic management strategies facilitate biomass recovery and CO2 absorption: evidence from three experiments in southern grasslands

Both biotic and abiotic management strategies facilitate biomass recovery and CO2 absorption: evidence from three experiments in southern grasslands | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Both biotic and abiotic management strategies facilitate biomass recovery and CO 2 absorption: evidence from three experiments in southern grasslands Jinghang Xu, Yi Xiong, Yan Li, Haibian Xu, Sicheng Li, Meng Xia, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6968136/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background The southern grasslands of China sustain substantial livestock but face functional degradation due to overuse. The carbon flux responses to restoration strategies remain unclear. Methods In this study, we implemented a three-year restoration experiment in artificial grasslands using three approaches: reseeding (D: Dactylis glomerata monoculture, T: Trifolium repens monoculture, DT: their mixture), chemical fertilization (NP L /NP M /NP H : nitrogen 2/4/8 g m⁻² + phosphorus 0.7/1.4/2.8 g m⁻²), and biofertilization with Burkholderia sp.(B 30 /B 60 /B 120 : 30/60/120 g m⁻²). Results All treatments increased grassland biomass. Specifically, NP H significantly enhanced above-ground biomass, while D, B 30, and NP L notably boosted below-ground biomass. D, NP L , NP H , and B 30 significantly increased total biomass. Restoration treatments usually had a negative effect on NEE, with DT (β = -0.383, P = 0.020), NP L (β = -0.350, P = 0.027), NP M (β = -0.422, P = 0.008), and B 60 (β = -0.341, P = 0.029) showing significant effects. However, they exhibited positive effects on both ER and GPP. Specifically, NP L , NP M , NP H , B 30 , B 60 , and B 120 significantly enhanced ER (β = 0.290 to 0.525, P = 0.001 to 0.049), while all measures except T significantly increased GPP (β = 0.297 to 0.613, P < 0.001 to 0.048). Dissolved organic carbon, available phosphorus, and biomass contributed to the changes in carbon sequestration. Conclusion Our results demonstrate that both traditional fertilization and alternative strategies like reseeding and biofertilization can effectively restore grassland productivity and carbon sequestration capacity, providing multiple pathways for sustainable grassland management. Grassland restoration CO2 flux Soil respiration Carbon sequestration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Grasslands cover approximately 40% of terrestrial ecosystems and are critical for human life (Buisson et al., 2022). They also store one-third of the global terrestrial carbon reserves, playing an important role in mitigating climate change (Bai and Cotrufo, 2022). However, about half of the world’s grasslands have begun to deteriorate to varying degrees (Bardgett et al., 2021). Moreover, grassland degradation greatly accelerates soil CO 2 emissions and thus diminishes the contribution of grasslands to the carbon sink (Li et al., 2015; Abdalla et al., 2018; Xu et al., 2022). Therefore, it is important to explore the dynamics of CO 2 flux under various grassland restoration strategies such as reseeding, chemical and biological fertilization, which help to understand the function of ecosystem carbon sink in grasslands. Reseeding is one of simple and feasible grassland restoration techniques (Mi et al., 2024), which improves grassland community productivity by seeding forage seeds directly on degraded grasslands (Li et al., 2017). On the one hand, reseeding can increase vegetation coverage, biomass, and the proportion of high-quality forage in degraded grasslands (Yang et al., 2018). On the other hand, tillage-based reseeding can also enhance the nutritional content of degraded grasslands (e.g., soil carbon) (Ji et al., 2022; Zhang et al., 2022). Different seed selections have various effects on the grassland yield, with grass-legume mixtures often producing higher productivity compared to monocultures (Finn et al., 2013). Therefore, reseeding restoration of degraded grasslands may enhance their contribution to carbon sinks. However, research on the relationship between reseeding restoration and CO 2 fluxes in degraded grassland is still limited. Fertilization is one of the effective methods to promote grassland restoration, which has an impact on severely degraded grasslands in the short term (Dong et al., 2020). Fertilization can significantly enhance biomass and soil nutrients in degraded grasslands (Li et al., 2023). Numerous studies have shown that nitrogen (N) addition can significantly enhance CO 2 absorption in grassland systems (Wu et al., 2021; Shi et al., 2022; Chen et al., 2023; Du et al., 2024; Wen et al., 2024). Phosphorus (P) is also a common limiting factor in soils (Elser et al., 2007; Fay et al., 2015), and combined nitrogen-phosphorus application typically had better restoration effects than added either nutrient alone (Wang et al., 2020). For example, NP addition experiments in temperate ecosystems suggested that NP addition increased biomass of rootlets and reduced heterotrophic respiration, thereby enhanced ecosystem carbon sequestration (Zeng and Wang, 2015). However, in grassland ecosystems, studies on fertilization and ecosystem carbon fluxes had mainly focused on nitrogen addition under the background of nitrogen deposition. Thus, it is important to explore the impact of combined nitrogen-phosphorus fertilization on carbon fluxes during grassland restoration. It is well known that Burkholderia sp. has positive effects on plant growth and tolerance (Pal et al., 2022). For example, inoculation with Burkholderia promoted the secretion of indole-3acetic acid (IAA) and 1-aminocyclopropane-1-carboxylate (ACC) in soybeans ( Glycine max ) and thus enhanced plant growth (Ratnaningsih et al., 2023); Burkholderia could trigger plants defense mechanisms, protecting them from pathogens (Kong et al., 2022); Burkholderia could also enhance the solubility of potassium and phosphorus, improving phosphorus availability (Peix et al., 2001; Baghel et al., 2020). Additionally, Burkholderia is a nitrogen-fixing bacterium that improves nitrogen uptake and utilization by plants (Reis et al., 2004). Furthermore, Burkholderia can degrade various pollutants and promote plant ACC production to enhance stress resistance (Lu et al., 2012; Chen et al., 2017). Therefore, Burkholderia has potential for restoring degraded grasslands and influencing carbon fluxes in such ecosystems. Although southern grasslands only account for 20% of the total grassland area in China (Fang et al., 2018), their adequate hydrothermal conditions and strong pasture regeneration ability (Teng et al., 2008), allowed these grasslands and related agricultural products to support nearly half of the China’s livestock (Huangfu et al., 2012). However, over 70% of China’s grasslands have already been experiencing degradation (Pan et al., 2023), which also severely affected the country’s grassland carbon sink. In addition, previous research on grassland restoration focused primarily on typical grasslands in northern China, how about the restoration effect on southern grasslands is still less understood. To fill the gap, we conducted the field experiments in the typical southern grasslands, Yunnan Province (Fang et al., 2018) and faces grassland degradation and soil erosion due to overgrazing (Deng, 2023). Therefore, we carried out a 3-year grassland restoration experiments by reseeding, inputting nitrogen-phosphorus chemical fertilization and adding biological organic fertilization with Burkholderia sp. We monitored grassland biomass, carbon effluxes, and their driving factors in order to answer the following hypotheses: (1) If management practices can promote plant growth, reseeding, fertilization, and addition Burkholderia sp. will enhance carbon sequestration. (2) Grassland degradation in southern regions is often driven by overgrazing, which leads to reduced community productivity and nutrient losses (Wang et al., 2015). Therefore, we hypothesize that soil nutrients after management practices, particularly soil phosphorus will be more important for improving grassland function. 2. Materials and methods 2.1. Site description and Experimental design The experiments were established in September 2020 at the Field Grassland Experiment Platform in Maolin Town, Zhaotong City, Yunnan Province (27°66′ N, 103°63′ E). The area is characterized by a plateau monsoon climate, where warm temperate and subtropical conditions coexist. The annual average temperature is 12.6°C, and the average precipitation is 760 mm. The region experiences frequent rain, snow, and fog throughout the year. The dominant species are Dactylis glomerata , Lolium perenne and Trifolium repens . The experiment was conducted from April 2021 to September 2023. Based on local vegetation surveys, Trifolium repens and Dactylis glomerata were selected as the reseeding species. The reseeding treatments included CK (no reseeding) and a seeding rate of 22.50 kg hm − 2 , with three different species combinations: D (monoculture of Dactylis glomerata ), T (monoculture of Trifolium repens ), and DT (mixed Trifolium repens and Dactylis glomerata at 2:3). Five replicates were set for each treatment, resulting in 20 plots in total. The seeds are sown in April each year for a total of three times. To minimize soil disturbance, no tillage reseeding was used. The fertilization treatments included CK (no fertilization) and three levels of nitrogen and phosphorus fertilization: NP L (2 g m − 2 year − 1 nitrogen + 0.7 g m − 2 year − 1 phosphorus), NP M (4 g m − 2 year − 1 nitrogen + 1.4 g m − 2 year − 1 phosphorus), and NP H (8 g m − 2 year − 1 nitrogen + 2.8 g m − 2 year − 1 phosphorus). The nitrogen fertilizer used was [CO(NH 2 ) 2 ] and the phosphorus fertilizer was [Ca(H 2 PO 4 ) 2 •H 2 O]. Five replicates were set for each treatment. The Burkholderia sp. treatments included three application levels: B 30 (30 g m − 2 year − 1 ), B 60 (60 g m − 2 year − 1 ), and B 120 (120 g m − 2 year − 1 ). The same CK were used for fertilization treatment. Five replicates were set for each treatment, resulting in 35 plots both Burkholderia sp. treatments and fertilization treatments. Each plot was 3 m × 3 m, with a 1 m buffer zone among them. Fertilization and Burkholderia sp. application were carried out in April and October each year, with a total of five times. 2.2. Measurement of ecosystem CO 2 flux, soil temperature, and soil moisture We measured soil carbon flux data during the growing season (April to September) from June 2021 to September 2023. Measurements were taken between 9:00 AM and 12:00 AM on clear days. Using a transparent assimilation tank (a cylinder with a base diameter of 20cm and a height of 25 cm made of plexiglass.) connected to the Li-8100a Automated Soil CO 2 Flux System monitored net ecosystem exchange of CO 2 (NEE) and ecosystem respiration (ER), and each measurement lasted 90 seconds (Due to COVID-19 restrictions in September 2022 and rainy weather during fertilization treatment in August 2023, data for these months were missed). After measuring NEE, the system was ventilated and purged for 30 seconds, and then blackout treatment was performed to measure ER (Niu et al., 2008). Gross primary productivity (GPP) was calculated using the formula GPP = ER - NEE (Ma et al., 2021). During CO 2 flux measurements, soil temperature (ST) and soil water content (SWC) were measured from 0–10 cm using the soil temperature and soil water content probes included with the Li8100a (SWC data for April 2022 and ST data for July 2023 were missing because the probes were damaged). 2.3. Sampling and measurement of grassland biomass and physicochemical properties In October 2022, above-ground biomass (AGB) and below-ground biomass (BGB) were collected from each plot. Three 20 cm × 20 cm subplots were randomly selected from each plot. The above-ground parts of all plants were collected using scissors and dried to a constant weight to determine AGB. Below-ground parts were collected using a 6 cm diameter soil auger with five sampling points at a depth of 0–20 cm. Roots were separated from the soil. Then they were cleaned and dried to a constant weight to determine BGB. Total biomass (TB) was sum of AGB and BGB. After separating the roots and rocks, soil samples were divided into two portions. One portion was analyzed using Cleverchem380 (DeChem-Tech GmbH, Hamburg, Germany) to measure ammonium nitrogen (NH 4 + -N) by Sodium Salicylate Method, nitrate nitrogen (NO 3 − -N) by Copper-zinc hydrazine reduction method, and available phosphorus (AP) by Olsen method, while another portion was analyzed with vario TOC cube (Elementar Analysensysteme GmbH, Langenselbold, Germany) to determine dissolved organic carbon (DOC) by high temperature catalytic combustion method, microbial biomass carbon (MBC) by fumigation method, and soil organic carbon (SOC) by dry combustion. All the above soil analysis methods were used from Liu (1996). Data were organized and Z-score transformations were performed using Microsoft Office Excel 2010. Statistical analysis and graphical visualizations were conducted using R 4.3.3. The effects of treatments on CO 2 flux, grassland biomass, and soil physicochemical properties were analyzed using the lme4 package. Fixed effects included D, T, DT, B 30 , B 60 , B 120 , NP L , NP M , and NP H , while random effects were block and sampling time (for grassland biomass and soil physicochemical properties, only block was treated as a random effect). A linear regression analysis was used to examine the relationship between carbon flux and soil physicochemical properties and biomass. 3. Results 3.1. Responses of plant biomass and soil physicochemical properties Reseeding treatments had non-significant effect on AGB (Fig. 1 a), but D had a significant positive effect on both BGB (β = 1.381, P = 0.033) and TB (β = 1.404, P = 0.027) (Fig. 1 b and 1 c). Except for NP M , fertilization treatments had a positive effect on AGB, among which NP H (β = 1.387, P = 0.023) showing significant positive effects (Fig. 1 d). Additionally, NP L (β = 1.398 P = 0.025) and NP M (β = 1.305, P = 0.035) had a significant positive effect on BGB (Fig. 1 e), while NP L (β = 1.362 P = 0.029) and NP H (β = 1.354 P = 0.030) significantly increased TB (Fig. 1 f). Burkholderia sp. treatments showed no significant effect on AGB (Fig. 1 g), but B 60 had a significant positive impact on both BGB (β = 1.519 P = 0.018) and TB (β = 1.430 P = 0.027) (Fig. 1 h and 1 i). Reseeding treatment showed a positive effect on DOC, with D (β = 1.665 P = 0.008) exhibiting a significant influence (Fig. 2 a). In the fertilization treatment, NP H (β = 1.445 P = 0.006) had a significant positive effect on NO 3 − -N (Fig. 2 b). All fertilization levels significantly increased AP (β = 1.440 to 1.692, P = 0.001 to P = 0.004) (Fig. 2 c). However, grassland management strategies had no significant effect on other soil physical and chemical properties (Fig. S2, S3 and S4). 3.2. Responses of NEE, ER, and GPP to experimental treatments Reseeding treatments had a negative effect on NEE, and the grass-legume mixture showed a significant negative effect (β = -0.383, P = 0.020) (Fig. 3 a). Reseeding treatments had non-significant effect on ER (Fig. 3 b). Reseeding treatments positively influenced GPP, where D (β = 0.345, P = 0.037) and DT (β = 0.414, P = 0.015) showed significant effects (Fig. 3 c). Fertilization treatments had a negative effect on NEE, among which NP L (β = -0.350, P = 0.027) and NP M (β = -0.422, P = 0.008) showing significant negative effects (Fig. 3 d). All levels of fertilization treatments significantly affected ER (β = 0.425 to 0.502, P = 0.005 to P = 0.015) and GPP (β = 0.418 to 0.613, P < 0.001 to P = 0.014) (Fig. 3 e and Fig. 3 f). Burkholderia sp. treatments had a positive effect on NEE under B 120 , but B 30 and B 60 showed negative effects, of which B 60 (β = -0.341, P = 0.029) being significant (Fig. 3 g). Burkholderia sp. treatments had positive effects on ER (β = 0.290 to 0.525, P = 0.001 to P = 0.049) and GPP (β = 0.297 to 0.556, P = 0.001 to P = 0.048) (Fig. 3 h and Fig. 3 i). 3.3. Relationships between soil physicochemical properties, biomass and CO 2 fluxes GPP was positively correlated with DOC under reseeding (Fig. 4 a). Under fertilization, GPP was positively correlated with AP (Fig. 4 b). No significant correlations were observed between other physicochemical properties and CO 2 fluxes (Fig. S9). Under reseeding treatments, TB showed a negative relationship with NEE (Fig. 5 a), but the relationships between AGB and BGB with NEE were not significant (Fig. S10). BGB and TB were positively correlated with GPP (Fig. 5 b and 5 c), and no significant relationships were observed between ER and AGB, BGB, or TB (Fig. S10). Under fertilization treatments, NEE was negatively related with TB (Fig. 5 d), while its relationship with AGB and BGB was not significant (Fig. S11). ER was positively correlated with BGB (Fig. 5 e), with no significant correlations observed between ER and BGB or TB (Fig. S11). BGB and TB were positively correlated with GPP (Fig. 5 f and 5 g). Under Burkholderia sp. additions, the effects of AGB, BGB, and TB on NEE and ER could not be determined (Fig. S12). However, BGB and TB were positively correlated with GPP (Fig. 5 h and 5 i). 4. Discussion 4.1 Effects of different managements on soil properties and grassland biomass Our results showed that reseeding improved the AGB, BGB, and TB of degraded grasslands, and D improved the BGB and TB significantly (Fig. 1 ). This effect may be due to reseeding increasing the number of viable seeds in the soil seed bank, thereby enhancing grassland productivity (Li et al., 2017). Similar findings have been observed in northern grasslands, including the Loess Plateau (Liu et al., 2023c), Mongolian Plateau (Zhang et al., 2024), and Qinghai-Tibet Plateau (Li et al., 2024). However, unlike previous studies (Yan et al., 2022), our results indicated that the biomass-promoting effect of grass-legume mixtures (DT) is weaker than that of D. This may be because, in DT, Dactylis glomerata allocates part of its nutrients to counteract the allelopathic effects of Trifolium repens (Zhang et al., 2020), thereby reducing its biomass accumulation. In addition, our data showed that fertilization significantly increased grassland biomass (Fig. 1 ), similarly with results from the Mongolian Plateau's typical steppes and the alpine meadows of the Qinghai-Tibet Plateau (Xu et al., 2015; Xiao et al., 2020). Fertilization usually enhanced soil nutrient levels and promoted the recovery of grassland productivity (Zong and Shi, 2019). Furthermore, similar to pot experiment results (da Costa et al., 2020), our study found that adding B 60 significantly increased BGB and TB (Fig. 1 i). However, addition of Burkholderia sp. had a significant impact on AGB (Fig. 1 g). On the one hand, Burkholderia sp. can promote plant growth by fixing nitrogen and resolving phosphorus. On the other hand, Burkholderia sp. can produce auxin to stimulate plant root growth (Zhang et al., 2021), so that grass biomass can be transferred to the ground. Combining the three management methods, we found that all the methods had a positive effect on the TB of degraded southern grasslands, which supported our hypothesis 1. However, only the NP H treatment significantly increased AGB (Fig. 1 ), while the other treatments increased TB primarily by enhancing BGB (Fig. 1 ). The intense grazing pressure in southern grasslands in China leads to the loss of viable grass seeds and the decline in soil nutrients. Plants general allocate more biomass to BGB, thus promoting plant access to soil nutrients (Hermans et al., 2006; Freschet et al., 2018). The analysis of soil properties in our experiment showed that reseeding increased DOC (Fig. 2 a), which was different from the findings from the Qinghai-Tibet Plateau (Wang et al., 2020), our results suggest that reseeding increased the active seed, which enhanced grass biomass and subsequently contributed to greater inputs of plant residues and rhizospheric carbon. Furthermore, consistent with previous studies (Xiao et al., 2021; Liu et al., 2023a), we found that fertilization had a significant positive effect on NO 3 − -N and AP (Fig. 2 c and 2 d). However, AP levels were generally low across all treatments (5.82 mg kg − 1 to 12.98 mg kg − 1 ) (Fig. S6, S7, and S8). This suggests that AP deficiency may be a key factor coursing the southern grassland degradation. For example, our reseeding treatments seem to cause AP depletion (Fig. S6) 4.2 Effects of different managements on carbon fluxes and potential drivers We found that all treatments had a positive effect on GPP in the grassland, while all except B 120 had a negative effect on NEE (Fig. 3 ). This result contradicted the hypothesis 1. Specifically, under reseeding treatment, our results showed that the grass-legume mixed reseeding had a significant positive effect on GPP (Fig. 3 c) and a significant negative effect on NEE (Fig. 3 a). The result was similar to the studies conducted in the eastern Pyrenees, because reseeding can restore biomass and promote carbon sequestration (Drewer et al., 2017; Ibañez et al., 2021). Additionally, studies on the Qinghai - Tibet Plateau have shown that NP fertilizer co-application significantly reduced NEE by 75.7% and increased GPP by 41.4% (Li et al., 2024). And the research on Inner Mongolia grasslands has revealed that fertilization significantly increases GPP and ER in the first two years, while significantly decreasing NEE (Niu et al., 2010). These findings are consistent with our results, indicating fertilization had a significant positive effect on both ER and GPP (Fig. 3 e and 3 f) and a significant negative effect on NEE (Fig. 3 d). The reason should be that fertilization promoted the growth of grassland and microbial activity as shown in our results and other studies (Chu et al., 2007; Zhang et al., 2024). The addition of Burkholderia sp. had a significant positive effect on ER and GPP (Fig. 3 h and 3 i). B 60 and B 30 showed a negative effect on NEE, and B 60 had a significant effect (Fig. 3 g). This suggests that Burkholderia sp. promoted CO 2 uptake by plants but also promoted CO 2 emissions through its own respiration, leading to a positive effect on ER. We proposed that at 30 g m − 2 year − 1 , the enhancement of CO 2 absorption by the ecosystem is limited, while at 60 g m − 2 year − 1 , it is most effective. However, at 120 g m − 2 year − 1 , the respiration of Burkholderia sp. covered the CO 2 absorption by plants, indicating that only lower application levels can improve degraded grasslands carbon sinks. Regression analysis results showed that different experimental measures influence CO 2 flux through both biotic and abiotic factors. Under reseeding treatments, DOC was significantly positively correlated with GPP (Fig. 4 a). The growth of reseeding plants resulted in an increase in root exudates and litter, which promoted the increase of DOC (Wu et al., 2013). These carbon inputs may change soil biotic and abiotic factors, leading to an increase in subsequent photosynthetic rates (van der Putten et al., 2013; Liu et al., 2023b), thereby enhancing GPP. Our results were similar to the previous studies (Shi et al., 2021), showing that AP was significantly positively correlated with GPP (Fig. 4 b). Phosphorus deficiency can disrupt the balance between the carboxylation capacity of leaves and the electron transport capacity supporting ribulose-1, 5-bisphosphate regeneration, thereby reducing photosynthesis (Ellsworth et al., 2022). Our results showed strong relationships between biomass and GPP, ER and NEE (Fig. 5 ), which were similar to previous studies (Wu et al., 2015; Oliver et al., 2019; Wang et al., 2021). Increased biomass is thought to improve GPP by increasing the leaf area index, which improves the plant's ability to capture light (Chu et al., 2019). However, unlike our results, GPP was significantly positively correlated with BGB and TB under all grassland restoration measures (Fig. 5 ), but not with AGB. This may be due to sampling at the end of the growing season, when plants transferred more biomass to the belowground (Garten et al., 2011). Additionally, our results showed that ER was significantly positively correlated with BGB only under fertilization treatments (Fig. 5 e). This is because nitrogen application not only negatively affects microbial abundance and composition, thereby reducing soil microbial respiration (Zhang et al., 2018), but also promotes root growth, which enhances autotrophic respiration (Gough and Seiler, 2004). Consequently, this strengthens the correlation between BGB and ER. Therefore, plant factors contributed more to ER under fertilization, but Burkholderia sp. addition likely affected soil microorganisms, masking the plant's contribution to ER. Furthermore, NEE was significantly negatively correlated with TB under both reseeding and fertilization treatments (Fig. 5 a and 5 d), indicating that reseeding and fertilization increased the carbon sink of degraded grasslands by increasing plant biomass. 5. Conclusion This study showed the effects of reseeding, fertilization, and the addition Burkholderia sp. on biomass production, soil nutrients, and carbon fluxes in southern grasslands. All management strategies increased total biomass and gross primary productivity, but they had different effects on biomass allocation, soil nutrients, and carbon sequestration. The addition of Burkholderia sp. had the strongest positive effect on below-ground biomass and total biomass, but excessive addition increased microbial respiration, thereby reducing carbon sequestration. Our study showed that phosphorus deficiency was the key factor of grassland degradation in southern China, so phosphorus fertilization had the strongest promotion effect on above-ground biomass and carbon sequestration. Reseeding increased biomass, dissolved organic carbon, and carbon sequestration, but it also exacerbated phosphorus limitation, which could lead to further grassland degradation with long-term use. Overall, future grassland managements should synthetically consider these strategies to enhance soil nutrients, increase biomass, and strengthen the carbon sink capacity in southern grasslands. Declarations Fundding This work was supported by the National Key R&D Program of China (No. 2023YFC2604502), the National Natural Science Foundation of China (32371733), the Xingdian Scholar Fund of Yunnan, the Project for Talent and Platform of Science and Technology in Yunnan Province Science and Technology Department (202205AM070005), the Agricultural joint fund (surface project) from science and technology department of Yunnan Province (No.202301BD070001-096). Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Authorship contribution Xinghong Wang: Writing– review & editing. Meiyan Zhang: Writing– review & editing. Yun Liu: Writing– review & editing. Juan Zhou: Writing– review & editing. Meng Xia: Writing – review & editing. Sicheng Li: Writing – review & editing, Investigation. Haibian Xu: Writing – review & editing, Data curation. Yan Li: Writing – review & editing, Investigation, Data curation. Yi Xiong: Writing – original draft, Visualization, Validation, Methodology, Investigation, Data curation. Jinghang Xu: Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation. Janping Wu: Writing – review & editing, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Data curation, Conceptualization. Data Availability The datasets generated by the current study are available from the corresponding author on reasonable request. Declaration of generative AI and AI-assisted technologies in the writing process. Statement: During the preparation of this work the authors used ChaGPT 4 in order to improve the readability and language. After using this tool, the authors reviewed and edited the content as needed and takes full responsibility for the content of the published article. 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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-6968136","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":476513872,"identity":"36c6aef7-81f4-4453-a101-0726cfd6368a","order_by":0,"name":"Jinghang Xu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jinghang","middleName":"","lastName":"Xu","suffix":""},{"id":476513873,"identity":"6363ca47-db18-4570-986a-1778aabeb7b4","order_by":1,"name":"Yi Xiong","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Xiong","suffix":""},{"id":476513874,"identity":"8c4bba3a-8d5b-463b-bfbc-dc09d296fa6b","order_by":2,"name":"Yan Li","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Li","suffix":""},{"id":476513875,"identity":"421c52d2-7f69-4282-b78c-dc9c81f7ba0e","order_by":3,"name":"Haibian Xu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Haibian","middleName":"","lastName":"Xu","suffix":""},{"id":476513876,"identity":"f5fad697-eb76-4791-be72-175cdecd1201","order_by":4,"name":"Sicheng Li","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Sicheng","middleName":"","lastName":"Li","suffix":""},{"id":476513877,"identity":"93679758-0c72-4a5a-9261-e4be326aa60e","order_by":5,"name":"Meng Xia","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Meng","middleName":"","lastName":"Xia","suffix":""},{"id":476513878,"identity":"568671c5-44b0-4f52-a297-2845e9551141","order_by":6,"name":"Juan Zhou","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Juan","middleName":"","lastName":"Zhou","suffix":""},{"id":476513879,"identity":"8bd0f63d-8c20-4685-9cb9-e71f411f71a4","order_by":7,"name":"Meiyan Zhang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Meiyan","middleName":"","lastName":"Zhang","suffix":""},{"id":476513880,"identity":"bf9a63e9-317d-4b39-8529-3ca03c9e7c48","order_by":8,"name":"Yun Liu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yun","middleName":"","lastName":"Liu","suffix":""},{"id":476513881,"identity":"1159af81-6f62-442e-ba15-b83f856d6f8c","order_by":9,"name":"Xinghong Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xinghong","middleName":"","lastName":"Wang","suffix":""},{"id":476513882,"identity":"e2b68015-7173-45df-94b5-71f94cf3af0b","order_by":10,"name":"Jianping Wu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIiWNgGAWjYBACxmYQaQBhP4CIJRCvhdmAKC3IgE2CKC3M7czPHhcU3LFrkMgxq+bdcZiBnz3HgOHnDnwOYzM3nmHwLBmk5TbvmcMMkj1vDBh7z+DTwmAmzWNwOJlBGqSl7TCDwY0cA2bGNnxa2L/BtRSDtNgT1sIDtsUOpIUZbIsEYS1l0jMMDiewyT8rlpzbls4jceZZwcFePFoM+49vky74c9ien+fwxg9v26zl+NuTNz74iU9LAzCggXQiSA0TDwMDD0j0AG4NDAzyDBAt9mBX/sCndBSMglEwCkYsAADdMkdijaO5OQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-5784-834X","institution":"Yunnan University","correspondingAuthor":true,"prefix":"","firstName":"Jianping","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2025-06-24 17:45:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6968136/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6968136/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85672465,"identity":"8cbd2d20-5427-4478-8c82-a86599c19c41","added_by":"auto","created_at":"2025-06-30 14:00:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":223987,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of experimental treatments on Z-Score transformed AGB, BGB, and TB. a, AGB under reseeding; b, BGB under reseeding; c, TB under reseeding; d, AGB under fertilization; e, BGB under fertilization; f, TB under fertilization; g, AGB under \u003cem\u003eBurkholderia\u003c/em\u003e sp. addition; h, BGB under \u003cem\u003eBurkholderia\u003c/em\u003e sp. addition; i, TB under \u003cem\u003eBurkholderia\u003c/em\u003e sp. addition. Linear mixed-effects models were applied to calculate the estimated effect size. Wald type II χ2 tests are applied to determine statistical significance. The estimated effect sizes are represented as mean values ± standard errors. Significant effects are denoted by asterisks: * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6968136/v1/e89f6cc5edf234327f3f663f.png"},{"id":85672466,"identity":"b9357ea0-08d9-496f-952c-5333c14c128b","added_by":"auto","created_at":"2025-06-30 14:00:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":173187,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of experimental treatments on Z-Score transformed Soil physicochemical. a, DOC under reseeding b, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N under fertilization; c, AP under fertilization. Linear mixed-effects models were applied to calculate the estimated effect size. Wald type II χ2 tests are applied to determine statistical significance. The estimated effect sizes are represented as mean values ± standard errors. Significant effects are denoted by asterisks: * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6968136/v1/6a83c0461951fbdcd7c7ada5.png"},{"id":85672468,"identity":"502c318c-b3a3-4880-ae70-3da34a26560f","added_by":"auto","created_at":"2025-06-30 14:00:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":241105,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of experimental treatments on Z-Score transformed NEE, ER, and GPP. a, NEE under reseeding; b, ER under reseeding; c, GPP under reseeding; d, NEE under fertilization; e, ER under fertilization; f, GPP under fertilization; g, NEE under \u003cem\u003eBurkholderia\u003c/em\u003e sp. addition; h, ER under \u003cem\u003eBurkholderia\u003c/em\u003e sp. addition; i, GPP under \u003cem\u003eBurkholderia\u003c/em\u003e sp. addition. Linear mixed-effects models were applied to calculate the estimated effect size. Wald type II χ2 tests are applied to determine statistical significance. The estimated effect sizes are represented as mean values ± standard errors. Significant effects are denoted by asterisks: * \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6968136/v1/f225cabd3b1fb3de50939c51.png"},{"id":85673137,"identity":"2ab654a4-84eb-4235-a0dd-887ba1afcaa4","added_by":"auto","created_at":"2025-06-30 14:08:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":127836,"visible":true,"origin":"","legend":"\u003cp\u003eRelationships between ecosystem carbon fluxes with soil physicochemical factors under different experimental treatments. a, Regression analysis of GPP and DOC under reseeding; b, Regression analysis of GPP and AP under fertilization. The grey area represents the 95% confidence interval.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6968136/v1/c5ad32e866f873fcf40be07e.png"},{"id":85672472,"identity":"49bd88f5-595d-46bf-8c5d-a52fafdc3391","added_by":"auto","created_at":"2025-06-30 14:00:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":701283,"visible":true,"origin":"","legend":"\u003cp\u003eRelationship between ecosystem carbon fluxes with grassland biomass under different experiment treatments. a, Regression analysis of NEE and TB under reseeding; b, Regression analysis of GPP and BGB under reseeding; c, Regression analysis of GPP and TB under reseeding; d, Regression analysis of NEE and TB under fertilization; e, Regression analysis of ER and BGB under fertilization; f, Regression analysis of GPP and BGB under fertilization; g, Regression analysis of GPP and TB under fertilization; h, Regression analysis of GPP and BGB under \u003cem\u003eBurkholderia\u003c/em\u003e sp. addition; i, Regression analysis of GPP and TB under \u003cem\u003eBurkholderia\u003c/em\u003e sp. addition. The grey area represents the 95% confidence interval.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6968136/v1/8ea4b7f62797e9abaeb0796a.png"},{"id":92639923,"identity":"bde32b3f-6cde-49a3-840a-8b7a2fe7da5d","added_by":"auto","created_at":"2025-10-02 07:51:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2192944,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6968136/v1/5896fea5-9d67-44ae-bd46-a313d8f3ec11.pdf"},{"id":85672473,"identity":"108f5505-2d08-4600-9cc5-aa1b83dfd915","added_by":"auto","created_at":"2025-06-30 14:00:10","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1354158,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6968136/v1/9f7a1c23f5cee01b9852ffb1.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eBoth biotic and abiotic management strategies facilitate biomass recovery and CO\u003csub\u003e2\u003c/sub\u003e absorption: evidence from three experiments in southern grasslands\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eGrasslands cover approximately 40% of terrestrial ecosystems and are critical for human life (Buisson et al., 2022). They also store one-third of the global terrestrial carbon reserves, playing an important role in mitigating climate change (Bai and Cotrufo, 2022). However, about half of the world\u0026rsquo;s grasslands have begun to deteriorate to varying degrees (Bardgett et al., 2021). Moreover, grassland degradation greatly accelerates soil CO\u003csub\u003e2\u003c/sub\u003e emissions and thus diminishes the contribution of grasslands to the carbon sink (Li et al., 2015; Abdalla et al., 2018; Xu et al., 2022). Therefore, it is important to explore the dynamics of CO\u003csub\u003e2\u003c/sub\u003e flux under various grassland restoration strategies such as reseeding, chemical and biological fertilization, which help to understand the function of ecosystem carbon sink in grasslands.\u003c/p\u003e \u003cp\u003eReseeding is one of simple and feasible grassland restoration techniques (Mi et al., 2024), which improves grassland community productivity by seeding forage seeds directly on degraded grasslands (Li et al., 2017). On the one hand, reseeding can increase vegetation coverage, biomass, and the proportion of high-quality forage in degraded grasslands (Yang et al., 2018). On the other hand, tillage-based reseeding can also enhance the nutritional content of degraded grasslands (e.g., soil carbon) (Ji et al., 2022; Zhang et al., 2022). Different seed selections have various effects on the grassland yield, with grass-legume mixtures often producing higher productivity compared to monocultures (Finn et al., 2013). Therefore, reseeding restoration of degraded grasslands may enhance their contribution to carbon sinks. However, research on the relationship between reseeding restoration and CO\u003csub\u003e2\u003c/sub\u003e fluxes in degraded grassland is still limited.\u003c/p\u003e \u003cp\u003eFertilization is one of the effective methods to promote grassland restoration, which has an impact on severely degraded grasslands in the short term (Dong et al., 2020). Fertilization can significantly enhance biomass and soil nutrients in degraded grasslands (Li et al., 2023). Numerous studies have shown that nitrogen (N) addition can significantly enhance CO\u003csub\u003e2\u003c/sub\u003e absorption in grassland systems (Wu et al., 2021; Shi et al., 2022; Chen et al., 2023; Du et al., 2024; Wen et al., 2024). Phosphorus (P) is also a common limiting factor in soils (Elser et al., 2007; Fay et al., 2015), and combined nitrogen-phosphorus application typically had better restoration effects than added either nutrient alone (Wang et al., 2020). For example, NP addition experiments in temperate ecosystems suggested that NP addition increased biomass of rootlets and reduced heterotrophic respiration, thereby enhanced ecosystem carbon sequestration (Zeng and Wang, 2015). However, in grassland ecosystems, studies on fertilization and ecosystem carbon fluxes had mainly focused on nitrogen addition under the background of nitrogen deposition. Thus, it is important to explore the impact of combined nitrogen-phosphorus fertilization on carbon fluxes during grassland restoration.\u003c/p\u003e \u003cp\u003eIt is well known that \u003cem\u003eBurkholderia\u003c/em\u003e sp. has positive effects on plant growth and tolerance (Pal et al., 2022). For example, inoculation with \u003cem\u003eBurkholderia\u003c/em\u003e promoted the secretion of indole-3acetic acid (IAA) and 1-aminocyclopropane-1-carboxylate (ACC) in soybeans (\u003cem\u003eGlycine max\u003c/em\u003e) and thus enhanced plant growth (Ratnaningsih et al., 2023); \u003cem\u003eBurkholderia\u003c/em\u003e could trigger plants defense mechanisms, protecting them from pathogens (Kong et al., 2022); \u003cem\u003eBurkholderia\u003c/em\u003e could also enhance the solubility of potassium and phosphorus, improving phosphorus availability (Peix et al., 2001; Baghel et al., 2020). Additionally, \u003cem\u003eBurkholderia\u003c/em\u003e is a nitrogen-fixing bacterium that improves nitrogen uptake and utilization by plants (Reis et al., 2004). Furthermore, \u003cem\u003eBurkholderia\u003c/em\u003e can degrade various pollutants and promote plant ACC production to enhance stress resistance (Lu et al., 2012; Chen et al., 2017). Therefore, \u003cem\u003eBurkholderia\u003c/em\u003e has potential for restoring degraded grasslands and influencing carbon fluxes in such ecosystems.\u003c/p\u003e \u003cp\u003eAlthough southern grasslands only account for 20% of the total grassland area in China (Fang et al., 2018), their adequate hydrothermal conditions and strong pasture regeneration ability (Teng et al., 2008), allowed these grasslands and related agricultural products to support nearly half of the China\u0026rsquo;s livestock (Huangfu et al., 2012). However, over 70% of China\u0026rsquo;s grasslands have already been experiencing degradation (Pan et al., 2023), which also severely affected the country\u0026rsquo;s grassland carbon sink. In addition, previous research on grassland restoration focused primarily on typical grasslands in northern China, how about the restoration effect on southern grasslands is still less understood.\u003c/p\u003e \u003cp\u003eTo fill the gap, we conducted the field experiments in the typical southern grasslands, Yunnan Province (Fang et al., 2018) and faces grassland degradation and soil erosion due to overgrazing (Deng, 2023). Therefore, we carried out a 3-year grassland restoration experiments by reseeding, inputting nitrogen-phosphorus chemical fertilization and adding biological organic fertilization with \u003cem\u003eBurkholderia\u003c/em\u003e sp. We monitored grassland biomass, carbon effluxes, and their driving factors in order to answer the following hypotheses: (1) If management practices can promote plant growth, reseeding, fertilization, and addition \u003cem\u003eBurkholderia\u003c/em\u003e sp. will enhance carbon sequestration. (2) Grassland degradation in southern regions is often driven by overgrazing, which leads to reduced community productivity and nutrient losses (Wang et al., 2015). Therefore, we hypothesize that soil nutrients after management practices, particularly soil phosphorus will be more important for improving grassland function.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Site description and Experimental design\u003c/h2\u003e \u003cp\u003eThe experiments were established in September 2020 at the Field Grassland Experiment Platform in Maolin Town, Zhaotong City, Yunnan Province (27\u0026deg;66\u0026prime; N, 103\u0026deg;63\u0026prime; E). The area is characterized by a plateau monsoon climate, where warm temperate and subtropical conditions coexist. The annual average temperature is 12.6\u0026deg;C, and the average precipitation is 760 mm. The region experiences frequent rain, snow, and fog throughout the year. The dominant species are \u003cem\u003eDactylis glomerata\u003c/em\u003e, \u003cem\u003eLolium perenne\u003c/em\u003e and \u003cem\u003eTrifolium repens\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe experiment was conducted from April 2021 to September 2023. Based on local vegetation surveys, \u003cem\u003eTrifolium repens\u003c/em\u003e and \u003cem\u003eDactylis glomerata\u003c/em\u003e were selected as the reseeding species. The reseeding treatments included CK (no reseeding) and a seeding rate of 22.50 kg hm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, with three different species combinations: D (monoculture of \u003cem\u003eDactylis glomerata\u003c/em\u003e), T (monoculture of \u003cem\u003eTrifolium repens\u003c/em\u003e), and DT (mixed \u003cem\u003eTrifolium repens\u003c/em\u003e and \u003cem\u003eDactylis glomerata\u003c/em\u003e at 2:3). Five replicates were set for each treatment, resulting in 20 plots in total. The seeds are sown in April each year for a total of three times. To minimize soil disturbance, no tillage reseeding was used.\u003c/p\u003e \u003cp\u003eThe fertilization treatments included CK (no fertilization) and three levels of nitrogen and phosphorus fertilization: NP\u003csub\u003eL\u003c/sub\u003e (2 g m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e year\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e nitrogen\u0026thinsp;+\u0026thinsp;0.7 g m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e year\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e phosphorus), NP\u003csub\u003eM\u003c/sub\u003e (4 g m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e year\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e nitrogen\u0026thinsp;+\u0026thinsp;1.4 g m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e year\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e phosphorus), and NP\u003csub\u003eH\u003c/sub\u003e (8 g m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e year\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e nitrogen\u0026thinsp;+\u0026thinsp;2.8 g m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e year\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e phosphorus). The nitrogen fertilizer used was [CO(NH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e] and the phosphorus fertilizer was [Ca(H\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;H\u003csub\u003e2\u003c/sub\u003eO]. Five replicates were set for each treatment.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eBurkholderia\u003c/em\u003e sp. treatments included three application levels: B\u003csub\u003e30\u003c/sub\u003e (30 g m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e year\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), B\u003csub\u003e60\u003c/sub\u003e (60 g m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e year\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and B\u003csub\u003e120\u003c/sub\u003e (120 g m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e year\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). The same CK were used for fertilization treatment. Five replicates were set for each treatment, resulting in 35 plots both \u003cem\u003eBurkholderia\u003c/em\u003e sp. treatments and fertilization treatments. Each plot was 3 m \u0026times; 3 m, with a 1 m buffer zone among them. Fertilization and \u003cem\u003eBurkholderia\u003c/em\u003e sp. application were carried out in April and October each year, with a total of five times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Measurement of ecosystem CO\u003csub\u003e2\u003c/sub\u003e flux, soil temperature, and soil moisture\u003c/h2\u003e \u003cp\u003eWe measured soil carbon flux data during the growing season (April to September) from June 2021 to September 2023. Measurements were taken between 9:00 AM and 12:00 AM on clear days. Using a transparent assimilation tank (a cylinder with a base diameter of 20cm and a height of 25 cm made of plexiglass.) connected to the Li-8100a Automated Soil CO\u003csub\u003e2\u003c/sub\u003e Flux System monitored net ecosystem exchange of CO\u003csub\u003e2\u003c/sub\u003e (NEE) and ecosystem respiration (ER), and each measurement lasted 90 seconds (Due to COVID-19 restrictions in September 2022 and rainy weather during fertilization treatment in August 2023, data for these months were missed). After measuring NEE, the system was ventilated and purged for 30 seconds, and then blackout treatment was performed to measure ER (Niu et al., 2008). Gross primary productivity (GPP) was calculated using the formula GPP\u0026thinsp;=\u0026thinsp;ER - NEE (Ma et al., 2021). During CO\u003csub\u003e2\u003c/sub\u003e flux measurements, soil temperature (ST) and soil water content (SWC) were measured from 0\u0026ndash;10 cm using the soil temperature and soil water content probes included with the Li8100a (SWC data for April 2022 and ST data for July 2023 were missing because the probes were damaged).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Sampling and measurement of grassland biomass and physicochemical properties\u003c/h2\u003e \u003cp\u003eIn October 2022, above-ground biomass (AGB) and below-ground biomass (BGB) were collected from each plot. Three 20 cm \u0026times; 20 cm subplots were randomly selected from each plot. The above-ground parts of all plants were collected using scissors and dried to a constant weight to determine AGB. Below-ground parts were collected using a 6 cm diameter soil auger with five sampling points at a depth of 0\u0026ndash;20 cm. Roots were separated from the soil. Then they were cleaned and dried to a constant weight to determine BGB. Total biomass (TB) was sum of AGB and BGB. After separating the roots and rocks, soil samples were divided into two portions. One portion was analyzed using Cleverchem380 (DeChem-Tech GmbH, Hamburg, Germany) to measure ammonium nitrogen (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N) by Sodium Salicylate Method, nitrate nitrogen (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N) by Copper-zinc hydrazine reduction method, and available phosphorus (AP) by Olsen method, while another portion was analyzed with vario TOC cube (Elementar Analysensysteme GmbH, Langenselbold, Germany) to determine dissolved organic carbon (DOC) by high temperature catalytic combustion method, microbial biomass carbon (MBC) by fumigation method, and soil organic carbon (SOC) by dry combustion. All the above soil analysis methods were used from Liu (1996).\u003c/p\u003e \u003cp\u003eData were organized and Z-score transformations were performed using Microsoft Office Excel 2010. Statistical analysis and graphical visualizations were conducted using R 4.3.3. The effects of treatments on CO\u003csub\u003e2\u003c/sub\u003e flux, grassland biomass, and soil physicochemical properties were analyzed using the lme4 package. Fixed effects included D, T, DT, B\u003csub\u003e30\u003c/sub\u003e, B\u003csub\u003e60\u003c/sub\u003e, B\u003csub\u003e120\u003c/sub\u003e, NP\u003csub\u003eL\u003c/sub\u003e, NP\u003csub\u003eM\u003c/sub\u003e, and NP\u003csub\u003eH\u003c/sub\u003e, while random effects were block and sampling time (for grassland biomass and soil physicochemical properties, only block was treated as a random effect). A linear regression analysis was used to examine the relationship between carbon flux and soil physicochemical properties and biomass.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Responses of plant biomass and soil physicochemical properties\u003c/h2\u003e \u003cp\u003eReseeding treatments had non-significant effect on AGB (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), but D had a significant positive effect on both BGB (β\u0026thinsp;=\u0026thinsp;1.381, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.033) and TB (β\u0026thinsp;=\u0026thinsp;1.404, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.027) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Except for NP\u003csub\u003eM\u003c/sub\u003e, fertilization treatments had a positive effect on AGB, among which NP\u003csub\u003eH\u003c/sub\u003e (β\u0026thinsp;=\u0026thinsp;1.387, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.023) showing significant positive effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Additionally, NP\u003csub\u003eL\u003c/sub\u003e (β\u0026thinsp;=\u0026thinsp;1.398 \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.025) and NP\u003csub\u003eM\u003c/sub\u003e (β\u0026thinsp;=\u0026thinsp;1.305, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.035) had a significant positive effect on BGB (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee), while NP\u003csub\u003eL\u003c/sub\u003e (β\u0026thinsp;=\u0026thinsp;1.362 \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.029) and NP\u003csub\u003eH\u003c/sub\u003e (β\u0026thinsp;=\u0026thinsp;1.354 \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.030) significantly increased TB (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). \u003cem\u003eBurkholderia\u003c/em\u003e sp. treatments showed no significant effect on AGB (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg), but B\u003csub\u003e60\u003c/sub\u003e had a significant positive impact on both BGB (β\u0026thinsp;=\u0026thinsp;1.519 \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.018) and TB (β\u0026thinsp;=\u0026thinsp;1.430 \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.027) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eReseeding treatment showed a positive effect on DOC, with D (β\u0026thinsp;=\u0026thinsp;1.665 \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.008) exhibiting a significant influence (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). In the fertilization treatment, NP\u003csub\u003eH\u003c/sub\u003e (β\u0026thinsp;=\u0026thinsp;1.445 \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.006) had a significant positive effect on NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). All fertilization levels significantly increased AP (β\u0026thinsp;=\u0026thinsp;1.440 to 1.692, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001 to \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). However, grassland management strategies had no significant effect on other soil physical and chemical properties (Fig. S2, S3 and S4).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Responses of NEE, ER, and GPP to experimental treatments\u003c/h2\u003e \u003cp\u003eReseeding treatments had a negative effect on NEE, and the grass-legume mixture showed a significant negative effect (β = -0.383, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.020) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Reseeding treatments had non-significant effect on ER (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Reseeding treatments positively influenced GPP, where D (β\u0026thinsp;=\u0026thinsp;0.345, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.037) and DT (β\u0026thinsp;=\u0026thinsp;0.414, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.015) showed significant effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Fertilization treatments had a negative effect on NEE, among which NP\u003csub\u003eL\u003c/sub\u003e (β = -0.350, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.027) and NP\u003csub\u003eM\u003c/sub\u003e (β = -0.422, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.008) showing significant negative effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). All levels of fertilization treatments significantly affected ER (β\u0026thinsp;=\u0026thinsp;0.425 to 0.502, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.005 to \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.015) and GPP (β\u0026thinsp;=\u0026thinsp;0.418 to 0.613, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 to \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.014) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). \u003cem\u003eBurkholderia\u003c/em\u003e sp. treatments had a positive effect on NEE under B\u003csub\u003e120\u003c/sub\u003e, but B\u003csub\u003e30\u003c/sub\u003e and B\u003csub\u003e60\u003c/sub\u003e showed negative effects, of which B\u003csub\u003e60\u003c/sub\u003e (β = -0.341, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.029) being significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg). \u003cem\u003eBurkholderia\u003c/em\u003e sp. treatments had positive effects on ER (β\u0026thinsp;=\u0026thinsp;0.290 to 0.525, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001 to \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.049) and GPP (β\u0026thinsp;=\u0026thinsp;0.297 to 0.556, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001 to \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.048) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Relationships between soil physicochemical properties, biomass and CO\u003csub\u003e2\u003c/sub\u003e fluxes\u003c/h2\u003e \u003cp\u003eGPP was positively correlated with DOC under reseeding (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Under fertilization, GPP was positively correlated with AP (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). No significant correlations were observed between other physicochemical properties and CO\u003csub\u003e2\u003c/sub\u003e fluxes (Fig. S9).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUnder reseeding treatments, TB showed a negative relationship with NEE (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea), but the relationships between AGB and BGB with NEE were not significant (Fig. S10). BGB and TB were positively correlated with GPP (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec), and no significant relationships were observed between ER and AGB, BGB, or TB (Fig. S10). Under fertilization treatments, NEE was negatively related with TB (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed), while its relationship with AGB and BGB was not significant (Fig. S11). ER was positively correlated with BGB (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee), with no significant correlations observed between ER and BGB or TB (Fig. S11). BGB and TB were positively correlated with GPP (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg). Under \u003cem\u003eBurkholderia\u003c/em\u003e sp. additions, the effects of AGB, BGB, and TB on NEE and ER could not be determined (Fig. S12). However, BGB and TB were positively correlated with GPP (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ei).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Effects of different managements on soil properties and grassland biomass\u003c/h2\u003e \u003cp\u003eOur results showed that reseeding improved the AGB, BGB, and TB of degraded grasslands, and D improved the BGB and TB significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This effect may be due to reseeding increasing the number of viable seeds in the soil seed bank, thereby enhancing grassland productivity (Li et al., 2017). Similar findings have been observed in northern grasslands, including the Loess Plateau (Liu et al., 2023c), Mongolian Plateau (Zhang et al., 2024), and Qinghai-Tibet Plateau (Li et al., 2024). However, unlike previous studies (Yan et al., 2022), our results indicated that the biomass-promoting effect of grass-legume mixtures (DT) is weaker than that of D. This may be because, in DT, \u003cem\u003eDactylis glomerata\u003c/em\u003e allocates part of its nutrients to counteract the allelopathic effects of \u003cem\u003eTrifolium repens\u003c/em\u003e (Zhang et al., 2020), thereby reducing its biomass accumulation.\u003c/p\u003e \u003cp\u003eIn addition, our data showed that fertilization significantly increased grassland biomass (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), similarly with results from the Mongolian Plateau's typical steppes and the alpine meadows of the Qinghai-Tibet Plateau (Xu et al., 2015; Xiao et al., 2020). Fertilization usually enhanced soil nutrient levels and promoted the recovery of grassland productivity (Zong and Shi, 2019). Furthermore, similar to pot experiment results (da Costa et al., 2020), our study found that adding B\u003csub\u003e60\u003c/sub\u003e significantly increased BGB and TB (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei). However, addition of \u003cem\u003eBurkholderia\u003c/em\u003e sp. had a significant impact on AGB (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg). On the one hand, \u003cem\u003eBurkholderia\u003c/em\u003e sp. can promote plant growth by fixing nitrogen and resolving phosphorus. On the other hand, \u003cem\u003eBurkholderia\u003c/em\u003e sp. can produce auxin to stimulate plant root growth (Zhang et al., 2021), so that grass biomass can be transferred to the ground.\u003c/p\u003e \u003cp\u003eCombining the three management methods, we found that all the methods had a positive effect on the TB of degraded southern grasslands, which supported our hypothesis 1. However, only the NP\u003csub\u003eH\u003c/sub\u003e treatment significantly increased AGB (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), while the other treatments increased TB primarily by enhancing BGB (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The intense grazing pressure in southern grasslands in China leads to the loss of viable grass seeds and the decline in soil nutrients. Plants general allocate more biomass to BGB, thus promoting plant access to soil nutrients (Hermans et al., 2006; Freschet et al., 2018).\u003c/p\u003e \u003cp\u003eThe analysis of soil properties in our experiment showed that reseeding increased DOC (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), which was different from the findings from the Qinghai-Tibet Plateau (Wang et al., 2020), our results suggest that reseeding increased the active seed, which enhanced grass biomass and subsequently contributed to greater inputs of plant residues and rhizospheric carbon. Furthermore, consistent with previous studies (Xiao et al., 2021; Liu et al., 2023a), we found that fertilization had a significant positive effect on NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N and AP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). However, AP levels were generally low across all treatments (5.82 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 12.98 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (Fig. S6, S7, and S8). This suggests that AP deficiency may be a key factor coursing the southern grassland degradation. For example, our reseeding treatments seem to cause AP depletion (Fig. S6)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Effects of different managements on carbon fluxes and potential drivers\u003c/h2\u003e \u003cp\u003eWe found that all treatments had a positive effect on GPP in the grassland, while all except B\u003csub\u003e120\u003c/sub\u003e had a negative effect on NEE (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This result contradicted the hypothesis 1. Specifically, under reseeding treatment, our results showed that the grass-legume mixed reseeding had a significant positive effect on GPP (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec) and a significant negative effect on NEE (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). The result was similar to the studies conducted in the eastern Pyrenees, because reseeding can restore biomass and promote carbon sequestration (Drewer et al., 2017; Iba\u0026ntilde;ez et al., 2021). Additionally, studies on the Qinghai - Tibet Plateau have shown that NP fertilizer co-application significantly reduced NEE by 75.7% and increased GPP by 41.4% (Li et al., 2024). And the research on Inner Mongolia grasslands has revealed that fertilization significantly increases GPP and ER in the first two years, while significantly decreasing NEE (Niu et al., 2010). These findings are consistent with our results, indicating fertilization had a significant positive effect on both ER and GPP (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef) and a significant negative effect on NEE (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). The reason should be that fertilization promoted the growth of grassland and microbial activity as shown in our results and other studies (Chu et al., 2007; Zhang et al., 2024). The addition of \u003cem\u003eBurkholderia\u003c/em\u003e sp. had a significant positive effect on ER and GPP (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei). B\u003csub\u003e60\u003c/sub\u003e and B\u003csub\u003e30\u003c/sub\u003e showed a negative effect on NEE, and B\u003csub\u003e60\u003c/sub\u003e had a significant effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg). This suggests that \u003cem\u003eBurkholderia\u003c/em\u003e sp. promoted CO\u003csub\u003e2\u003c/sub\u003e uptake by plants but also promoted CO\u003csub\u003e2\u003c/sub\u003e emissions through its own respiration, leading to a positive effect on ER. We proposed that at 30 g m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e year\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the enhancement of CO\u003csub\u003e2\u003c/sub\u003e absorption by the ecosystem is limited, while at 60 g m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e year\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, it is most effective. However, at 120 g m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e year\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the respiration of \u003cem\u003eBurkholderia\u003c/em\u003e sp. covered the CO\u003csub\u003e2\u003c/sub\u003e absorption by plants, indicating that only lower application levels can improve degraded grasslands carbon sinks.\u003c/p\u003e \u003cp\u003eRegression analysis results showed that different experimental measures influence CO\u003csub\u003e2\u003c/sub\u003e flux through both biotic and abiotic factors. Under reseeding treatments, DOC was significantly positively correlated with GPP (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). The growth of reseeding plants resulted in an increase in root exudates and litter, which promoted the increase of DOC (Wu et al., 2013). These carbon inputs may change soil biotic and abiotic factors, leading to an increase in subsequent photosynthetic rates (van der Putten et al., 2013; Liu et al., 2023b), thereby enhancing GPP. Our results were similar to the previous studies (Shi et al., 2021), showing that AP was significantly positively correlated with GPP (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Phosphorus deficiency can disrupt the balance between the carboxylation capacity of leaves and the electron transport capacity supporting ribulose-1, 5-bisphosphate regeneration, thereby reducing photosynthesis (Ellsworth et al., 2022).\u003c/p\u003e \u003cp\u003eOur results showed strong relationships between biomass and GPP, ER and NEE (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), which were similar to previous studies (Wu et al., 2015; Oliver et al., 2019; Wang et al., 2021). Increased biomass is thought to improve GPP by increasing the leaf area index, which improves the plant's ability to capture light (Chu et al., 2019). However, unlike our results, GPP was significantly positively correlated with BGB and TB under all grassland restoration measures (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), but not with AGB. This may be due to sampling at the end of the growing season, when plants transferred more biomass to the belowground (Garten et al., 2011). Additionally, our results showed that ER was significantly positively correlated with BGB only under fertilization treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). This is because nitrogen application not only negatively affects microbial abundance and composition, thereby reducing soil microbial respiration (Zhang et al., 2018), but also promotes root growth, which enhances autotrophic respiration (Gough and Seiler, 2004). Consequently, this strengthens the correlation between BGB and ER. Therefore, plant factors contributed more to ER under fertilization, but \u003cem\u003eBurkholderia\u003c/em\u003e sp. addition likely affected soil microorganisms, masking the plant's contribution to ER. Furthermore, NEE was significantly negatively correlated with TB under both reseeding and fertilization treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed), indicating that reseeding and fertilization increased the carbon sink of degraded grasslands by increasing plant biomass.\u003c/p\u003e \u003c/div\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study showed the effects of reseeding, fertilization, and the addition \u003cem\u003eBurkholderia sp.\u003c/em\u003e on biomass production, soil nutrients, and carbon fluxes in southern grasslands. All management strategies increased total biomass and gross primary productivity, but they had different effects on biomass allocation, soil nutrients, and carbon sequestration. The addition of \u003cem\u003eBurkholderia sp.\u003c/em\u003e had the strongest positive effect on below-ground biomass and total biomass, but excessive addition increased microbial respiration, thereby reducing carbon sequestration. Our study showed that phosphorus deficiency was the key factor of grassland degradation in southern China, so phosphorus fertilization had the strongest promotion effect on above-ground biomass and carbon sequestration. Reseeding increased biomass, dissolved organic carbon, and carbon sequestration, but it also exacerbated phosphorus limitation, which could lead to further grassland degradation with long-term use. Overall, future grassland managements should synthetically consider these strategies to enhance soil nutrients, increase biomass, and strengthen the carbon sink capacity in southern grasslands.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFundding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Key R\u0026amp;D Program of China (No. 2023YFC2604502), the National Natural Science Foundation of China (32371733), the Xingdian Scholar Fund of Yunnan, the Project for Talent and Platform of Science and Technology in Yunnan Province Science and Technology Department (202205AM070005), the Agricultural joint fund (surface project) from science and technology department of Yunnan Province (No.202301BD070001-096).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship contribution\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXinghong Wang: Writing\u0026ndash; review \u0026amp; editing. Meiyan Zhang: Writing\u0026ndash; review \u0026amp; editing. Yun Liu: Writing\u0026ndash; review \u0026amp; editing. Juan Zhou: Writing\u0026ndash; review \u0026amp; editing. Meng Xia: Writing \u0026ndash; review \u0026amp; editing. Sicheng Li: Writing \u0026ndash; review \u0026amp; editing, Investigation. Haibian Xu: Writing \u0026ndash; review \u0026amp; editing, Data curation. Yan Li: Writing \u0026ndash; review \u0026amp; editing, Investigation, Data curation. Yi Xiong: Writing \u0026ndash; original draft, Visualization, Validation, Methodology, Investigation, Data curation. Jinghang Xu: Writing \u0026ndash; review \u0026amp; editing, Writing \u0026ndash; original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation. Janping Wu: Writing \u0026ndash; review \u0026amp; editing, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Data curation, Conceptualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated by the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of generative AI and AI-assisted technologies in the writing process.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatement: During the preparation of this work the authors used ChaGPT 4 in order to improve the readability and language. 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Sustainability 11: 106361. https://doi.org/10.3390/su11072023.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Grassland restoration, CO2 flux, Soil respiration, Carbon sequestration","lastPublishedDoi":"10.21203/rs.3.rs-6968136/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6968136/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe southern grasslands of China sustain substantial livestock but face functional degradation due to overuse. The carbon flux responses to restoration strategies remain unclear.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eIn this study, we implemented a three-year restoration experiment in artificial grasslands using three approaches: reseeding (D: \u003cem\u003eDactylis glomerata\u003c/em\u003e monoculture, T: \u003cem\u003eTrifolium repens\u003c/em\u003e monoculture, DT: their mixture), chemical fertilization (NP\u003csub\u003eL\u003c/sub\u003e/NP\u003csub\u003eM\u003c/sub\u003e/NP\u003csub\u003eH\u003c/sub\u003e: nitrogen 2/4/8 g m⁻\u0026sup2; + phosphorus 0.7/1.4/2.8 g m⁻\u0026sup2;), and biofertilization with \u003cem\u003eBurkholderia\u003c/em\u003e sp.(B\u003csub\u003e30\u003c/sub\u003e/B\u003csub\u003e60\u003c/sub\u003e/B\u003csub\u003e120\u003c/sub\u003e: 30/60/120 g m⁻\u0026sup2;).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eAll treatments increased grassland biomass. Specifically, NP\u003csub\u003eH\u003c/sub\u003e significantly enhanced above-ground biomass, while D, B\u003csub\u003e30,\u003c/sub\u003e and NP\u003csub\u003eL\u003c/sub\u003e notably boosted below-ground biomass. D, NP\u003csub\u003eL\u003c/sub\u003e, NP\u003csub\u003eH\u003c/sub\u003e, and B\u003csub\u003e30\u003c/sub\u003e significantly increased total biomass. Restoration treatments usually had a negative effect on NEE, with DT (β = -0.383, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.020), NP\u003csub\u003eL\u003c/sub\u003e (β = -0.350, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.027), NP\u003csub\u003eM\u003c/sub\u003e (β = -0.422, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.008), and B\u003csub\u003e60\u003c/sub\u003e (β = -0.341, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.029) showing significant effects. However, they exhibited positive effects on both ER and GPP. Specifically, NP\u003csub\u003eL\u003c/sub\u003e, NP\u003csub\u003eM\u003c/sub\u003e, NP\u003csub\u003eH\u003c/sub\u003e, B\u003csub\u003e30\u003c/sub\u003e, B\u003csub\u003e60\u003c/sub\u003e, and B\u003csub\u003e120\u003c/sub\u003e significantly enhanced ER (β\u0026thinsp;=\u0026thinsp;0.290 to 0.525, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001 to 0.049), while all measures except T significantly increased GPP (β\u0026thinsp;=\u0026thinsp;0.297 to 0.613, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 to 0.048). Dissolved organic carbon, available phosphorus, and biomass contributed to the changes in carbon sequestration.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOur results demonstrate that both traditional fertilization and alternative strategies like reseeding and biofertilization can effectively restore grassland productivity and carbon sequestration capacity, providing multiple pathways for sustainable grassland management.\u003c/p\u003e","manuscriptTitle":"Both biotic and abiotic management strategies facilitate biomass recovery and CO2 absorption: evidence from three experiments in southern grasslands","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-30 14:00:05","doi":"10.21203/rs.3.rs-6968136/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"534887d5-0ce5-45fd-9192-b492b5bcfb93","owner":[],"postedDate":"June 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-02T07:43:06+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-30 14:00:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6968136","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6968136","identity":"rs-6968136","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}