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  • RESEARCH ARTICLE
    Bianka Guadalupe Salas-Enriquez, Héctor Iván Bedolla-Rivera, María de la Luz Xochilt Negrete-Rodríguez, Aidé Minerva Torres-Huerta, Miguel Antonio Domínguez-Crespo, Ángeles Iveth Licona-Aguilar, Eloy Conde-Barajas
    Soil Ecology Letters, 2024, 6(4): 240234. https://doi.org/10.1007/s42832-024-0234-6

    ● Biosolids boost OM mineralization, enhancing soil health.

    ● Moderate biosolid doses improve soil conditions effectively.

    SQI w, with Nmin, efficiently gauges soil quality, simplifying monitoring.

    Economic development triggers environmental pollution. To address this issue and mitigate its consequences on the environment and human health, urban wastewater treatment plants are commonly employed to produce treated water and biosolids. However, biosolid disposals pose issues due to space limits and leachate contamination. This study investigates the potential of using biosolids as an organic amendment to remediate soil contaminated with leachate from an open dump in Mexico. Treatments with different doses of biosolids were tested (control, without addition of biosolids; high, medium, and low doses, with a C/N = 8, 10, 12 respectively). The physicochemical and biological characteristics of the soil and biosolids were analyzed, and the dynamics of carbon and nitrogen mineralization over time were studied. The developed soil quality index, primarily based on the mineralized nitrogen indicator, differentiated soil quality among treatments, showing values of moderate quality for the treatments (high, medium, and low doses (0.56, 0.48, 0.40, respectively) and low quality for the control (0.34)). The use of biosolids as an organic amendment improved soil quality by increasing organic matter and microbial growth. Soil quality indices emerges as a practical tool for monitoring the remediation of leachate-contaminated open dump soils in Mexico and similar contexts worldwide.

  • RESEARCH ARTICLE
    Shuobing He, Yuying Ma, Teng Yang, Xiao Fu, Li Nie, Jiasui Li, Daozhong Wang, Yanhua Su, Haiyan Chu
    Soil Ecology Letters, 2024, 6(4): 240227. https://doi.org/10.1007/s42832-024-0227-5

    ● Soil pH was the key factor influencing the phoD -harboring bacterial networks.

    ● Identification of a cluster positively linked to ALP activity and plant P uptake.

    ● Low soil pH resulted in a severe loss of phoD -harboring bacterial core cluster.

    Fertilization treatments profoundly influence the bacterial communities associated with soil organic phosphorus (P) mineralization and alkaline phosphatase (ALP) activity. However, the relationships among the phoD-harboring bacterial communities associated with soil organic P mineralization, soil ALP activity, and plant P uptake under long-term fertilization remain unexplored. This study investigated these associations at the wheat rapid growth stage in a 40-year fertilization experiment. NPK fertilization led to a significant decrease in the diversity of phoD-harboring bacteria, which could be partially mitigated by the addition of organic materials. Soil pH emerged as the key factor influencing the structure and diversity of the phoD-harboring bacterial community. Furthermore, fertilizations involving manure additions resulted in more stable and cooperative phoD-harboring bacterial co-occurrence networks, compared to NPK fertilization. A functional phoD-harboring bacterial cluster, comprising genera Nostoc, Bradyrhizobium, and Pseudomonas, was identified, showing a positive association with soil ALP activity and plant P uptake. In summary, our study highlights the significant role of the identified core cluster of phoD-harboring bacteria in maintaining soil ALP activity and promoting plant P uptake, in decades of fertilization. Moreover, this study inferred a list of phoD-harboring bacterial genera from the core cluster, with established links to both plant P uptake and soil organic P mineralization. These findings offer valuable insights for sustainable agricultural practices.

  • RESEARCH ARTICLE
    Li Chen, Xiaosan Luo, Haoran He, Ting Duan, Ying Zhou, Lequn Yang, Yi Zeng, Hansong Chen, Linchuan Fang
    Soil Ecology Letters, 2024, 6(4): 240233. https://doi.org/10.1007/s42832-024-0233-7

    ● Agricultural activities may promote the conversion of inorganic Hg to MeHg in soil.

    ● Hg and As present an extremely and a moderately contaminated level, respectively.

    ● The human health risks posed by As, Hg, and Ni merit more attention.

    ● Pokeweed may be considered as a potential Hg hyperaccumulator.

    Soil pollution caused by potentially toxic metal(loid)s (PTMs) near mercury (Hg) mines has attracted extensive attention, yet the status and potential health risks of PTM contamination in soils near Hg mining sites have rarely been investigated on a large scale. Global data on methylmercury (MeHg), Hg, Cd, Cr, As, Pb, Cu, Zn, Mn, and Ni concentrations in soils from Hg mining areas were obtained from published research articles (1999–2023). Based on the database, pollution levels, spatial distributions, and potential health risks were investigated. Results indicated that the average percentage of MeHg to total Hg in agricultural soils (0.19%) was significantly higher than that in non-agricultural soils (0.013%). Indeed, 72.4% of these study sites were extremely contaminated with Hg. Approximately 45% of the examined sites displayed a moderate level of As contamination or even more. Meanwhile, the examined sites in Spain and Turkey exhibited considerably higher pollution levels of Hg and As than other regions. The mean hazard indices of the nine PTMs were 2.91 and 0.59 for children and adults, with 85.6% and 13.3% of non-carcinogenic risks for children and adults that exceeded the safe level of 1, respectively. In addition, 70.2% and 56.7% of the total cancer risks through exposure to five carcinogenic PTMs in children and adults, respectively, exceeded the safety level. As and Hg showed a high exceedance of non-carcinogenic risks, while As and Ni were the leading contributors to carcinogenic risks. This study demonstrates the urgent necessity for controlling PTM pollution and reducing the health risks in soils near Hg mining sites and provides an important basis for soil remediation.

  • PERSPECTIVE
    Yi Zhao, Rui Xu, Siobhan F. Cox, Min Qiao, Huaming Guo
    Soil Ecology Letters, 2024, 6(4): 240244. https://doi.org/10.1007/s42832-024-0244-4

    ● Metals are increasingly important risk factors for the evolution of antibiotic resistance in environments.

    The rapid development of antibiotic resistance is occurring at a global scale. We therefore stride into the post-antibiotic era and have to battle antibiotic resistance in the Anthropocene. Metals are widely used and their pollution is widespread worldwide. More importantly, metal-induced co-selection greatly expands the environmental resistomes and increases the health risk of antibiotic resistance in environments. Here, we reviewed the metal-induced co-selection and their increasingly important roles in the development of antibiotic resistance. In particular, we highlight the metal-rich environments that maintain reservoirs for high-risk antibiotic resistance genes with horizontally transferable potentials. We also call for considerations and further investigations of other co-selective agents and the efficacy of metal-based interventions to better manage and combat the global antibiotic resistance crisis within the One Health framework.

  • RESEARCH ARTICLE
    Tessa Camenzind, Heike Haslwimmer, Matthias C. Rillig, Liliane Ruess, Damien R. Finn, Christoph C. Tebbe, Stefan Hempel, Sven Marhan
    Soil Ecology Letters, 2024, 6(4): 240243. https://doi.org/10.1007/s42832-024-0243-5

    ● Refined conversion factors for soil fungal biomarkers are proposed.

    ● High interspecific variability is present in all fungal biomarkers.

    ● A modeling approach supports the validity of biomarker estimates in diverse soils.

    ● ITS1 copies vary strongly, but are fungal-specific with least phylogenetic bias.

    ● A combination of fungal biomarkers will reveal soil fungal physiology and activity.

    The abundances of fungi and bacteria in soil are used as simple predictors for carbon dynamics, and represent widely available microbial traits. Soil biomarkers serve as quantitative estimates of these microbial groups, though not quantifying microbial biomass per se. The accurate conversion to microbial carbon pools, and an understanding of its comparability among soils is therefore needed. We refined conversion factors for classical fungal biomarkers, and evaluated the application of quantitative PCR (qPCR, rDNA copies) as a biomarker for soil fungi. Based on biomarker contents in pure fungal cultures of 30 isolates tested here, combined with comparable published datasets, we propose average conversion factors of 95.3 g fungal C g−1 ergosterol, 32.0 mg fungal C µmol−1 PLFA 18:2ω6,9 and 0.264 pg fungal C ITS1 DNA copy−1. As expected, interspecific variability was most pronounced in rDNA copies, though qPCR results showed the least phylogenetic bias. A modeling approach based on exemplary agricultural soils further supported the hypothesis that high diversity in soil buffers against biomarker variability, whereas also phylogenetic biases impact the accuracy of comparisons in biomarker estimates. Our analyses suggest that qPCR results cover the fungal community in soil best, though with a variability only partly offset in highly diverse soils. PLFA 18:2ω6,9 and ergosterol represent accurate biomarkers to quantify Ascomycota and Basidiomycota. To conclude, the ecological interpretation and coverage of biomarker data prior to their application in global models is important, where the combination of different biomarkers may be most insightful.

  • RESEARCH ARTICLE
    Haoran Mao, Guilong Li, Ke Leng, Luyuan Sun, Kailou Liu, Yongxin Lin, Jia Liu, Xingjia Xiang
    Soil Ecology Letters, 2024, 6(4): 240241. https://doi.org/10.1007/s42832-024-0241-7

    ● Core taxa play an important role in regulating soil carbon metabolism.

    ● Ecological cluster with oligotrophic made key contributions to soil carbon metabolism.

    ● Microbial cluster characteristics link microorganisms to carbon metabolism.

    Characterizing the ecological roles of core soil microbial species in soil carbon metabolism is critically important for enhancing carbon sequestration in agricultural systems; however, no studies to date have determined the effects of core soil microbial taxa on carbon metabolism under various long-term fertilization practices. Here, we collected soil samples from field plots that had been subjected to different fertilization practices for nearly 30 years and examined the long-term effects of fertilization on the preferences of core soil bacterial taxa for different carbon sources. We also examined the relative contribution of core soil bacterial taxa in utilization of different carbon source types in Biolog Eco microplates. Long-term fertilization treatment had a significant effect on soil properties and bacterial community structure. The core taxa were closely related to soil carbon source utilization. The co-occurrence network showed that the major ecological clusters containing core taxa made key contributions to soil carbon source utilization. The organic fertilization increased the abundance of a core cluster with a low weighted average rrn copy number. This ecological cluster was the most important factor affecting soil carbon source utilization even among soil physicochemical factors considered. Our findings indicate that core taxa characterized by oligotrophic bacteria have a major effect on carbon source utilization in Ultisols.

  • RESEARCH ARTICLE
    Maria Ludovica Saccà, Caputo Francesco, Ceotto Enrico, Fornasier Flavio
    Soil Ecology Letters, 2024, 6(4): 240238. https://doi.org/10.1007/s42832-024-0238-2

    ● Soils from Poplar, Willow, Black locust plantations were compared to arable soil.

    ● Among five tested C cycle functional genes, three discriminated between treatments.

    ● Fungi contributed more than bacteria to the β-glucosidase enzyme activity.

    ● Fungal β-glucosidase gene may be considered an indicator of increased C storage.

    Soil carbon sequestration is regulated by microbial extracellular enzymes. Insight into this process can be gained by studying the relationship between enzyme activity, soil organic carbon and microbial functional genes. The genetic potential of microorganisms to produce carbon cycling enzymes was evaluated in unmanaged plantations of Poplar, Willow, and Black locust, compared with a nearby arable soil. Bacterial and fungal functional genes encoding for cellulase, endoglucanase, endoxylanase and β-glucosidase enzymes were quantified by real-time PCR. The abundance of three out of five genes differed between the treatments. The fungal gene encoding β-glucosidase contributed to the corresponding enzyme activity more than the bacterial one, as evidenced by a positive correlation between gene abundance and enzyme activity (r = 0.42). This gene exhibited a positive correlation with soil organic carbon content (r = 0.42), with higher values in Willow (9 × 102 gene copies µL−1 and 1.4% SOC). These results suggest that the fungal β-glucosidase gene abundance can be regarded as an indicator of increased carbon storage, similarly to the corresponding enzyme activity. The integrated analysis of soil carbon enzyme activities and DNA-based techniques enhanced our comprehension of carbon dynamics by revealing distinct contributions of microbial taxonomic groups to carbon accrual.

  • RESEARCH ARTICLE
    Shan Xu, Xiaoyu Song, Hui Zeng, Junjian Wang
    Soil Ecology Letters, 2024, 6(4): 240237. https://doi.org/10.1007/s42832-024-0237-3

    ● Boreal and temperate forests had higher MNC and FNC/BNC than other forest biomes.

    ● Mixed forests had higher MNC and lower FNC/BNC than other forest types.

    ● The dependence of MNC on forest type varied among forest biomes.

    ● MAT and soil total N were the important factors on MNC and MNC/SOC.

    ● MAT, soil pH, and clay content were identified as direct factors on FNC/BNC.

    Soil microbial necromass carbon (MNC) is an important contributor to soil organic carbon (SOC) and plays a vital role in carbon sequestration and climate change mitigation. However, it remains unclear whether the content, contribution to SOC (MNC/SOC), and fungal-to-bacterial necromass carbon ratio (FNC/BNC) of MNC vary across forest biomes and types. By summarizing data from 1704 points across 93 forest sites, we explored the spatial patterns of MNC, MNC/SOC, and FNC/BNC in the surface layer of 0–20 cm of forest soils, as well as the controlling factors involved. Overall, boreal and temperate forests had higher MNC and FNC/BNC values than tropical, subtropical, and Mediterranean forests, whereas both boreal and Mediterranean forests had low MNC/SOC values. Mixed forests had higher MNC and lower FNC/BNC than broadleaved and coniferous forests, whereas MNC/SOC was higher in broad-leaved forests than that in coniferous forests. Interestingly, the dependence of MNC on forest type also varies among forest biomes. Regression analyses identified soil total N as one of the most important factors affecting MNC and MNC/SOC; whereas MAT, soil pH, and clay content were identified as the important factors affecting FNC/BNC. This synthesis is critical for managing soil MNC to mitigate climate change in forests.

  • RESEARCH ARTICLE
    Zhibo Zhou, Yongyong Ding, Kexin Li, Fengge Zhang
    Soil Ecology Letters, 2024, 6(4): 240236. https://doi.org/10.1007/s42832-024-0236-4

    P. frumentum biomass could be improved by appropriating returning measures.

    P. frumentum biomass was excellent in 75% alfalfa returning amount.

    ● Key species of bacteria differed among the alfalfa returning amounts

    ● The relationship of core bacteria and their potential ecological functions are more close to biomass.

    The use of green manure returning to field is a common practice in conservation tillage. However, there is limited research on how different amounts of alfalfa can affect saline-alkali soil properties, bacterial community characteristics, and subsequent productivity. In this study, five different amounts of alfalfa return were investigated to understand the biological relationships between rhizospheres soil properties, bacterial communities, potential functions, and the Purus frumentum biomass. The results showed that the biomass was highest when 75% of the alfalfa was returned to the field. This particular amount was associated with relatively low soil pH and electrical conductivity. Additionally, it increased the relative abundance of beneficial bacterial taxa in both core and non-core bacteria. Statistical analysis revealed significant differences in both core (RANOSIM = 0.871, P = 0.001) and non-core (RANOSIM = 0.947, P = 0.001) bacterial communities among the different amounts of alfalfa return based on non-metric multidimensional scaling analysis. Core bacterial taxa and their potential ecological functions were more closely related to plant biomass compared to non-core bacteria based on correlation analysis and multiple regression analysis. Therefore, our results indicate that optimizing the amount of alfalfa return can improve subsequent plant biomass. Regulating soil physicochemical properties and influencing core microbial community structure are of great significance for soil functional stability and crop productivity sustainability.

  • RESEARCH ARTICLE
    An Yang, Dong Zhu, Tingting Zhu, Bowen Liu, Hongtao Wang, Yuanhu Shao, Weixin Zhang, Yulong Wang, Shenglei Fu
    Soil Ecology Letters, 2024, 6(4): 240235. https://doi.org/10.1007/s42832-024-0235-5

    ● Arsenic characteristics in earthworms and soils across 47 sites in China were studied.

    ● Earthworm tissues showed lower arsenic levels than surrounding soils.

    ● Higher arsenite to arsenate ratio was observed in earthworm tissues.

    ● Positive correlation of arsenic levels in earthworm tissues with soil nitrate.

    The total arsenic (As) and As species of earthworm body tissues and surrounding soils were investigated in 47 locations (16 forested lands and 31 agricultural lands) at a national scale across China using inductively coupled plasma-mass spectrometer (ICP-MS) and high-performance liquid chromatography-inductively coupled plasma-mass spectrometer (HPLC-ICP-MS). Earthworm body tissues had an average total As concentration of 6.21 mg kg−1, significantly lower than the soil As concentration of 12.99 mg kg−1. The ratio of arsenite to arsenate (AsIII/AsV ratio) in earthworm body tissues (67%) was significantly higher compared to that in surrounding soils (19%). HPLC-ICP-MS analysis detected small amounts of organic As forms, such as arsenobetaine (2.9%), dimethylarsinic acid (1%), and monomethylarsonic acid (0.3%), mainly in earthworm tissues from certain locations. The total As content and AsIII/AsV ratio in earthworm tissues exhibited a strong positive correlation with soil NO3 content. This field study enhances our understanding of As concentration and speciation in earthworm body tissues across China, contributing valuable insights into the biogeochemical cycle of As and its biological risks in diverse soil ecosystems. These findings provide crucial evidence for policymakers to formulate strategies addressing and mitigating soil As pollution and associated health risks.

  • RAPID REPORT
    Xianjin He, Shengjie Liu, Akihiro Nakamura, M.D. Farnon Ellwood, Shanyi Zhou, Shuang Xing, Yue Li, Dazhi Wen
    Soil Ecology Letters, 2024, 6(4): 240230. https://doi.org/10.1007/s42832-024-0230-x

    ● Ant morphological traits (dry mass, head length, body size and leg length) increased with elevation.

    ● Ant δ13C increased with elevation, whereas δ15N did not.

    ● Ant δ13C values correlated positively with soil C:N ratio.

    Understanding the responses of species to changing climates is becoming increasingly urgent. Investigating the effects of climate change on the functional traits of species at the intraspecific level is particularly important. We used elevation gradients as proxies for climate change to explore the intraspecific responses of two ground-dwelling ant species, Ectomomyrmex javanus and Odontoponera transversa, from 100 to 700 m.a.s.l. within a subtropical evergreen broadleaf forest. Our study addressed the specific relationships among environmental factors, trait variations, and trophic levels. Key functional traits such as dry mass, head length, body size, and leg length exhibited a general increase with elevation. Using stable isotope signatures (δ13C and δ15N), we quantified shifts in diets and trophic positions along the elevation gradients. Notably, our data revealed a significant elevation-related increase in Ant δ13C, whereas δ15N exhibited no such correlation. Moreover, Ant δ13C values of E. javanus demonstrated a negative correlation with mean annual temperature (MAT), and the δ13C values of both species correlated positively with soil C:N ratio. Having revealed that the individual traits and δ13C signatures of ground-dwelling ants exhibit significant negative correlations with temperature, our findings suggest that climate warming has the potential to cause intraspecific variation in the functional traits and diets of ground-dwelling ants and possibly other insect species.

  • ERRATUM
    Sabrina M. Pittroff, Stefan Olsson, Ashlea Doolette, Ralf Greiner, Alan E. Richardson, Mette Haubjerg Nicolaisen
    Soil Ecology Letters, 2024, 6(4): 240239. https://doi.org/10.1007/s42832-024-0239-1
  • RESEARCH ARTICLE
    M. Fernanda Chiappero, María V. Vaieretti, Norma Gallardo, Andrea E. Izquierdo
    Soil Ecology Letters, 2024, 6(4): 240242. https://doi.org/10.1007/s42832-024-0242-6

    ● Under warming soil respiration was higher, but soil microbial biomass was lower.

    ● Warming effect on soil respiration was higher in soil from the highest elevation.

    ● Soil respiration was higher in soil with higher soil carbon content.

    ● Warming increased biomass-specific respiration and enzyme activity.

    ● The Q 10 did not differ among soils from different elevations.

    Global warming is expected to increase the rate of soil carbon (C) efflux through enhanced soil microbial processes, mainly in systems, such as high elevation wetlands, storing large quantities of soil organic C. Here, we assessed the impact of experimental warming on respiration and microbial communities of high Andean wetland soils of the Puna region located at three different elevations (3793, 3862, 4206 m a.s.l.). We incubated soils at 10°C and 25°C for 68 days and measured the soil respiration rate and its temperature sensitivity (Q10). Furthermore, we measured biomass and composition and enzymatic activity of soil microbial communities, and initial and final soil C content. Although warming increased soil respiration rates, with more pronounced effect in soils sampled from 4206 m a.s.l., Q10 did not differ between elevations. Soil C content was higher at the highest elevation. Soil microbial biomass, but not enzymatic activity, was lower for warmed soil samples. However, the biomass-specific respiration and biomass-specific enzymatic activity were higher under warming, and in soil from the highest elevation wetland. These results suggest that, in the short-term, warming could stimulate resource allocation to respiration rather than microbial growth, probably related to a reduction in the microbial carbon use efficiency. Simultaneously, soils with higher soil C concentrations could release more CO2, despite the similar Q10 in the different wetlands. Overall, the soil of these high Andean wetlands could become C sources instead of C sinks, in view of forecasted increasing temperatures, with C-losses at regional scale.

  • REVIEW
    Hexon Angel Contreras-Cornejo, Monika Schmoll, Blanca Alicia Esquivel-Ayala, Carlos E. González-Esquivel, Victor Rocha-Ramírez, John Larsen
    Soil Ecology Letters, 2024, 6(4): 240240. https://doi.org/10.1007/s42832-024-0240-8

    ● Ascomycetes of the genus Trichoderma are beneficial fungi that promote plant growth.

    ● Several fungal species can mitigate abiotic stress in plants.

    Trichoderma spp. induce salt stress tolerance and drought protection in plants.

    ● Soil contamination by heavy metals can be bioremediated by Trichoderma .

    Trichoderma can detoxify pesticides and other pollutants in soils.

    Plants drive both carbon and nitrogen cycling and mediate complex biotic interactions with soil microorganisms. Climate change and the resulting temperature variations, altered precipitation, and water shortages in soils, affect the performance of plants. Negative effects of abiotic stress are reflected in changes of plant morphology associated with biochemical alterations and inadequate adaptation to rapid ecological change. Accumulation of chemical agents, derived from pesticides, salinity due to chemical fertilization, and accumulation of heavy metals, are recurrent problems in agricultural soils. Trichoderma spp. are soil fungi interacting with roots and in this way helping plants to cope with abiotic stresses by increasing root branching, shoot growth and productivity. In part, such fungal effects on the host plant are consequences of the activation of fine-tuned molecular mechanisms mediated by phytohormones, by profound biochemical changes that include production of osmolytes, by the activity of the redox-enzymatic machinery, as well by as complex processes of detoxification. Here, we summarize the most recent advances regarding the beneficial effects of Trichoderma in mitigating the negative effects on plant performance caused by different environmental and chemical factors associated with global change and agricultural practices that provoke abiotic stress. Additionally, we present new perspectives and propose further research directions in the field of Trichoderma-plant interactions when the two types of organism cooperate.

  • RESEARCH ARTICLE
    Li Wang, Jianhong Ji, Fei Zhou, Bibo Wu, Yingying Zhong, Lin Qi, Miao Wang, Yuping Wu, Xintao Cui, Tida Ge, Zhenke Zhu
    Soil Ecology Letters, 2024, 6(4): 240232. https://doi.org/10.1007/s42832-024-0232-8

    ● Loquat orchard location was the main driver of microbial communities and loquat fruit quality.

    ● The average fruit weight was correlated with the ɑ- and β-diversity of bacteria and protists.

    ● Soil bacterial and protistan communities drove the multiple nutrient cycling.

    The role of the soil microbiome in fruit quality within loquat orchards remains largely unknown. In this study, we collected soil samples from various loquat orchards in Ningbo, Zhejiang Province, China and investigated bacterial, fungal, and protist communities. The results showed that soil physicochemical conditions, the microbial community, and loquat fruit quality were significantly related to orchard location but unrelated to cultivation time and fertilization. The heterogeneity of the bacterial community was driven by soil pH, available phosphorus, and available potassium (AK). The fungal community was driven by soil electrical conductivity and AK. The protist community was driven by soil dissolved organic nitrogen and AK. The average fruit weight was significantly correlated with the ɑ- and β-diversity of bacteria and protists as well as the soil multiple nutrient cycling index. Several microbial phyla were related to average fruit weight, while other fruit quality indicators could not be explained by the soil microbiome. Our results reveal that bacterial and protist communities in loquat orchards drive the cycling of multiple nutrients that are related to fruit weight. These insights shed light on the relationship among the soil microbiome, nutrient cycling, and fruit quality, offering valuable scientific guidance for orchard management practices.