Microbial Stimulation in Apple Orchards of Different Ages on the Loess Plateau: Poor Predictability of Increased Soil N2O Emissions

Microbial Stimulation in Apple Orchards of Different Ages on the Loess Plateau: Poor Predictability of Increased Soil N2O Emissions | 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 Microbial Stimulation in Apple Orchards of Different Ages on the Loess Plateau: Poor Predictability of Increased Soil N2O Emissions Man Zhang, Cui Li, Weixin Wang, Xin Tong, Kaixuan Wang, Minmin Qiang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5201652/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Aims The continuously expanding apple plantation and excessive nitrogen input have made it a major source of nitrous oxide (N 2 O) emissions over the past 40 years in the Loess Plateau, China. However, the difference in N 2 O emissions from different stand ages of orchards and its key driving factors remain unclear. Methods A three-year field study was set up to evaluate the soil N 2 O emissions and the soil properties in apple orchards of two different stand ages (young orchard: 5 years and old orchard: 25 years), and soil bacteria, fungi, ammonia oxidizing bacteria (AOB) and denitrification bacteria ( nir S) were determined via amplicon sequencing. Results The higher N 2 O emissions and emission factors (EFs) were recorded in the old apple orchard under the conventional nitrogen (N) strategy. The microbial community composition in topsoil was obviously shifted by stand age (22.2% interpretation, P = 0.022) and stand age and fertilization also had a combined effect (36.6% interpretation, P = 0.003). The relative abundances of Firmicutes and Basidiomycota involved in the decomposition of plant residues increased with stand age. Nonetheless, N 2 O fluxes were not significantly correlated with soil nitrifiers and denitrifiers, but were strongly correlated with NO 3 − -N, NH 4 + -N, soil moisture and enzyme activity. In general, abiotic factors, especially mineral N availability, resulted in differences in N 2 O emissions between orchards of different stand ages. Conclusions The selection of future N 2 O emissions mitigation strategies for apple orchards should take into account both nonbiological processes and biological processes, and the assessment of N 2 O emissions in apple orchards should consider stand age. apple orchard N2O emission stand ages microbial community nitrification denitrification Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Agricultural soils have been identified as one of the major nitrous oxide (N 2 O) sources, and excessive and repeated input of nitrogen (N) fertilizer (Gao et al., 2021 ; Zhu et al., 2022 ) and reactive N gas production, resulting in many environmental problems, such as increased N 2 O emissions (Fowler et al., 2013 ; Tian et al., 2020 ). In particular, N 2 O emissions from orchards have received widespread attention recently (Cheng et al., 2017 ; Escanhoela et al., 2019 ; Zhou et al., 2022 ). China occupies a leading position among the apple producers in the world and China has the highest apple orchard acreage (accounting for 41.4%) and production (accounting for 46.9%) of the world’s total output, which has expanded nearly 3 times over the past 40 years and reached approximately 1.91 million hectares in 2020 due to the economic benefits of apple production FAO (2022). The Loess Plateau has been recognized as one of the most suitable apple production areas in the world; however, the expansion of fruit orchards raises serious concerns about the risk of high N 2 O emissions due to intensive management methods (Cheng et al., 2017 ; Wu et al., 2017 ; Pang et al., 2019 ). Field measurements have shown that the N 2 O emission factor from apple orchards on the Loess Plateau are as high as 0.658% (Pang et al., 2009 ), which is greater than that of cropland (0.58%) in nearby regions (Zhang et al., 2017a ). However, the limited available orchard measurements contribute to high uncertainties in the estimates of N 2 O emissions from the agricultural sector (Cui et al., 2021 ). This limitation is all the more challenging due to the vast expansion of fruit cropping systems across the globe in the last decade. Therefore, a better understanding of the pattern and sources of N 2 O emissions from apple orchards is urgently needed to improve the global budget and formulate practical mitigation strategies, which could therefore strongly contribute to the mitigation of anthropogenic N 2 O emissions at national and global scales. Compared with other upland orchards, apple orchards have gained more attention in investigations of plant-soil-microbial interactions because of their unique perennial cropping systems, chemical composition and nutrient use efficiency of root exudates depending on stand age, which can profoundly affect soil C and N availability and other related soil properties (Haney et al., 2010 ). As a consequence, the changes in C and N turnover may affect the diversity of soil microbial communities, which govern the rate of C and N mineralization and concomitant greenhouse gas (GHG) release, including N 2 O fluxes (Ceja-Navarro et al., 2010 ). Previous results showed that stand ages can significantly affect soil conditions and thus lead to changes in soil bacterial community structure (Zheng et al., 2022 ), and it is speculated that the feedback responses of the associated microorganisms related to N 2 O emissions from stand age growth may be different. Therefore, research on apple orchards should be based on different stand ages. In addition, the perennial nature of the communities in the rows of trees means that soil management is constrained by time and space, and above and belowground ecosystems have greater stability than those associated with annual cropping. The low frequency of physical disturbance also means there are ample opportunities for the development of multiple bidirectional soil-microbial interactions (Deakin et al., 2018 ). N 2 O emissions from northern calcareous soils (well-aerated and high pH conditions) primarily result from the microbial nitrification process (Wolf and Russow, 2000 ; Wang et al., 2019 ). Ammonia oxidation is the primary and rate-limiting step in nitrification by ammonia-oxidizing bacteria (AOB) and archaea (AOA) (He et al., 2012 ), which are major contributors to N 2 O production in soil. Moreover, AOB prefer N-rich or fertilized soils (Di et al., 2009 ), and recent studies have also shown that AOB play a more important role than AOA in NH 3 oxidation in high N input and fertilized calcareous field soils (Zhong et al., 2016 ; Tao et al., 2017 ). In addition, nitrite is converted to NO or N 2 O by nitrite reductase (NIR) in denitrification. With the increasing age of perennial crops was reported to shift both nir S- and nir K-denitrifying bacterial communities under fertilization conditions in Ontario, Canada (Thompson et al., 2016 ). Deakin et al., ( 2018 ) also found that most of the differences in soil microbial community structure were due to large-scale differences between orchards, even within-orchards, and spatial relationships in microbial community structure differed between orchards and were not predictable. Xu et al., ( 2010 ) demonstrated a positive correlation between the enhancement of soil biological conditions and the time required for vegetation recovery. Xue et al., ( 2006 ) showed that the nitrification rate of tea garden soil showed an increasing trend in 8–50 years, and then a decreasing trend in 50–90 years. Plassart et al., ( 2008 ) reported that fungal genetic diversity was closely related to grassland age. However, few studies have offered a rigorous assessment of the microbial community coupled with a rigorous assessment of N 2 O production rates from different stand ages from apple orchards. In particular, it is necessary to explore the contributions of AOB and nir S to N 2 O emissions in apple orchards on the Loess Plateau of China. To elucidate how the different stand ages of orchards affect potential N 2 O emission rates and the related microbiological mechanism, we conducted a nearly 3-year field experiment to study N 2 O emissions and whether the relative abundance and composition of bacteria, fungi, AOB and nir S were changed by different stand ages in the Loess Plateau of China. We hypothesized that the change in stand age would alter the microbial community structure by affecting the soil moisture, carbon and nitrogen levels, thereby impacting N 2 O emissions. Our objectives were to i) investigate the effects of stand age and fertilizer in the presence or absence on N 2 O emissions and the community composition of microorganisms; ii) evaluate the relative contribution of microorganisms to N 2 O emissions. Materials and methods Site description and experimental design The study was located in the Wangdonggou Catchment, Changwu County, Shaanxi Province, China (35°13′N, 107°40′E; altitude 1200 m), which is a typical apple growing area in the Loess Plateau (Fig. 1 ). In the region, the average annual temperature and precipitation were 9.1°C and 582 mm, respectively. The annual average cumulative sunshine time is 2115 hours, and the annual average solar radiation is 5077 MJ m –2 , which is characterized by a warm temperate subhumid continental monsoon climate. The soil type at the experimental site is dark loessial soil from the Chinese Soil Classification System and Cumulic Haplustoll from the United States Department of Agriculture (Zhang et al., 2019a ), which was developed from Malan loess parent material. Meteorological data were obtained from the Changwu State Key Agri-Ecological Station on Loess Plateau, Chinese Academy of Sciences (Fig. S1 ). This study started in July 2017, and the experimental field was a conventional cropland (rotation of winter wheat and summer maize) prior to apple planting. The two apple ( Malus pumila Mill) orchards of different stand ages were selected as research objects, as follows: 5 years old (established in 2012) and 25 years old (established in 1992). The apple trees in the two orchards are spaced 5 m apart in rows and 2 m apart in plants. The basic soil properties (0–20 cm) of the two orchards soils at the beginning of the experiment are shown in Table 1 . Each treatment was replicated 3 times and included fertilized or unfertilized plots in both orchards: (1) conventional fertilization of the young orchard (5F), (2) conventional fertilization of the old orchard (25F), (3) no fertilizer in the young orchard (5CK), (4) no fertilizer in the old orchard (25CK). Five well-grown apple trees with no diseases or insect pests were selected in each experimental plot and every plot area was approximately 50 m 2 . During the research period, no irrigation and tillage events were provided, while necessary weeding and pest control were carried out in accordance with local management practices. The fertilization method involved digging ditches (width 20 cm and depth 20 cm), and the fertilization bands were at a distance of 1 m on both sides of the tree row (Fig. 1 ). Fertilization details are shown in Table S1 . Table 1 Topsoil (0–20 cm) basic properties in the plots from different stand ages. Sample Organic C (g kg –1 ) Total N (g kg –1 ) C/N NO 3 − -N (mg kg –1 ) NH 4 + -N (mg kg –1 ) pH Bulk density (g cm –3 ) Sand (%) Silt (%) Clay (%) 5yr 8.4 1.2 6.9 65.2 0.7 8.3 1.3 10.0 73.0 17.0 25yr 10.3 1.7 6.2 66.6 1.0 8.5 1.3 9.4 73.2 17.4 N 2 O analysis determination N 2 O emissions were measured from July 2017 to December 2019 by static chamber-gas chromatographic techniques. N 2 O samples were collected from 9:00 to 11:00 a.m. every week and the interval was extended to 10 days after the soil froze, and continuous sampling every other day for one week if fertilization was encountered. Insulated sample chambers size 0.25 m × 0.25 m × 0.5 m and a stainless-steel base (0.25 m × 0.25 m × 0.1 m) with groove is equipped. A small electric fan was fixed on the top of the chamber to evenly mix the air during sampling. Specific sampling details and soil temperature (ST) monitoring are consistent with those described by (Han et al., 2022 ). N 2 O gases were measured by a gas chromatograph instrument (7890A, Agilent US). According to (Zhang et al., 2010 ), the N 2 O concentration was quantified by an electron capture detector (ECD, 300°C). The chromatographic column was an 80/100 mesh SS-2 m × 2 mm Porapak Q, and N 2 with a flow rate of 40 cm 3 ·min –1 was used as a carrier gas. According to the change in gas concentrations in four continuous samples over time, the soil N 2 O flux was calculated (µg N m –2 h –1 ). The formula is as follows: $$\:\text{Flux}\text{=}\frac{\text{M}}{\text{22.4}}\text{×}\text{H}\text{×}\frac{{\text{d}}_{\text{c}}}{{\text{d}}_{\text{t}}}\text{×}\frac{\text{273}}{\text{273+}\text{T}}$$ 1 where M is the molar mass (12 g mol –1 ) of the N atom in N 2 O, H is the chamber height (m), d c /d t (µL L –1 min –1 ) is the slope of the regression curve of the N 2 O concentration over time, and T is the air temperature in the chamber. The total N 2 O emissions during the monitoring period were obtained from the average N 2 O flux and the monitoring period (Zou et al., 2005 ). Considering uneven fertilization in apple orchards, N 2 O emissions in the different age plots should be calculated by the following equation (Zhang et al., 2019b ). where F and nF are cumulative N 2 O emissions (kg N ha –1 ) from fertilized and unfertilized regions, respectively, and a and b are the areas (m 2 ) of fertilized and unfertilized plots, respectively. Sampling and soil physicochemical properties and enzyme activity analysis Topsoil (0–20 cm) around the chamber was collected by a soil auger (diameter: 5 cm), which was synchronized with the greenhouse gas collection. Three samples were collected and mixed into one sample for each plot, and each treatment was repeated three times. Soil samples were brought back to the laboratory in Ziplock bags, which were used for soil physical and chemical property analysis. After air drying, the soil was passed through a 2 mm sieve to measure pH (soil-water ratio was 1:2.5) and a 0.15 mm sieve to measure soil organic carbon (Walkley-Black potassium dichromate), total N (Kjeldahl methods), hydrogen peroxidase, cellulase, invertase and urease. At the same time, topsoil samples were collected to monitor the water content and ammonium nitrate N during greenhouse gas collection. The mass moisture content of the soil was determined by the drying method and fresh soil was used to determine the contents of nitrate and ammonium nitrogen, which were extracted with 1 mol/L KCl and analyzed by a continuous flow analyzer (SEAL AutoAnalyzer 3, Germany). Water filled pore space (WFPS) was calculated according to (Han et al., 2022 ). The determination methods of soil physical and chemical properties and enzymes mentioned above refer to Bao ( 2008 ) and Guan ( 1986 ). Microbial analysis Soil samples (0–20 cm) for microbial analysis were collected by a soil auger (diameter: 5 cm, autoclave sterilization) during the N 2 O flux peak after fertilization (10 April 2018, 2 days after fertilization) at the fertilizer bands and non-fertilized bands, and the samples were stored in a -80℃ refrigerator for DNA coextraction. Three soil samples were taken from each plot and mixed into one soil sample to be tested. Microbial DNA was obtained from fresh soil samples, and the concentration and purity were checked by agarose gel electrophoresis. Then, sterile water was used to dilute the DNA sample and the diluted genomic DNA was used as a template. Specific primers (bacteria: 515 F 5'- GTGCCAGCMGCCGCGGTAA-3' and 806R 5'-GGACTACNNGGGTATCTAAT-3' (Caporaso et al., 2011 ); fungi: ITS5 5'-GGAAGTAAAAGTCGTAACAAGG-3' and ITS2 5'-GCTGCGTTCTTCATCGATGC-3') (Bellemain et al., 2010 ); nir S: cd3aF 5'-GTSAACGTSAAGGARACSGG-3' and R3cd 5'-GASTTCGGRTGSGTCTTGA-3' (Michotey et al., 2000 ); AOB- amo A: amo A1F 5'-GGGGTTTCTACTGGTGGT-3' and amo A2R 5'-CCCCTCKGSAAAGCCTTCTTC-3' (Rotthauwe et al., 1997 ) and high-fidelity enzymes were used for PCR amplification according to the selected regions. The Ion Plus Fragment Library Kit 48 rxns (Thermo Fisher) was used to construct the library and sequencing was performed using the Ion S5 platform (Thermo Fisher) at Novogene Bioinformatics Technology Co., Ltd., Beijing, China. Raw sequences were obtained by cutting off barcodes and primers and using FLASH spliced reads (Magoc and Salzberg, 2011 ); then the filtered data were screened out via a strict filtering process (Caporaso et al., 2011 ). The extracted clean sequences were clustered to estimated operational taxonomic unit (OTU) numbers at 97% identity. OTU sequences were annotated with the SILVA (bacteria), UNITE (fungi), and FGPR functional gene (AOB and nir S) databases. Data analysis and statistics Microsoft Office Excel 2019 (Microsoft, USA) was used to calculate the N 2 O fluxes and cumulative emissions, averages, standard deviations and significance analyses were calculated using IBM SPSS 25 (IBM, USA). Figures were designed by ArcGIS 10.6 (ESRI, USA), Origin Pro 2018 (OriginLab, USA), SigmaPlot (version 15.0) and R software (version 4.1.1). Hierarchical clustering of bacteria and fungi was carried out by QIIME (version 1.9.1) based on unweighted UniFrac distances. Principal coordinates component analysis (PCoA, by WGCNA, stats and ggplot2 packages), Mantel test (by vegan package), Spearman correlation analysis of various soil indices (by psych package) and Redundancy analysis of environmental factors and microbial communities (RDA, by vegan, ggplot2 and ggrepe1 packages) were performed using R software. R software was used to determine the effects of stand years and fertilization on the microbial community via permutational multivariate analysis of variance (PERMANOVA, by vegan packages). Results Dynamic changes in soil temperature, WFPS and mineral nitrogen ST was recorded from July 2017 to December 2020, which showed a similar dynamic trend and an obvious seasonal variation characteristic (Fig. 2 a). During the experimental periods the ST ranged from − 7.1℃ to 22.7℃, with the highest and lowest ST occurring in July and January, respectively. In addition, topsoil WFPS monitored during nonfreezing periods changed with precipitation and began to increase in April (Fig. 2 b), which varied from 15.5–82.5% and showed a general trend of low in winter and spring and high in summer and autumn, with mean values of 50.0% (5CK), 48.3% (5F), 51.2% (25CK) and 55.0% (25F) in different plots, respectively. The soil mineral N content was influenced by N inputs, and higher contents of mineral N were present at the fertilization site (5F and 25F, Fig. 2 c and d). The soil NH 4 + -N content generally changed slightly most of the time (Fig. 2 c), except in April 2018 and April 2019, when urea was applied, and both fertilized orchard NH 4 + -N contents showed sharp fluctuations and peaked (95.8 mg N kg –1 -112.3 mg N kg –1 ) within 3–5 days. However, compound fertilizer addition did not cause the rapid increase in NH 4 + -N contents. Soil nitrate N (NO 3 − -N) was sensitive to compound fertilizer and urea, and NO 3 − -N contents in 5F and 25F increased sharply after fertilization and generally lasted a month before falling back (Fig. 2 d). During the experimental period, the average NO 3 − -N contents of fertilized soil (78.6 mg N kg –1 -79.4 mg N kg –1 ) were significantly higher than those of non-fertilized soil (38.4 mg N kg –1 -47.4 mg N kg –1 ). In addition, during the trial period, NH 4 + -N increased by 45.9% and 104.2%, and NO 3 − -N increased by 48.6% and 80.2%, respectively, in 5a and 25a apple orchards after fertilization according to the weighted effect size of each category (Fig. S2). Our global meta-analysis of field observations revealed significant increases in NH 4 + -N across orchards aged 0–5 years, 5–15 years, and 15–25 years by 61.63%, 310.08%, and 151.4%, respectively. Similarly, NO 3 − -N levels showed significant increases of 92.5%, 171.2%, and 279.1% across the same age categories (Fig. 3 ). N 2 O emissions from young and old apple orchards During the study period, the average N 2 O flux ranged from 16.7 µg N m –2 h –1 to 71.9 µg N m –2 h –1 among the different treatments (Fig. 4 a), and different stand ages showed obvious seasonal variation. As shown in Fig. 4 b, soil N 2 O fluxes were mostly distributed below 50 µg N m –2 h –1 , accounting for 91.9%, 73.3%, 93.3% and 65.9% of the total monitoring events in 5CK, 5F, 25CK and 25F, respectively. A pulse emission peak was recorded within a week after top dressing in summer, the N 2 O fluxes from 5F and 25F peaked at 320.3 µg N m –2 h –1 and 1067.0 µg N m –2 h –1 in July 2018 and 319.3 µg N m –2 h –1 and 469.8 µg N m –2 h –1 in July 2019, respectively. N 2 O emissions from old orchards were significantly higher than those from young orchards with N addition (252.7%, P < 0.05). The cumulative N 2 O emissions of fertilized plots were observably higher than that in nonfertilized plots and the highest emissions occurred in fertilized old orchards (Table 2 , P < 0.05). However, the differences in N 2 O efflux rates and cumulative N 2 O emissions between 5CK and 25CK were not significant. The N 2 O emission factors of the young orchard and old orchard were 0.37% and 0.54%, respectively. Our global meta-analysis consistently showed an increasing trend in N 2 O emissions following nitrogen fertilizer application across orchards aged 0–5 years, 5–15 years, and 15–25 years. Specifically, N 2 O increased significantly by 89.0%, 39.7%, and 93.6%, respectively (Fig. 3 ). Table 2 The average N 2 O fluxes, annual cumulative N 2 O emissions and its emission factors (EF) in different treatments during experimental period. Treatments N 2 O fluxes (μg N m –2 h –1 ) N 2 O cumulative emission (kg N ha –1 yr –1 ) N 2 O Emission factor (%) 5CK 20.72 ± 4.13 c 1.38 ± 0.03 c - 5N 46.78 ± 4.34 b 3.92 ± 0.09 b 0.37±0.02 25CK 23.94 ± 2.77 c 1.57 ± 0.11 c - 25N 65.23 ± 3.95 a 5.60 ± 0.22 a 0.54±0.01 Different lowercase letters indicate significant difference between treatments ( P < 0.05). Table 3 Alpha diversity index of bacteria, fungi, AOB and nir S. Types Treatments Shannon Simpson Chao1 Bacteria 5CK 9.41±0.25 a 0.99±0.003 a 3383.17±1013.88 a 5N 9.26±0.34 a 0.99±0.004 a 2636.59±227.91 a 25CK 9.49±0.33 a 0.99±0.003 a 2998.73±74.08 a 25N 8.76±1.37 a 0.98±0.029 a 2921.15±198.51 a Fungi 5CK 7.06±0.22 a 0.98±0.005 a 924.88±53.99 a 5N 6.77±0.34 a 0.97±0.016 a 983.90±77.06 a 25CK 6.40±0.30 a 0.96±0.013 a 892.71±36.79 a 25N 6.68±0.51 a 0.97±0.008 a 870.27±87.57 a AOB 5CK 6.45±0.26 b 0.92±0.007 b 4443.29±1012.30 a 5N 6.56±0.22 b 0.93±0.021 b 5342.00±1246.57 a 25CK 7.82±0.12 a 0.98±0.003 a 5096.25±256.04 a 25N 7.41±0.29 a 0.97±0.002 a 5641.00±1732.34 a nir S 5CK 10.02±0.41 a 0.99±0.002 a 8311.17±3414.64 a 5N 9.66±0.16 a 0.99±0.001 a 7325.60±1647.34 a 25CK 10.14±0.29 a 0.99±0.001 a 10833.69±178.40 a 25N 9.88±0.09 a 0.99±0.001 a 8358.06±2952.16 a Different lowercase letters indicate significant difference between treatments ( P < 0.05) Bacterial and fungal analysis The average numbers of excellent-quality base sequences from bacteria and fungi were 76482–79825 and 80130–80212, respectively. The results indicated that the composition of bacteria was similar under different treatments, but there were some differences in the relative abundance of dominant bacteria in the soil of the young and old orchards (Fig. 5 a). Proteobacteria (25.7%-37.2%), Bacteroidetes (15.4%-19.2%), Actinobacteria (15.3%-22.2%) and Acidobacteria (9.1%-11.7%) were the dominant phyla in the 5CK, 5F and 25CK treatments, accounting for 76.4%, 75.4% and 81.6% of the total bacterial abundance, respectively. It should be noted that Firmicutes was a major dominant phylum in the 25F treatment, contributing 21.9% of total bacterial abundance, which is quite different from other treatments. The hierarchical clustering of bacterial phyla also significantly suggested that the bacterial community composition of 25F was independent of other treatments, and the abundance of Firmicutes in 25F obviously increased. The relative abundance of some phyla of bacteria of 25F was also different from that of the young orchard (Fig. 6 a). The relative abundance of the top ten fungi at the phylum level accounted for 60.1% (5CK), 56.2% (5F), 61.2% (25CK) and 66.9% (25F) of the total abundance, respectively (Fig. 5 b). Ascomycota was the most abundant fungus in the young apple orchard, accounting for 45.4% and 44.8% in 5CK and 5F, while the proportion of other fungi was low. In the old apple orchard, Ascomycota and Basidiomycota were the top two fungal phyla, and the proportions reached 35.9%-40.0% and 19.6%-27.4%, respectively. As illustrated in Fig. 6 b, UPGMA clustering indicated that the soil fungal communities of young and old orchards were divided into two branches at the phylum level, Ascomycota decreased and Basidiomycota increased in old orchard soil. In this study, alpha and beta diversity analyses were used to determine the differences in soil bacterial and fungal community compositions and the number of species between young and old apple orchard soils. The results of alpha diversity analysis showed that the Shannon, Simpson and Chao1 indices were not significantly different between the different treatments (Table 3 ), indicating that the number and composition of soil bacteria and fungi in orchards of different stand ages were similar. According to the OTU tables of the bacterial and fungal communities, PCoA analysis obtained Fig. 7 a and b. The result of PCoA showed that the elliptical confidence of bacterial communities in 5F deviates from that of 25CK and 25F. Combined with Adonis analysis, there were significant differences in bacterial communities between young orchards with fertilizer and old orchards ( P < 0.001), and the bacterial communities of orchards of the same stand ages were similar regardless of fertilizer inputs. The composition of the fungal community composition between young and old orchards was relatively dispersed without overlap, and the significant differences were found between 5CK and 25F, 5F and 25CK and 5F and 25F via the results of Adonis analyses (Fig. 7 b, P < 0.001). Table 4 Environmental factors corresponding to microorganisms. Treatment TN (g kg -1 ) pH WC (%) SOC (g kg -1 ) N 2 O fluxes (μg N m –2 h –1 ) NO 3 - -N (mg kg −1 ) NH 4 + -N (mg kg −1 ) HPA (mg g −1 h −1 ) CA (μg g –1 d –1 ) IA (mg g –1 d –1 ) UA (mg NH 3 -N g –1 d –1 ) 5CK 1.19±0.10b 8.20±0.06a 11.53±0.95b 10.36±1.20b 8.17±2.23c 28.64±3.49b 2.01±0.43b 45.22±0.34a 82.87±3.91b 43.12±2.02a 1.01±0.02c 5N 1.27±0.15b 8.00±0.28a 11.18±1.93b 10.13±0.40b 95.41±24.50b 152.63±70.59a 5.93±1.50b 43.68±0.27a 89.83±4.61b 45.00±0.48a 1.04±0.03c 25CK 1.46±0.25ab 8.19±0.16a 13.89±1.54b 12.65±1.54ab 39.87±0.18c 56.13±25.29b 5.56±2.94b 35.78±0.23b 82.47±1.88b 26.90±0.62c 1.65±0.02b 25N 1.70±0.07a 8.06±0.10a 18.88±0.89a 13.70±1.91a 233.12±47.70a 233.09±26.00a 15.59±2.89a 26.91±1.59c 103.80±4.51a 31.28±1.26b 1.95±0.08a Different lowercase letters indicate significant difference between treatments ( P < 0.05). TN: Soil total nitrogen; WC: Mass moisture content of soil; SOC: Soil organic carbon; NO 3 - -N: Soil nitrate nitrogen; NH 4 + -N: Soil ammonium nitrogen; HPA: Hydrogen peroxidase activity; CA: Cellulase activity; IA: Invertase activity; UA: Urease activity. Ammonia oxidizing and denitrifying bacterial community As shown in Fig. 5 c, d, many sequences came from uncultured strains. The dominant AOB species were Uncultured_bacterium , Uncultured_ammonia_oxidizing_bacterium and Nitrosospira_sp_Nv6 , and the dominant species of denitrifying bacteria were uncultured_bacterium , Pseudomonas_marginalis and uncultured_organism . It is clear that a higher proportion of annotated AOB species in older orchard soil was higher, and the relative abundance of Uncultured_bacterium in the old orchard was significantly increased ( P < 0.001). Alpha diversity indices showed that Shannon and Simpson showed an increasing trend with increasing planting years, suggesting that the increase in AOB community diversity may be related to stand age (Table 3 , P < 0.01). However, the Chao1 index of each treatment showed no significant difference, indicating that there was no difference in the number of AOB species in orchards of different stand ages. Furthermore, there were no significant differences in the Shannon, Simpson and Chao1 indices of nir S ( P > 0.05, Table 3 ). PCoA analysis showed that the elliptical confidence of AOB communities in the new orchard deviated from that in the old orchard, while the distribution of AOB community composition in the same-age orchard relatively concentrated, indicating that the stand ages of orchards caused the difference in AOB community structure (Fig. 7 c). Adonis analysis further confirmed the significant difference in AOB diversity between 5F and 25CK and 5F and 25F ( P < 0.001). According to the results of PCoA, the nir S bacterial community composition distribution of each treatment was concentrated, and the confidence ellipses had obvious overlapping areas. PCoA and Adonis combined were used to analyze the nir S bacterial diversity of all treatments, and there was a significant difference between 5F and 25CK ( P 0.05). In general, permutational multivariate analysis of variance (PERMANOVA) showed that the soil microbial community in apple orchards was dramatically influenced by stand age and the interaction between stand age and fertilization addition, which explained 22.2% ( P < 0.022) and 36.6% ( P < 0.003) of the variation, respectively. However, the effect of fertilization alone on the microbial community was not significant, and there was still a 36.8% unexplained portion (Fig. 8 ). Correlation among N 2 O emission, environmental factors and microorganisms As shown in Table 4 , the soil properties except pH were altered greatly among the different treatments, and 25F had the highest Soil organic carbon (SOC), Soil total nitrogen (TN), soil water content (WC), NH 4 + -N, NO 3 − -N, cellulase activity (CA) and urease activity (UA). Pairwise correlation analysis based on environmental factors (Fig. 9 a) indicated that N 2 O emissions were obviously positively correlated with NH 4 + -N ( P < 0.001), NO 3 − -N ( P < 0.001), CA ( P < 0.001), and UA ( P < 0.05), but significantly negatively correlated with hydrogen peroxidase activity (HPA) ( P < 0.01) and invertase activity (IA) ( P < 0.05). In addition, bacterial composition (phylum level), fungal composition (phylum level), AOB species and nir S species were correlated with the environmental factors via the Mantel test, as shown in Fig. 9 a. SOC ( P = 0.04 and P = 0.01), WFPS ( P = 0.03 and P = 0.02), CA ( P = 0.03 and P = 0.001), IA ( P = 0.004 and P = 0.001) and UA ( P = 0.002 and P = 0.002) all had the strongest associations with bacterial and fungal composition, respectively. TN contents were also the key factors affecting fungal communities ( P = 0.02), but not bacteria. Meanwhile, IA ( P = 0.04) also influenced the AOB communities. However, no significant correlation was found between nir S species and all environmental factors. Furthermore, to identify the drivers of N 2 O emissions in apple orchards of different stand ages, redundancy analysis (RDA) was conducted to analyze the correlation between key environmental factors and microbial community compositions (Fig. 9 b and c). According to the correlation results in Fig. 9 a, environmental factors strongly correlated with N 2 O emissions were selected for RDA. Both the RDA1 and RDA2 axes explained 76.2% and 88.7% of the bacterial, fungal, AOB and nir S community variations in young and old apple orchards, respectively. For the young apple orchard, bacteria, CA, NH 4 + -N and NO 3 − -N were the main influencing factors on N 2 O emissions and were oriented towards the right side of RDA axis 1. However, bacteria, fungi, CA, NH 4 + -N and NO 3 − -N had a strong correlation with N 2 O emissions in the old apple orchard. Discussion N 2 O emissions in orchards under different stand ages Dynamic variation in N 2 O fluxes was observed in this study (Fig. 4 a), which could be explained by the positive correlation between ST and N 2 O emissions (Figs. 2 a and 4 a), and agreed with previous results (Cui et al., 2016 ; Pärn et al., 2018 ). The amount of fertilizer input may be the main factor causing different N 2 O emissions under the same climate conditions, soil types and management practices. Previous findings suggested that N 2 O emissions in orchards were strongly influenced by fertilization (Pang et al., 2009 ) and increased linearly with N input rates (Cheng et al., 2017 ; Gu et al., 2019 ). N 2 O emissions peaks induced by fertilization events accounted for more than 62.5%-67.6% of the total annual emissions along with high mineral N content, especially after urea addition in April 2019. Therefore, we speculated that nitrification might be the main mechanism of N 2 O production in apple orchard soil on the Loess Plateau, which agreed with the changes in soil mineral N (Figs. 2 d and 4 a). In addition, emissions peaks occurred following fertilization and were more obvious between July and August (Fig. 4 a), which could be explained by the dramatic increase in mineral N (Duan et al., 2019 ) and precipitation (Pang et al., 2019 ; Zhang et al., 2021 ). A previous study confirmed that fertilization during rainy periods can induce much higher N 2 O emissions (Groffman et al., 2000 ; Pang et al., 2019 ). In addition, the annual cumulative N 2 O emissions and EFs of different stand ages showed significant differences, and the old orchard had noticeably higher N 2 O emissions, which were 42.86% higher than those of the new orchards (Table 2 ). The EF in the old orchard (0.54%) was significantly higher than that in the young orchard (0.37%), suggesting that more N was lost as N 2 O gas in older apple orchard. Referring to the results of our previous study (Han et al., 2022 ), a 15-year apple orchard in this region showed that the EF was 0.34%-0.45%, and the EF was lower than that from the 25-year orchard but higher than that from the 5-year orchard in this study. This indicated that EF of apple orchards was affected by stand age, thus, estimating the EF of perennial crops should consider the different stand ages. However, compared with the EF (0.72%) of orchards in continental climate regions (Gu et al., 2019 ), the results of this study were relatively low. This is probably because of uncertainty in the EF, including soil properties, farmland management, and climate being key factors affecting the EF (Zhou et al., 2014 ). The EF in orchard soil related to climate types, temperature and continental climate had a lower EF (Gu et al., 2019 ). This study region has a typical temperature continental monsoon climate, and the EF was 60%-72% lower than that of a peach orchard (EF = 1.32%) in subtropical monsoon climate zone where had more precipitation and higher temperature (Cheng et al., 2017 ), despite the similar N application rate. Moreover, soil texture also significantly influenced the EF and fine-textured soils could result in larger N 2 O emissions (Stehfest and Bouwman, 2006 ). However, the medium-textured soil texture in this study had difficulty forming an anaerobic environment to provide conditions for denitrification (Ball, 2013 ), resulting in a low EF. At present, the evidence that orchard age affects N 2 O emissions is still insufficient, but in Chinese tea plantations, there is evidence that different stand ages will lead to significantly different N 2 O releases (Yao et al., 2018 ; Zhang et al., 2020 ). The establishment of woodland can form a soil organic layer that promotes the release of N 2 O (Merino et al., 2004 ; Peichl et al., 2010 ), and SOC and TN in the topsoil play a key role in promoting N 2 O emissions (Pang et al., 2019 ). Older orchard soils with higher SOC and TN could provide more abundant substrates for nitrification and denitrification processes. Microbial community in orchards under different stand ages The alpha diversity of the bacteria, fungal and nir S communities did not change with the stand age of the apple orchard, but the alpha diversity of AOB significantly increased with stand age (Table 3 ). This result disagrees with the conclusion of (Wang et al., 2022 ) that the soil bacterial alpha diversity increased with forest stand age. The possible reason is that agricultural management, such as weeding, has resulted in a simple and stable vegetation community since the apple orchard was established, which created a stable bacterial community (Qiao et al., 2021 ). A previous study reported that plant communities limited soil bacterial communities (Dang et al., 2017 ) due to diverse substrates from different plants being provided to bacteria (Liu et al., 2018 ). However, long-term weeding in our orchards led to a single vegetation species and directional bacterial community succession due to single root exudates was provided. Fungi have strong adaptability (Corneo et al., 2013 ), and their response to soil nutrient changes is weaker than that of bacteria (Delgado-Baquerizo et al., 2016 ). Therefore, the alpha diversity of fungi did not change with stand age in apple orchard. The increase in AOB diversity in the old apple orchard was consistent with the findings of our meta-analysis, potentially influenced by long-term fertilization and alkaline soil conditions. Several studies have indicated that fertilizers alter the community structure of nitrogen-fixing bacteria, with higher levels of nitrification occurring in alkaline and neutral soils compared to acidic soils (Jiang et al., 2015 ; Cui et al., 2016 ; Meng et al., 2023 ). Soil microbes participate in nutrient cycling and affect the development of soil plants in terrestrial ecosystems (Li et al., 2015 ). Numerous findings indicated that soil microbial communities in apple orchards are influenced by edaphic factors (Zheng et al., 2018 ; Yang et al., 2020 , 2022 ). A report from a citrus orchard indicated that with plant growing and development, changes in the understory microenvironment and root system led to changes in microbial community composition (Qiang et al., 2020 ). Thus, it is instructive to study soil microorganisms of different stand ages for long-term apple orchard planting management. In this study, the community structure composition of bacteria and fungi was significantly affected by stand age and the interaction between fertilization and stand age. However, fertilization had minimal impact on the composition of the microbial community (Figs. 6 , 7 , and 8 ). This finding was supported by our meta-analysis results, which contrasted with previous reports indicating significant changes in the soil microbial community due to nitrogen fertilizer in crop fields (Ullah et al., 2019 ; Hu et al., 2022 ). The important driving factor may be changes in soil nutrients (Fig. 9 a), and some studies in orchards showed a strongly positive link between soil bacteria/fungi and soil nutrients, especially TN and SOC (Qiang et al., 2020 ; Zheng et al., 2021 ; Yang et al., 2022 ). As shown in Table S1 , there were higher TN and SOC contents in the old apple orchard, particularly in the N-amendment plots. The results agreed with previous research showing that the soil microbial community changed with stand age in orchards (Zhang et al., 2017b ; Wu et al., 2020 ). However, different opinions also existed in orange orchards, that stand ages increased soil C and N but decreased microbial activity due to soil acidification (Wan et al., 2017 ). With increasing stand age, the relative abundances of some phyla of bacteria and fungi changed obviously (Fig. 5 ), and a classification phenomenon related to tree age occurred (Fig. 6 ). Notably, the relative abundance of Firmicutes obviously increased in the 25F treatment because the long-term accumulation of dead leaves and N provide substrates for bacteria in old orchards, and Firmicutes is closely related to the degradation of plant residues (Tiwari et al., 2016 ; Verzeaux et al., 2016 ). Meanwhile, Basidiomycota as saprophytic fungi (Yang et al., 2022 ), and their relative abundance was significantly increased in old orchards that had abundant plant residues (Wan et al., 2017 ). Basidiomycota have an advantage in degrading stubborn lignin (Lundell et al., 2010 ) and its increase could be driven by the accumulation of soil recalcitrant C components in old stands (Wang et al., 2022 ). Soil moisture was also an important factor limiting bacterial and fungal communities (Fig. 9 a), agreeing with the conclusion of (Wu et al., 2020 ). Higher vegetation coverage and more litter coverage on soil surface in older stands would better maintain soil moisture (Qiao et al., 2021 ). Thus, the relative abundance of Gemmatimonadetes , which like dry environment (Fawaz, 2013 ), was lower in the old orchard. Influencing factors of N 2 O emissions in orchards under different stand ages Numerous studies have strongly proven that stand age affects N 2 O emissions from the soil in tea plantations (Yao et al., 2018 ; Zhang et al., 2020 ), arbor forests (Christiansen and Gundersen, 2011 ; Shrestha et al., 2014 ; Yin et al., 2016 ; Ishizuka et al., 2021 ) and sisal plantations (Wachiye et al., 2021 ) which is attributed to differences in soil properties, including carbon and nitrogen availability, pH, C/N, mineral N level, temperature and WFPS. Moreover the plant N requirement and N use efficiency are also considered to be responsible for the differences in N 2 O emissions (Shrestha et al., 2014 ). However, there is currently insufficient evidence on the effect of stand ages on N 2 O emissions in fruit orchards, especially in apple orchards. In apple orchards, the differences in soil abiotic properties in young and old orchards may be the more important inducing factors to N 2 O emissions (Fig. 9 a, b and c). Precipitation influences soil water and oxygen availability, which are closely linked to soil N 2 O emissions (Saggar et al., 2013 ; Song et al., 2019 ). Comprehensive analysis reveals a significant correlation between mean annual precipitation (MAP) and N 2 O emissions (Fig. S4), with N 2 O release particularly sensitive to mineral N content and WFPS (Fig. 9 a). Hence, the predominant role of chemical processes in N 2 O production is paramount and should not be overlooked (Zhang et al., 2021 ). First, the availability of soil N was the most critical driver of N 2 O emissions (Levy-Booth et al., 2014 ; Pärn et al., 2018 ) and with the growth of apple orchard stand ages, mineral N in orchard soil was strongly accumulated (Wan et al., 2017 ; Liu et al., 2019 ). The meta-analysis results indicate that orchards aged 15–25 years have NH 4 + -N levels 89.74% higher than orchards aged 0–5 years (Fig. 3 ). Second, WFPS also play an important role in N 2 O emissions, and a certain range of WFPS (50%-80%) created the optimal conditions for N 2 O production (Pärn et al., 2018 ). The range of this region could be considered to be 35%-75% because the N 2 O fluxes larger than the average flux were mostly distributed in this range (Fig. S3). Soil moisture influenced the soil oxygen availability (Song et al., 2019 ), and nitrification and denitrification were synchronized to produce higher N 2 O under moderate soil WFPS conditions (Pärn et al., 2018 ). Above all, soil NO 3 − -N contents and moisture explained 72% of N 2 O emissions (Pärn et al., 2018 ). Meanwhile, the higher NH 4 + -N contents could induce more N 2 O fluxes. Because NH 4 + -N is involved in the nitrification process (Levy-Booth et al., 2014 ) to produce N 2 O fluxes, this process is particularly pronounced in calcareous soils with well-aerated and high pH conditions (Wolf and Russow, 2000 ; Wang et al., 2019 ). However, the pH had no impact on N 2 O emissions which was different from a previous study (Cui et al., 2016 ; Deng et al., 2019 ; Zhang et al., 2021 ). The reason may be that the pH value of orchard soil in this study was relatively stable and had not induced a drastic effect on N 2 O. Moreover, this study was conducted in arid and semiarid regions, and previous researchers confirmed that mineral N contents and soil water content were the main factors affecting N 2 O emissions in semiarid areas (Galbally et al., 2010 ). The RDA indicated that N 2 O emission was positively associated with bacteria and fungi in young and old orchards (Fig. 9 b and c). Several reports (Baggs, 2011 ; Chen et al., 2014 ) confirmed that bacterial and fungal processes were important contributions to N 2 O emissions; fungi in particular have been increasingly identified as major contributors to N 2 O emissions in recent years (Mothapo et al., 2015 ; Wankel et al., 2017 ) because the final product of fungal denitrification is N 2 O instead of N 2 (Baggs, 2011 ; Mothapo et al., 2015 ). Considering that Ascomycota and basidiomycota contain many N 2 O-producing fungi and their strong N 2 O-producing activity (Mothapo et al., 2015 ), the higher relative abundance of Ascomycota and Basidiomycota may be an important microbiological reason for the higher N 2 O emissions in old orchards. The results of this field study were different from laboratory experiments, which concluded that ammonia oxidizer and denitrifier abundances dominated N 2 O emissions (Hink et al., 2018 ; Qiu et al., 2019 ). This may be related to the time of sampling; the microbial activity varies with season, and the abundances over a period of time indicate the relative number of microorganisms but are not a complete representation of activity (Shrewsbury et al., 2016 ). Another reason for the low correlation between N 2 O and soil microbes may be that the key role of soil properties masked the impact of ammonia oxidizers and denitrifiers (Graham et al., 2014 ; Levy-Booth et al., 2014 ; Pärn et al., 2018 ). In conclusion, soil abiotic properties, rather than nitrification and denitrification bacteria, caused the difference in N 2 O emissions in apple orchards of different stand ages. This result also agrees with the previous study showing that compared to denitrifiers, environmental attributes were more important influencers of N 2 O emissions in larger landscapes (Shrewsbury et al., 2016 ). A meta-analysis also reported similar results that N loading induced soil N 2 O emissions rather than ammonia oxidizer and denitrifier abundance (Zhang et al., 2021 ); thus, abiotic factors were the main drivers of the changes in soil N 2 O emissions. Conclusion This study showed that higher N 2 O emissions occurred in older apple orchards and that the main contributors were soil abiotic properties rather than soil bacteria, fungi, AOB and nir S genes. The richness and diversity of soil bacteria, fungi and nir S showed no significant differences between the young and old apple orchards, but that of AOB was higher in the old orchard. The soil microbial community composition was obviously affected by the stand age of the apple orchard. Stand age and the combination of stand age and fertilization explained most of the variation in microbial communities, but the effect of fertilizer addition was not significant. Nonetheless, microbial processes were not the key drivers of N 2 O release, instead, soil reactive N availability, soil moisture and enzyme activity were more direct factors. Therefore, in future research on soil N cycling and transformation of apple orchards, soil abiotic processes should not be ignored when considering soil microbial processes. Declarations Funding This work was supported by the Natural Science Foundation of Shaanxi Province (grant numbers 2023-JC-QN-0356, 2022JM-154); the Open Foundation of Key Laboratory in Jiangxi Academy of Water Science and Engineering (grant numbers 2021SKTR02, 2022SKTR02); the National Natural Science Foundation of China (grant numbers 41601321, 42177327, 42007063). Competing Interests The authors declare that they have no conflict of interest. Author Contributions All authors contributed to the study conception and design. Material preparation, data curation, and analysis were performed by Man Zhang and Cui Li. The first draft of the manuscript was written by Man Zhang and Cui Li. Weixin Wang, Xin Tong, Kaixuan Wang, Minmin Qiang, Qiong Zhang contributed to the review and editing of the manuscript. All authors read and approved the final manuscript. References Baggs, E. M. (2011). Soil microbial sources of nitrous oxide: recent advances in knowledge, emerging challenges and future direction. Current Opinion in Environmental Sustainability 3, 321–327. doi: 10.1016/j.cosust.2011.08.011 Ball, B. C. (2013). Soil structure and greenhouse gas emissions: a synthesis of 20 years of experimentation. European Journal of Soil Science 64, 357–373. doi: 10.1111/ejss.12013 Bao, S., 2008. Soil and Agro-chemical Analysis. 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Global Biogeochemical Cycles 19. doi: 10.1029/2004GB002401 Supplementary Files Supplementarymaterial.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-5201652","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":377060114,"identity":"54c40c31-b090-4197-90c5-3d4546b5ced2","order_by":0,"name":"Man Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABA0lEQVRIiWNgGAWjYBADHn5m9oMPwEzmA0AuEVrkJNt7kg3ATLYE4rQYG5w5YCZBlBb5aYcfPvhRYZPYcCMhrbqwrS6xv42B8cHbNgZ5cxxaGGenGRv2nElLbJyReOz2zLbDiTOOMTAbzm1jMNzZgF0Ls3QOmzQjUGWzRELabd5tBxIb7jewSfO2MSQYHMCuhQ2mpU0iwayYd1td4vxjDOy/8WnhgWox5uE5YMbMu405ccMxBjZmfFokpCF+kZNg70mW5v132HjjMcZmyTnnJAw34NAiPzsZHGI89ofZD37mOVMnO+8Y88EPb8ps5HHZgg0wNoCsJ179KBgFo2AUjAIMAACAmVh4PkmNgQAAAABJRU5ErkJggg==","orcid":"","institution":"College of Soil and Water Conservation Science and Engineering, Northwest A\u0026F University","correspondingAuthor":true,"prefix":"","firstName":"Man","middleName":"","lastName":"Zhang","suffix":""},{"id":377060115,"identity":"49cb74dd-48b3-4038-954a-3c9bc6ec207b","order_by":1,"name":"Cui Li","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Cui","middleName":"","lastName":"Li","suffix":""},{"id":377060116,"identity":"1d2be6b0-afb1-4789-ba8e-bc6cc8481023","order_by":2,"name":"Weixin Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Weixin","middleName":"","lastName":"Wang","suffix":""},{"id":377060117,"identity":"4b17c71d-eb0a-4447-8c43-06c7107f2816","order_by":3,"name":"Xin Tong","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Tong","suffix":""},{"id":377060118,"identity":"aa2d74dc-00c5-47b9-b3a6-6d1605962c1f","order_by":4,"name":"Kaixuan Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Kaixuan","middleName":"","lastName":"Wang","suffix":""},{"id":377060119,"identity":"9ed7eb89-1937-484f-828a-28bc02eaf71b","order_by":5,"name":"Minmin Qiang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Minmin","middleName":"","lastName":"Qiang","suffix":""},{"id":377060120,"identity":"99dc4ce6-4097-4b18-a419-76abaaa64157","order_by":6,"name":"Qiong Zhang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Qiong","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2024-10-04 05:41:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5201652/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5201652/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":70199942,"identity":"d7d25369-fc36-4840-8dfa-de912ace196a","added_by":"auto","created_at":"2024-11-29 12:19:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2710125,"visible":true,"origin":"","legend":"\u003cp\u003eLocation of research site in the Loess Plateau and a sketch map of the experiment.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-5201652/v1/7ed9b4ee4b4724c745eaf687.png"},{"id":70199727,"identity":"4ff67815-d2b2-4535-8f13-43f93732a467","added_by":"auto","created_at":"2024-11-29 12:11:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":364472,"visible":true,"origin":"","legend":"\u003cp\u003eThe dynamic of soil temperature (a), water filled pore space (b), soil ammonium nitrogen contents (c) and soil nitrous nitrogen contents (d) during monitoring period. The downward black arrow indicated fertilization events.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-5201652/v1/fd62b7bf26a05ba6600a98c7.png"},{"id":70199729,"identity":"17c7ba51-644b-4527-85fa-b166535da54d","added_by":"auto","created_at":"2024-11-29 12:11:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":302041,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of nitrogen fertilizer on soil N\u003csub\u003e2\u003c/sub\u003eO (a), NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N (b), NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N (c) and bacterial (d) community in orchards of different ages. The error bar represents a 95% confidence interval (CI). The number of observations is shown next to the right Y-axis. a, age; N\u003csub\u003e2\u003c/sub\u003eO: nitrous oxide; NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N: nitrate nitrogen; NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N: ammonium nitrogen.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-5201652/v1/bcf284821686c05d75a8d9f0.png"},{"id":70200722,"identity":"1eba05b2-b4f4-4040-841c-9c786d056772","added_by":"auto","created_at":"2024-11-29 12:27:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":139113,"visible":true,"origin":"","legend":"\u003cp\u003eN\u003csub\u003e2\u003c/sub\u003eO fluxes and its dynamic change with time from different treatments (a), the black downward arrows indicate fertilization time. Distribution of N\u003csub\u003e2\u003c/sub\u003eO flux values under different treatments (b).\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-5201652/v1/e1c16578da8d2369e4633538.png"},{"id":70199943,"identity":"884b118a-43b8-453d-b8f1-56a6a2415fc0","added_by":"auto","created_at":"2024-11-29 12:19:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":245723,"visible":true,"origin":"","legend":"\u003cp\u003eThe bacteria (a) and fungi (b) relative abundance at the phylum level and AOB (c) and \u003cem\u003enir\u003c/em\u003eS (d) relative abundance at the species level.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-5201652/v1/efb86052ac2985c1def8e409.png"},{"id":70199734,"identity":"ac68e1bf-b2e7-42ac-9a7d-96ad4583a362","added_by":"auto","created_at":"2024-11-29 12:11:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":718277,"visible":true,"origin":"","legend":"\u003cp\u003eHierarchical clustering of bacteria (a) and fungi (b) using Bray-Curtis dissimilarity indices at the phylum level by the unweighted unifrac distances.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-5201652/v1/1e6a5dd3a78de9b7a7911ae9.png"},{"id":70199730,"identity":"f32dee8b-cbd3-41c3-86c9-b98e0773db96","added_by":"auto","created_at":"2024-11-29 12:11:34","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1306790,"visible":true,"origin":"","legend":"\u003cp\u003eThe PCoA analysis and Adonis analysis of soil bacteria (a), fungi (b), AOB (c) and \u003cem\u003enir\u003c/em\u003eS (d) from different treatments.\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-5201652/v1/09ce04be61cd066bde71b237.png"},{"id":70199944,"identity":"21dd802e-ba04-4548-a894-9dfc613951ed","added_by":"auto","created_at":"2024-11-29 12:19:34","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":268243,"visible":true,"origin":"","legend":"\u003cp\u003eHierarchical clustering of bacteria (a) and fungi (b) using Bray-Curtis dissimilarity indices at the phylum level by the unweighted unifrac distances.\u003c/p\u003e","description":"","filename":"Fig.8.png","url":"https://assets-eu.researchsquare.com/files/rs-5201652/v1/59c2b1e9f855b47d5a41a0d7.png"},{"id":70200723,"identity":"b4cc467b-0dff-4d28-a432-2ac219c1c155","added_by":"auto","created_at":"2024-11-29 12:27:34","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":3853703,"visible":true,"origin":"","legend":"\u003cp\u003eA heat-network map (a) was based on pairwise comparisons of environmental factors, with different colors representing spearman correlation coefficients. Relationships between bacteria, fungi, AOB and \u003cem\u003enir\u003c/em\u003eS and environmental factors were demonstrated by Mantel tests. Redundancy analysis and mapping were performed based on partial soil indexes and the absolute abundance of bacteria phylum, fungi phylum, AOB species and \u003cem\u003enir\u003c/em\u003eS species in young (b) and old (c) apple orchards. TN: total nitrogen; WC: soil water content; SOC: soil organic carbon; N\u003csub\u003e2\u003c/sub\u003eO: nitrous oxide; NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N: nitrate nitrogen; NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N: ammonium nitrogen; HPA: Hydrogen peroxidase activity; CA: cellulase activity; IA: invertase activity; UA: urease activity.\u003c/p\u003e","description":"","filename":"Fig.9.png","url":"https://assets-eu.researchsquare.com/files/rs-5201652/v1/fedd4807b92fb744c4f5dbee.png"},{"id":72061375,"identity":"57d2f925-b113-4a59-b1d2-22311c1d961b","added_by":"auto","created_at":"2024-12-21 10:44:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10357194,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5201652/v1/64278173-1f07-4928-a6ef-993145fb9128.pdf"},{"id":70199736,"identity":"e3459abb-fcb6-4e16-a4b2-c834a1a061fc","added_by":"auto","created_at":"2024-11-29 12:11:35","extension":"docx","order_by":15,"title":"","display":"","copyAsset":false,"role":"supplement","size":610845,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-5201652/v1/e0e8d7cf065fefcf025d12a3.docx"}],"financialInterests":"","formattedTitle":"Microbial Stimulation in Apple Orchards of Different Ages on the Loess Plateau: Poor Predictability of Increased Soil N2O Emissions","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAgricultural soils have been identified as one of the major nitrous oxide (N\u003csub\u003e2\u003c/sub\u003eO) sources, and excessive and repeated input of nitrogen (N) fertilizer (Gao et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhu et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and reactive N gas production, resulting in many environmental problems, such as increased N\u003csub\u003e2\u003c/sub\u003eO emissions (Fowler et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Tian et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In particular, N\u003csub\u003e2\u003c/sub\u003eO emissions from orchards have received widespread attention recently (Cheng et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Escanhoela et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zhou et al., \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eChina occupies a leading position among the apple producers in the world and China has the highest apple orchard acreage (accounting for 41.4%) and production (accounting for 46.9%) of the world\u0026rsquo;s total output, which has expanded nearly 3 times over the past 40 years and reached approximately 1.91\u0026nbsp;million hectares in 2020 due to the economic benefits of apple production FAO (2022). The Loess Plateau has been recognized as one of the most suitable apple production areas in the world; however, the expansion of fruit orchards raises serious concerns about the risk of high N\u003csub\u003e2\u003c/sub\u003eO emissions due to intensive management methods (Cheng et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Wu et al., \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Pang et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Field measurements have shown that the N\u003csub\u003e2\u003c/sub\u003eO emission factor from apple orchards on the Loess Plateau are as high as 0.658% (Pang et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), which is greater than that of cropland (0.58%) in nearby regions (Zhang et al., \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2017a\u003c/span\u003e). However, the limited available orchard measurements contribute to high uncertainties in the estimates of N\u003csub\u003e2\u003c/sub\u003eO emissions from the agricultural sector (Cui et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This limitation is all the more challenging due to the vast expansion of fruit cropping systems across the globe in the last decade. Therefore, a better understanding of the pattern and sources of N\u003csub\u003e2\u003c/sub\u003eO emissions from apple orchards is urgently needed to improve the global budget and formulate practical mitigation strategies, which could therefore strongly contribute to the mitigation of anthropogenic N\u003csub\u003e2\u003c/sub\u003eO emissions at national and global scales.\u003c/p\u003e \u003cp\u003eCompared with other upland orchards, apple orchards have gained more attention in investigations of plant-soil-microbial interactions because of their unique perennial cropping systems, chemical composition and nutrient use efficiency of root exudates depending on stand age, which can profoundly affect soil C and N availability and other related soil properties (Haney et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). As a consequence, the changes in C and N turnover may affect the diversity of soil microbial communities, which govern the rate of C and N mineralization and concomitant greenhouse gas (GHG) release, including N\u003csub\u003e2\u003c/sub\u003eO fluxes (Ceja-Navarro et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Previous results showed that stand ages can significantly affect soil conditions and thus lead to changes in soil bacterial community structure (Zheng et al., \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and it is speculated that the feedback responses of the associated microorganisms related to N\u003csub\u003e2\u003c/sub\u003eO emissions from stand age growth may be different. Therefore, research on apple orchards should be based on different stand ages. In addition, the perennial nature of the communities in the rows of trees means that soil management is constrained by time and space, and above and belowground ecosystems have greater stability than those associated with annual cropping. The low frequency of physical disturbance also means there are ample opportunities for the development of multiple bidirectional soil-microbial interactions (Deakin et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eN\u003csub\u003e2\u003c/sub\u003eO emissions from northern calcareous soils (well-aerated and high pH conditions) primarily result from the microbial nitrification process (Wolf and Russow, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Ammonia oxidation is the primary and rate-limiting step in nitrification by ammonia-oxidizing bacteria (AOB) and archaea (AOA) (He et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), which are major contributors to N\u003csub\u003e2\u003c/sub\u003eO production in soil. Moreover, AOB prefer N-rich or fertilized soils (Di et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), and recent studies have also shown that AOB play a more important role than AOA in NH\u003csub\u003e3\u003c/sub\u003e oxidation in high N input and fertilized calcareous field soils (Zhong et al., \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Tao et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In addition, nitrite is converted to NO or N\u003csub\u003e2\u003c/sub\u003eO by nitrite reductase (NIR) in denitrification. With the increasing age of perennial crops was reported to shift both \u003cem\u003enir\u003c/em\u003eS- and \u003cem\u003enir\u003c/em\u003eK-denitrifying bacterial communities under fertilization conditions in Ontario, Canada (Thompson et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Deakin et al., (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) also found that most of the differences in soil microbial community structure were due to large-scale differences between orchards, even within-orchards, and spatial relationships in microbial community structure differed between orchards and were not predictable. Xu et al., (\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) demonstrated a positive correlation between the enhancement of soil biological conditions and the time required for vegetation recovery. Xue et al., (\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) showed that the nitrification rate of tea garden soil showed an increasing trend in 8\u0026ndash;50 years, and then a decreasing trend in 50\u0026ndash;90 years. Plassart et al., (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) reported that fungal genetic diversity was closely related to grassland age. However, few studies have offered a rigorous assessment of the microbial community coupled with a rigorous assessment of N\u003csub\u003e2\u003c/sub\u003eO production rates from different stand ages from apple orchards. In particular, it is necessary to explore the contributions of AOB and \u003cem\u003enir\u003c/em\u003eS to N\u003csub\u003e2\u003c/sub\u003eO emissions in apple orchards on the Loess Plateau of China.\u003c/p\u003e \u003cp\u003eTo elucidate how the different stand ages of orchards affect potential N\u003csub\u003e2\u003c/sub\u003eO emission rates and the related microbiological mechanism, we conducted a nearly 3-year field experiment to study N\u003csub\u003e2\u003c/sub\u003eO emissions and whether the relative abundance and composition of bacteria, fungi, AOB and \u003cem\u003enir\u003c/em\u003eS were changed by different stand ages in the Loess Plateau of China. We hypothesized that the change in stand age would alter the microbial community structure by affecting the soil moisture, carbon and nitrogen levels, thereby impacting N\u003csub\u003e2\u003c/sub\u003eO emissions. Our objectives were to i) investigate the effects of stand age and fertilizer in the presence or absence on N\u003csub\u003e2\u003c/sub\u003eO emissions and the community composition of microorganisms; ii) evaluate the relative contribution of microorganisms to N\u003csub\u003e2\u003c/sub\u003eO emissions.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eSite description and experimental design\u003c/h2\u003e\n \u003cp\u003eThe study was located in the Wangdonggou Catchment, Changwu County, Shaanxi Province, China (35\u0026deg;13\u0026prime;N, 107\u0026deg;40\u0026prime;E; altitude 1200 m), which is a typical apple growing area in the Loess Plateau (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). In the region, the average annual temperature and precipitation were 9.1\u0026deg;C and 582 mm, respectively. The annual average cumulative sunshine time is 2115 hours, and the annual average solar radiation is 5077 MJ m\u003csup\u003e\u0026ndash;2\u003c/sup\u003e, which is characterized by a warm temperate subhumid continental monsoon climate. The soil type at the experimental site is dark loessial soil from the Chinese Soil Classification System and Cumulic Haplustoll from the United States Department of Agriculture (Zhang et al., \u003cspan class=\"CitationRef\"\u003e2019a\u003c/span\u003e), which was developed from Malan loess parent material. Meteorological data were obtained from the Changwu State Key Agri-Ecological Station on Loess Plateau, Chinese Academy of Sciences (Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eThis study started in July 2017, and the experimental field was a conventional cropland (rotation of winter wheat and summer maize) prior to apple planting. The two apple (\u003cem\u003eMalus pumila\u003c/em\u003e Mill) orchards of different stand ages were selected as research objects, as follows: 5 years old (established in 2012) and 25 years old (established in 1992). The apple trees in the two orchards are spaced 5 m apart in rows and 2 m apart in plants. The basic soil properties (0\u0026ndash;20 cm) of the two orchards soils at the beginning of the experiment are shown in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Each treatment was replicated 3 times and included fertilized or unfertilized plots in both orchards: (1) conventional fertilization of the young orchard (5F), (2) conventional fertilization of the old orchard (25F), (3) no fertilizer in the young orchard (5CK), (4) no fertilizer in the old orchard (25CK). Five well-grown apple trees with no diseases or insect pests were selected in each experimental plot and every plot area was approximately 50 m\u003csup\u003e2\u003c/sup\u003e. During the research period, no irrigation and tillage events were provided, while necessary weeding and pest control were carried out in accordance with local management practices. The fertilization method involved digging ditches (width 20 cm and depth 20 cm), and the fertilization bands were at a distance of 1 m on both sides of the tree row (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Fertilization details are shown in Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e. \u0026nbsp;\u003c/p\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eTopsoil (0\u0026ndash;20 cm) basic properties in the plots from different stand ages.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSample\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eOrganic C\u003c/p\u003e\n \u003cp\u003e(g kg\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTotal N\u003c/p\u003e\n \u003cp\u003e(g kg\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eC/N\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N\u003c/p\u003e\n \u003cp\u003e(mg kg\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N\u003c/p\u003e\n \u003cp\u003e(mg kg\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003epH\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBulk density\u003c/p\u003e\n \u003cp\u003e(g cm\u003csup\u003e\u0026ndash;3\u003c/sup\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSand (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSilt (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eClay (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5yr\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e65.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e73.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25yr\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e66.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e73.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eN\u003csub\u003e2\u003c/sub\u003eO analysis determination\u003c/h3\u003e\n\u003cp\u003eN\u003csub\u003e2\u003c/sub\u003eO emissions were measured from July 2017 to December 2019 by static chamber-gas chromatographic techniques. N\u003csub\u003e2\u003c/sub\u003eO samples were collected from 9:00 to 11:00 a.m. every week and the interval was extended to 10 days after the soil froze, and continuous sampling every other day for one week if fertilization was encountered. Insulated sample chambers size 0.25 m \u0026times; 0.25 m \u0026times; 0.5 m and a stainless-steel base (0.25 m \u0026times; 0.25 m \u0026times; 0.1 m) with groove is equipped. A small electric fan was fixed on the top of the chamber to evenly mix the air during sampling. Specific sampling details and soil temperature (ST) monitoring are consistent with those described by (Han et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e). N\u003csub\u003e2\u003c/sub\u003eO gases were measured by a gas chromatograph instrument (7890A, Agilent US). According to (Zhang et al., \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e), the N\u003csub\u003e2\u003c/sub\u003eO concentration was quantified by an electron capture detector (ECD, 300\u0026deg;C). The chromatographic column was an 80/100 mesh SS-2 m \u0026times; 2 mm Porapak Q, and N\u003csub\u003e2\u003c/sub\u003e with a flow rate of 40 cm\u003csup\u003e3\u003c/sup\u003e\u0026middot;min\u003csup\u003e\u0026ndash;1\u003c/sup\u003e was used as a carrier gas.\u003c/p\u003e\n\u003cp\u003eAccording to the change in gas concentrations in four continuous samples over time, the soil N\u003csub\u003e2\u003c/sub\u003eO flux was calculated (\u0026micro;g N m\u003csup\u003e\u0026ndash;2\u003c/sup\u003e h\u003csup\u003e\u0026ndash;1\u003c/sup\u003e). The formula is as follows:\u003c/p\u003e\n\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e$$\\:\\text{Flux}\\text{=}\\frac{\\text{M}}{\\text{22.4}}\\text{\u0026times;}\\text{H}\\text{\u0026times;}\\frac{{\\text{d}}_{\\text{c}}}{{\\text{d}}_{\\text{t}}}\\text{\u0026times;}\\frac{\\text{273}}{\\text{273+}\\text{T}}$$\u003c/div\u003e\n \u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\n\u003c/div\u003e\n\u003cp\u003ewhere \u003cem\u003eM\u003c/em\u003e is the molar mass (12 g mol\u003csup\u003e\u0026ndash;1\u003c/sup\u003e) of the N atom in N\u003csub\u003e2\u003c/sub\u003eO, \u003cem\u003eH\u003c/em\u003e is the chamber height (m), d\u003cem\u003ec\u003c/em\u003e/d\u003cem\u003et\u003c/em\u003e (\u0026micro;L L\u003csup\u003e\u0026ndash;1\u003c/sup\u003e min\u003csup\u003e\u0026ndash;1\u003c/sup\u003e) is the slope of the regression curve of the N\u003csub\u003e2\u003c/sub\u003eO concentration over time, and \u003cem\u003eT\u003c/em\u003e is the air temperature in the chamber.\u003c/p\u003e\n\u003cp\u003eThe total N\u003csub\u003e2\u003c/sub\u003eO emissions during the monitoring period were obtained from the average N\u003csub\u003e2\u003c/sub\u003eO flux and the monitoring period (Zou et al., \u003cspan class=\"CitationRef\"\u003e2005\u003c/span\u003e). Considering uneven fertilization in apple orchards, N\u003csub\u003e2\u003c/sub\u003eO emissions in the different age plots should be calculated by the following equation (Zhang et al., \u003cspan class=\"CitationRef\"\u003e2019b\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\u003cimg src=\"https://myfiles.space/user_files/122228_c8a1650c59388082/122228_custom_files/img1732881185.png\"\u003e\u003cbr\u003e\u003c/div\u003e\n\u003c/div\u003e\n\u003cp\u003ewhere \u003cem\u003eF\u003c/em\u003e and \u003cem\u003enF\u003c/em\u003e are cumulative N\u003csub\u003e2\u003c/sub\u003eO emissions (kg N ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e) from fertilized and unfertilized regions, respectively, and \u003cem\u003ea\u003c/em\u003e and \u003cem\u003eb\u003c/em\u003e are the areas (m\u003csup\u003e2\u003c/sup\u003e) of fertilized and unfertilized plots, respectively.\u003c/p\u003e\n\u003ch3\u003eSampling and soil physicochemical properties and enzyme activity analysis\u003c/h3\u003e\n\u003cp\u003eTopsoil (0\u0026ndash;20 cm) around the chamber was collected by a soil auger (diameter: 5 cm), which was synchronized with the greenhouse gas collection. Three samples were collected and mixed into one sample for each plot, and each treatment was repeated three times. Soil samples were brought back to the laboratory in Ziplock bags, which were used for soil physical and chemical property analysis. After air drying, the soil was passed through a 2 mm sieve to measure pH (soil-water ratio was 1:2.5) and a 0.15 mm sieve to measure soil organic carbon (Walkley-Black potassium dichromate), total N (Kjeldahl methods), hydrogen peroxidase, cellulase, invertase and urease. At the same time, topsoil samples were collected to monitor the water content and ammonium nitrate N during greenhouse gas collection. The mass moisture content of the soil was determined by the drying method and fresh soil was used to determine the contents of nitrate and ammonium nitrogen, which were extracted with 1 mol/L KCl and analyzed by a continuous flow analyzer (SEAL AutoAnalyzer 3, Germany). Water filled pore space (WFPS) was calculated according to (Han et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e). The determination methods of soil physical and chemical properties and enzymes mentioned above refer to Bao (\u003cspan class=\"CitationRef\"\u003e2008\u003c/span\u003e) and Guan (\u003cspan class=\"CitationRef\"\u003e1986\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eMicrobial analysis\u003c/h3\u003e\n\u003cp\u003eSoil samples (0\u0026ndash;20 cm) for microbial analysis were collected by a soil auger (diameter: 5 cm, autoclave sterilization) during the N\u003csub\u003e2\u003c/sub\u003eO flux peak after fertilization (10 April 2018, 2 days after fertilization) at the fertilizer bands and non-fertilized bands, and the samples were stored in a -80℃ refrigerator for DNA coextraction. Three soil samples were taken from each plot and mixed into one soil sample to be tested. Microbial DNA was obtained from fresh soil samples, and the concentration and purity were checked by agarose gel electrophoresis. Then, sterile water was used to dilute the DNA sample and the diluted genomic DNA was used as a template. Specific primers (bacteria: 515 F 5\u0026apos;- GTGCCAGCMGCCGCGGTAA-3\u0026apos; and 806R 5\u0026apos;-GGACTACNNGGGTATCTAAT-3\u0026apos; (Caporaso et al., \u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e); fungi: ITS5 5\u0026apos;-GGAAGTAAAAGTCGTAACAAGG-3\u0026apos; and ITS2 5\u0026apos;-GCTGCGTTCTTCATCGATGC-3\u0026apos;) (Bellemain et al., \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e); \u003cem\u003enir\u003c/em\u003eS: cd3aF 5\u0026apos;-GTSAACGTSAAGGARACSGG-3\u0026apos; and R3cd 5\u0026apos;-GASTTCGGRTGSGTCTTGA-3\u0026apos; (Michotey et al., \u003cspan class=\"CitationRef\"\u003e2000\u003c/span\u003e); AOB-\u003cem\u003eamo\u003c/em\u003eA: \u003cem\u003eamo\u003c/em\u003eA1F 5\u0026apos;-GGGGTTTCTACTGGTGGT-3\u0026apos; and \u003cem\u003eamo\u003c/em\u003eA2R 5\u0026apos;-CCCCTCKGSAAAGCCTTCTTC-3\u0026apos; (Rotthauwe et al., \u003cspan class=\"CitationRef\"\u003e1997\u003c/span\u003e) and high-fidelity enzymes were used for PCR amplification according to the selected regions. The Ion Plus Fragment Library Kit 48 rxns (Thermo Fisher) was used to construct the library and sequencing was performed using the Ion S5 platform (Thermo Fisher) at Novogene Bioinformatics Technology Co., Ltd., Beijing, China. Raw sequences were obtained by cutting off barcodes and primers and using FLASH spliced reads (Magoc and Salzberg, \u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e); then the filtered data were screened out via a strict filtering process (Caporaso et al., \u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e). The extracted clean sequences were clustered to estimated operational taxonomic unit (OTU) numbers at 97% identity. OTU sequences were annotated with the SILVA (bacteria), UNITE (fungi), and FGPR functional gene (AOB and \u003cem\u003enir\u003c/em\u003eS) databases.\u003c/p\u003e\n\u003ch3\u003eData analysis and statistics\u003c/h3\u003e\n\u003cp\u003eMicrosoft Office Excel 2019 (Microsoft, USA) was used to calculate the N\u003csub\u003e2\u003c/sub\u003eO fluxes and cumulative emissions, averages, standard deviations and significance analyses were calculated using IBM SPSS 25 (IBM, USA). Figures were designed by ArcGIS 10.6 (ESRI, USA), Origin Pro 2018 (OriginLab, USA), SigmaPlot (version 15.0) and R software (version 4.1.1). Hierarchical clustering of bacteria and fungi was carried out by QIIME (version 1.9.1) based on unweighted UniFrac distances. Principal coordinates component analysis (PCoA, by WGCNA, stats and ggplot2 packages), Mantel test (by vegan package), Spearman correlation analysis of various soil indices (by psych package) and Redundancy analysis of environmental factors and microbial communities (RDA, by vegan, ggplot2 and ggrepe1 packages) were performed using R software. R software was used to determine the effects of stand years and fertilization on the microbial community via permutational multivariate analysis of variance (PERMANOVA, by vegan packages).\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\"\u003e\n \u003ch2\u003eDynamic changes in soil temperature, WFPS and mineral nitrogen\u003c/h2\u003e\n \u003cp\u003eST was recorded from July 2017 to December 2020, which showed a similar dynamic trend and an obvious seasonal variation characteristic (Fig. \u003cspan\u003e2\u003c/span\u003ea). During the experimental periods the ST ranged from \u0026minus;\u0026thinsp;7.1℃ to 22.7℃, with the highest and lowest ST occurring in July and January, respectively. In addition, topsoil WFPS monitored during nonfreezing periods changed with precipitation and began to increase in April (Fig. \u003cspan\u003e2\u003c/span\u003eb), which varied from 15.5\u0026ndash;82.5% and showed a general trend of low in winter and spring and high in summer and autumn, with mean values of 50.0% (5CK), 48.3% (5F), 51.2% (25CK) and 55.0% (25F) in different plots, respectively.\u003c/p\u003e\n \u003cp\u003eThe soil mineral N content was influenced by N inputs, and higher contents of mineral N were present at the fertilization site (5F and 25F, Fig. \u003cspan\u003e2\u003c/span\u003ec and d). The soil NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N content generally changed slightly most of the time (Fig. \u003cspan\u003e2\u003c/span\u003ec), except in April 2018 and April 2019, when urea was applied, and both fertilized orchard NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N contents showed sharp fluctuations and peaked (95.8 mg N kg\u003csup\u003e\u0026ndash;1\u003c/sup\u003e-112.3 mg N kg\u003csup\u003e\u0026ndash;1\u003c/sup\u003e) within 3\u0026ndash;5 days. However, compound fertilizer addition did not cause the rapid increase in NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N contents. Soil nitrate N (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N) was sensitive to compound fertilizer and urea, and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N contents in 5F and 25F increased sharply after fertilization and generally lasted a month before falling back (Fig. \u003cspan\u003e2\u003c/span\u003ed). During the experimental period, the average NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N contents of fertilized soil (78.6 mg N kg\u003csup\u003e\u0026ndash;1\u003c/sup\u003e-79.4 mg N kg\u003csup\u003e\u0026ndash;1\u003c/sup\u003e) were significantly higher than those of non-fertilized soil (38.4 mg N kg\u003csup\u003e\u0026ndash;1\u003c/sup\u003e-47.4 mg N kg\u003csup\u003e\u0026ndash;1\u003c/sup\u003e). In addition, during the trial period, NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N increased by 45.9% and 104.2%, and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N increased by 48.6% and 80.2%, respectively, in 5a and 25a apple orchards after fertilization according to the weighted effect size of each category (Fig. S2). Our global meta-analysis of field observations revealed significant increases in NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N across orchards aged 0\u0026ndash;5 years, 5\u0026ndash;15 years, and 15\u0026ndash;25 years by 61.63%, 310.08%, and 151.4%, respectively. Similarly, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N levels showed significant increases of 92.5%, 171.2%, and 279.1% across the same age categories (Fig. \u003cspan\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eN\u003csub\u003e2\u003c/sub\u003eO emissions from young and old apple orchards\u003c/h3\u003e\n\u003cp\u003eDuring the study period, the average N\u003csub\u003e2\u003c/sub\u003eO flux ranged from 16.7 \u0026micro;g N m\u003csup\u003e\u0026ndash;2\u003c/sup\u003e h\u003csup\u003e\u0026ndash;1\u003c/sup\u003e to 71.9 \u0026micro;g N m\u003csup\u003e\u0026ndash;2\u003c/sup\u003e h\u003csup\u003e\u0026ndash;1\u003c/sup\u003e among the different treatments (Fig. \u003cspan\u003e4\u003c/span\u003ea), and different stand ages showed obvious seasonal variation. As shown in Fig. \u003cspan\u003e4\u003c/span\u003eb, soil N\u003csub\u003e2\u003c/sub\u003eO fluxes were mostly distributed below 50 \u0026micro;g N m\u003csup\u003e\u0026ndash;2\u003c/sup\u003e h\u003csup\u003e\u0026ndash;1\u003c/sup\u003e, accounting for 91.9%, 73.3%, 93.3% and 65.9% of the total monitoring events in 5CK, 5F, 25CK and 25F, respectively. A pulse emission peak was recorded within a week after top dressing in summer, the N\u003csub\u003e2\u003c/sub\u003eO fluxes from 5F and 25F peaked at 320.3 \u0026micro;g N m\u003csup\u003e\u0026ndash;2\u003c/sup\u003e h\u003csup\u003e\u0026ndash;1\u003c/sup\u003e and 1067.0 \u0026micro;g N m\u003csup\u003e\u0026ndash;2\u003c/sup\u003e h\u003csup\u003e\u0026ndash;1\u003c/sup\u003e in July 2018 and 319.3 \u0026micro;g N m\u003csup\u003e\u0026ndash;2\u003c/sup\u003e h\u003csup\u003e\u0026ndash;1\u003c/sup\u003e and 469.8 \u0026micro;g N m\u003csup\u003e\u0026ndash;2\u003c/sup\u003e h\u003csup\u003e\u0026ndash;1\u003c/sup\u003e in July 2019, respectively. N\u003csub\u003e2\u003c/sub\u003eO emissions from old orchards were significantly higher than those from young orchards with N addition (252.7%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The cumulative N\u003csub\u003e2\u003c/sub\u003eO emissions of fertilized plots were observably higher than that in nonfertilized plots and the highest emissions occurred in fertilized old orchards (Table \u003cspan\u003e2\u003c/span\u003e, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, the differences in N\u003csub\u003e2\u003c/sub\u003eO efflux rates and cumulative N\u003csub\u003e2\u003c/sub\u003eO emissions between 5CK and 25CK were not significant. The N\u003csub\u003e2\u003c/sub\u003eO emission factors of the young orchard and old orchard were 0.37% and 0.54%, respectively. Our global meta-analysis consistently showed an increasing trend in N\u003csub\u003e2\u003c/sub\u003eO emissions following nitrogen fertilizer application across orchards aged 0\u0026ndash;5 years, 5\u0026ndash;15 years, and 15\u0026ndash;25 years. Specifically, N\u003csub\u003e2\u003c/sub\u003eO increased significantly by 89.0%, 39.7%, and 93.6%, respectively (Fig. \u003cspan\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eTable 2 The average N\u003csub\u003e2\u003c/sub\u003eO fluxes, annual cumulative N\u003csub\u003e2\u003c/sub\u003eO emissions and its emission factors (EF) in different treatments during experimental period.\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eTreatments\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eN\u003csub\u003e2\u003c/sub\u003eO fluxes (\u0026mu;g N m\u003csup\u003e\u0026ndash;2\u003c/sup\u003e h\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eN\u003csub\u003e2\u003c/sub\u003eO cumulative emission (kg N ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003eyr\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eN\u003csub\u003e2\u003c/sub\u003eO Emission factor (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5CK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e20.72 \u0026plusmn; 4.13 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.38 \u0026plusmn; 0.03 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5N\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e46.78 \u0026plusmn; 4.34 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3.92 \u0026plusmn; 0.09 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.37\u0026plusmn;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e25CK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e23.94 \u0026plusmn; 2.77 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.57 \u0026plusmn; 0.11 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e25N\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e65.23 \u0026plusmn; 3.95 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5.60 \u0026plusmn; 0.22 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.54\u0026plusmn;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eDifferent lowercase letters indicate significant difference between treatments (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e\n\u003cdiv\u003e\u003cbr\u003e\u003c/div\u003e\n\u003cdiv\u003e\n \u003cp\u003eTable 3 Alpha diversity index of bacteria, fungi, AOB and \u003cem\u003enir\u003c/em\u003eS.\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eTypes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eTreatments\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eShannon\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSimpson\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eChao1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\"\u003e\n \u003cp\u003eBacteria\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5CK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e9.41\u0026plusmn;0.25 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.99\u0026plusmn;0.003 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3383.17\u0026plusmn;1013.88 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e5N\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e9.26\u0026plusmn;0.34 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.99\u0026plusmn;0.004 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2636.59\u0026plusmn;227.91 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e25CK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e9.49\u0026plusmn;0.33 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.99\u0026plusmn;0.003 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2998.73\u0026plusmn;74.08 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e25N\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e8.76\u0026plusmn;1.37 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.98\u0026plusmn;0.029 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2921.15\u0026plusmn;198.51 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\"\u003e\n \u003cp\u003eFungi\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5CK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.06\u0026plusmn;0.22 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.98\u0026plusmn;0.005 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e924.88\u0026plusmn;53.99 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e5N\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6.77\u0026plusmn;0.34 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.97\u0026plusmn;0.016 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e983.90\u0026plusmn;77.06 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e25CK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6.40\u0026plusmn;0.30 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.96\u0026plusmn;0.013 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e892.71\u0026plusmn;36.79 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e25N\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6.68\u0026plusmn;0.51 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.97\u0026plusmn;0.008 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e870.27\u0026plusmn;87.57 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\"\u003e\n \u003cp\u003eAOB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5CK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6.45\u0026plusmn;0.26 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.92\u0026plusmn;0.007 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4443.29\u0026plusmn;1012.30 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e5N\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6.56\u0026plusmn;0.22 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.93\u0026plusmn;0.021 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5342.00\u0026plusmn;1246.57 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e25CK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.82\u0026plusmn;0.12 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.98\u0026plusmn;0.003 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5096.25\u0026plusmn;256.04 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e25N\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.41\u0026plusmn;0.29 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.97\u0026plusmn;0.002 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5641.00\u0026plusmn;1732.34 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\"\u003e\n \u003cp\u003e\u003cem\u003enir\u003c/em\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5CK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10.02\u0026plusmn;0.41 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.99\u0026plusmn;0.002 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e8311.17\u0026plusmn;3414.64 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e5N\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e9.66\u0026plusmn;0.16 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.99\u0026plusmn;0.001 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7325.60\u0026plusmn;1647.34 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e25CK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10.14\u0026plusmn;0.29 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.99\u0026plusmn;0.001 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10833.69\u0026plusmn;178.40 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e25N\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e9.88\u0026plusmn;0.09 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.99\u0026plusmn;0.001 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e8358.06\u0026plusmn;2952.16 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003eDifferent lowercase letters indicate significant difference between treatments (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05)\n\u003c/div\u003e\n\u003cdiv\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv id=\"Sec11\"\u003e\n \u003ch2\u003eBacterial and fungal analysis\u003c/h2\u003e\n \u003cp\u003eThe average numbers of excellent-quality base sequences from bacteria and fungi were 76482\u0026ndash;79825 and 80130\u0026ndash;80212, respectively. The results indicated that the composition of bacteria was similar under different treatments, but there were some differences in the relative abundance of dominant bacteria in the soil of the young and old orchards (Fig. \u003cspan\u003e5\u003c/span\u003ea). \u003cem\u003eProteobacteria\u003c/em\u003e (25.7%-37.2%), \u003cem\u003eBacteroidetes\u003c/em\u003e (15.4%-19.2%), \u003cem\u003eActinobacteria\u003c/em\u003e (15.3%-22.2%) and \u003cem\u003eAcidobacteria\u003c/em\u003e (9.1%-11.7%) were the dominant phyla in the 5CK, 5F and 25CK treatments, accounting for 76.4%, 75.4% and 81.6% of the total bacterial abundance, respectively. It should be noted that \u003cem\u003eFirmicutes\u003c/em\u003e was a major dominant phylum in the 25F treatment, contributing 21.9% of total bacterial abundance, which is quite different from other treatments. The hierarchical clustering of bacterial phyla also significantly suggested that the bacterial community composition of 25F was independent of other treatments, and the abundance of \u003cem\u003eFirmicutes\u003c/em\u003e in 25F obviously increased. The relative abundance of some phyla of bacteria of 25F was also different from that of the young orchard (Fig. \u003cspan\u003e6\u003c/span\u003ea). The relative abundance of the top ten fungi at the phylum level accounted for 60.1% (5CK), 56.2% (5F), 61.2% (25CK) and 66.9% (25F) of the total abundance, respectively (Fig. \u003cspan\u003e5\u003c/span\u003eb). \u003cem\u003eAscomycota\u003c/em\u003e was the most abundant fungus in the young apple orchard, accounting for 45.4% and 44.8% in 5CK and 5F, while the proportion of other fungi was low. In the old apple orchard, \u003cem\u003eAscomycota\u003c/em\u003e and \u003cem\u003eBasidiomycota\u003c/em\u003e were the top two fungal phyla, and the proportions reached 35.9%-40.0% and 19.6%-27.4%, respectively. As illustrated in Fig. \u003cspan\u003e6\u003c/span\u003eb, UPGMA clustering indicated that the soil fungal communities of young and old orchards were divided into two branches at the phylum level, \u003cem\u003eAscomycota\u003c/em\u003e decreased and \u003cem\u003eBasidiomycota\u003c/em\u003e increased in old orchard soil.\u003c/p\u003e\n \u003cp\u003eIn this study, alpha and beta diversity analyses were used to determine the differences in soil bacterial and fungal community compositions and the number of species between young and old apple orchard soils. The results of alpha diversity analysis showed that the Shannon, Simpson and Chao1 indices were not significantly different between the different treatments (Table \u003cspan\u003e3\u003c/span\u003e), indicating that the number and composition of soil bacteria and fungi in orchards of different stand ages were similar. According to the OTU tables of the bacterial and fungal communities, PCoA analysis obtained Fig. \u003cspan\u003e7\u003c/span\u003ea and b. The result of PCoA showed that the elliptical confidence of bacterial communities in 5F deviates from that of 25CK and 25F. Combined with Adonis analysis, there were significant differences in bacterial communities between young orchards with fertilizer and old orchards (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and the bacterial communities of orchards of the same stand ages were similar regardless of fertilizer inputs. The composition of the fungal community composition between young and old orchards was relatively dispersed without overlap, and the significant differences were found between 5CK and 25F, 5F and 25CK and 5F and 25F via the results of Adonis analyses (Fig. \u003cspan\u003e7\u003c/span\u003eb, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\n \u003cdiv\u003e\n \u003cp\u003eTable 4 Environmental factors corresponding to microorganisms.\u003c/p\u003e\n \u003cdiv align=\"center\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"924\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eTN\u003c/p\u003e\n \u003cp\u003e(g kg\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003epH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eWC\u003c/p\u003e\n \u003cp\u003e(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSOC\u003c/p\u003e\n \u003cp\u003e(g kg\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eN\u003csub\u003e2\u003c/sub\u003eO fluxes\u003c/p\u003e\n \u003cp\u003e(\u0026mu;g N m\u003csup\u003e\u0026ndash;2\u003c/sup\u003eh\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N\u003c/p\u003e\n \u003cp\u003e(mg kg\u003csup\u003e\u0026minus;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N\u003c/p\u003e\n \u003cp\u003e(mg kg\u003csup\u003e\u0026minus;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eHPA\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(mg g\u003csup\u003e\u0026minus;1\u003c/sup\u003eh\u003csup\u003e\u0026minus;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eCA\u003c/p\u003e\n \u003cp\u003e(\u0026mu;g g\u003csup\u003e\u0026ndash;1\u003c/sup\u003ed\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eIA\u003c/p\u003e\n \u003cp\u003e(mg g\u003csup\u003e\u0026ndash;1\u003c/sup\u003ed\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eUA\u003c/p\u003e\n \u003cp\u003e(mg NH\u003csub\u003e3\u003c/sub\u003e-N g\u003csup\u003e\u0026ndash;1\u003c/sup\u003ed\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e5CK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.19\u0026plusmn;0.10b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e8.20\u0026plusmn;0.06a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e11.53\u0026plusmn;0.95b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10.36\u0026plusmn;1.20b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e8.17\u0026plusmn;2.23c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e28.64\u0026plusmn;3.49b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.01\u0026plusmn;0.43b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e45.22\u0026plusmn;0.34a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e82.87\u0026plusmn;3.91b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e43.12\u0026plusmn;2.02a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.01\u0026plusmn;0.02c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e5N\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.27\u0026plusmn;0.15b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e8.00\u0026plusmn;0.28a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e11.18\u0026plusmn;1.93b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10.13\u0026plusmn;0.40b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e95.41\u0026plusmn;24.50b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e152.63\u0026plusmn;70.59a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.93\u0026plusmn;1.50b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e43.68\u0026plusmn;0.27a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e89.83\u0026plusmn;4.61b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e45.00\u0026plusmn;0.48a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.04\u0026plusmn;0.03c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e25CK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.46\u0026plusmn;0.25ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e8.19\u0026plusmn;0.16a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e13.89\u0026plusmn;1.54b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e12.65\u0026plusmn;1.54ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e39.87\u0026plusmn;0.18c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e56.13\u0026plusmn;25.29b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.56\u0026plusmn;2.94b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e35.78\u0026plusmn;0.23b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e82.47\u0026plusmn;1.88b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e26.90\u0026plusmn;0.62c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.65\u0026plusmn;0.02b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e25N\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.70\u0026plusmn;0.07a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e8.06\u0026plusmn;0.10a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e18.88\u0026plusmn;0.89a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e13.70\u0026plusmn;1.91a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e233.12\u0026plusmn;47.70a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e233.09\u0026plusmn;26.00a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e15.59\u0026plusmn;2.89a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e26.91\u0026plusmn;1.59c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e103.80\u0026plusmn;4.51a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e31.28\u0026plusmn;1.26b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.95\u0026plusmn;0.08a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eDifferent lowercase letters indicate significant difference between treatments (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05). TN: Soil total nitrogen; WC: Mass moisture content of soil; SOC: Soil organic carbon; NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N: Soil nitrate nitrogen; NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N: Soil ammonium nitrogen; HPA: Hydrogen peroxidase activity; CA: Cellulase activity; IA: Invertase activity; UA: Urease activity.\u003c/p\u003e\u0026nbsp;\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\"\u003e\n \u003ch2\u003eAmmonia oxidizing and denitrifying bacterial community\u003c/h2\u003e\n \u003cp\u003eAs shown in Fig. \u003cspan\u003e5\u003c/span\u003ec, d, many sequences came from uncultured strains. The dominant AOB species were \u003cem\u003eUncultured_bacterium\u003c/em\u003e, \u003cem\u003eUncultured_ammonia_oxidizing_bacterium\u003c/em\u003e and \u003cem\u003eNitrosospira_sp_Nv6\u003c/em\u003e, and the dominant species of denitrifying bacteria were \u003cem\u003euncultured_bacterium\u003c/em\u003e, \u003cem\u003ePseudomonas_marginalis\u003c/em\u003e and \u003cem\u003euncultured_organism\u003c/em\u003e. It is clear that a higher proportion of annotated AOB species in older orchard soil was higher, and the relative abundance of \u003cem\u003eUncultured_bacterium\u003c/em\u003e in the old orchard was significantly increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Alpha diversity indices showed that Shannon and Simpson showed an increasing trend with increasing planting years, suggesting that the increase in AOB community diversity may be related to stand age (Table \u003cspan\u003e3\u003c/span\u003e, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). However, the Chao1 index of each treatment showed no significant difference, indicating that there was no difference in the number of AOB species in orchards of different stand ages. Furthermore, there were no significant differences in the Shannon, Simpson and Chao1 indices of \u003cem\u003enir\u003c/em\u003eS (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Table \u003cspan\u003e3\u003c/span\u003e). PCoA analysis showed that the elliptical confidence of AOB communities in the new orchard deviated from that in the old orchard, while the distribution of AOB community composition in the same-age orchard relatively concentrated, indicating that the stand ages of orchards caused the difference in AOB community structure (Fig. \u003cspan\u003e7\u003c/span\u003ec). Adonis analysis further confirmed the significant difference in AOB diversity between 5F and 25CK and 5F and 25F (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). According to the results of PCoA, the \u003cem\u003enir\u003c/em\u003eS bacterial community composition distribution of each treatment was concentrated, and the confidence ellipses had obvious overlapping areas. PCoA and Adonis combined were used to analyze the \u003cem\u003enir\u003c/em\u003eS bacterial diversity of all treatments, and there was a significant difference between 5F and 25CK (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), but no difference among the other treatments (Fig. \u003cspan\u003e7\u003c/span\u003ed, P\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\n \u003cp\u003eIn general, permutational multivariate analysis of variance (PERMANOVA) showed that the soil microbial community in apple orchards was dramatically influenced by stand age and the interaction between stand age and fertilization addition, which explained 22.2% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.022) and 36.6% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.003) of the variation, respectively. However, the effect of fertilization alone on the microbial community was not significant, and there was still a 36.8% unexplained portion (Fig. \u003cspan\u003e8\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\"\u003e\n \u003ch2\u003eCorrelation among N\u003csub\u003e2\u003c/sub\u003eO emission, environmental factors and microorganisms\u003c/h2\u003e\n \u003cp\u003eAs shown in Table \u003cspan\u003e4\u003c/span\u003e, the soil properties except pH were altered greatly among the different treatments, and 25F had the highest Soil organic carbon (SOC), Soil total nitrogen (TN), soil water content (WC), NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, cellulase activity (CA) and urease activity (UA). Pairwise correlation analysis based on environmental factors (Fig. \u003cspan\u003e9\u003c/span\u003ea) indicated that N\u003csub\u003e2\u003c/sub\u003eO emissions were obviously positively correlated with NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), CA (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and UA (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), but significantly negatively correlated with hydrogen peroxidase activity (HPA) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and invertase activity (IA) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In addition, bacterial composition (phylum level), fungal composition (phylum level), AOB species and \u003cem\u003enir\u003c/em\u003eS species were correlated with the environmental factors via the Mantel test, as shown in Fig. \u003cspan\u003e9\u003c/span\u003ea. SOC (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.04 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.01), WFPS (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.03 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.02), CA (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.03 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001), IA (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001) and UA (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002) all had the strongest associations with bacterial and fungal composition, respectively. TN contents were also the key factors affecting fungal communities (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.02), but not bacteria. Meanwhile, IA (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.04) also influenced the AOB communities. However, no significant correlation was found between \u003cem\u003enir\u003c/em\u003eS species and all environmental factors.\u003c/p\u003e\n \u003cdiv\u003e\u003c/div\u003e\n \u003cp\u003eFurthermore, to identify the drivers of N\u003csub\u003e2\u003c/sub\u003eO emissions in apple orchards of different stand ages, redundancy analysis (RDA) was conducted to analyze the correlation between key environmental factors and microbial community compositions (Fig. \u003cspan\u003e9\u003c/span\u003eb and c). According to the correlation results in Fig. \u003cspan\u003e9\u003c/span\u003ea, environmental factors strongly correlated with N\u003csub\u003e2\u003c/sub\u003eO emissions were selected for RDA. Both the RDA1 and RDA2 axes explained 76.2% and 88.7% of the bacterial, fungal, AOB and \u003cem\u003enir\u003c/em\u003eS community variations in young and old apple orchards, respectively. For the young apple orchard, bacteria, CA, NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N were the main influencing factors on N\u003csub\u003e2\u003c/sub\u003eO emissions and were oriented towards the right side of RDA axis 1. However, bacteria, fungi, CA, NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N had a strong correlation with N\u003csub\u003e2\u003c/sub\u003eO emissions in the old apple orchard.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eN\u003csub\u003e2\u003c/sub\u003eO emissions in orchards under different stand ages\u003c/h2\u003e \u003cp\u003eDynamic variation in N\u003csub\u003e2\u003c/sub\u003eO fluxes was observed in this study (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), which could be explained by the positive correlation between ST and N\u003csub\u003e2\u003c/sub\u003eO emissions (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), and agreed with previous results (Cui et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; P\u0026auml;rn et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The amount of fertilizer input may be the main factor causing different N\u003csub\u003e2\u003c/sub\u003eO emissions under the same climate conditions, soil types and management practices. Previous findings suggested that N\u003csub\u003e2\u003c/sub\u003eO emissions in orchards were strongly influenced by fertilization (Pang et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) and increased linearly with N input rates (Cheng et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Gu et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). N\u003csub\u003e2\u003c/sub\u003eO emissions peaks induced by fertilization events accounted for more than 62.5%-67.6% of the total annual emissions along with high mineral N content, especially after urea addition in April 2019. Therefore, we speculated that nitrification might be the main mechanism of N\u003csub\u003e2\u003c/sub\u003eO production in apple orchard soil on the Loess Plateau, which agreed with the changes in soil mineral N (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). In addition, emissions peaks occurred following fertilization and were more obvious between July and August (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), which could be explained by the dramatic increase in mineral N (Duan et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and precipitation (Pang et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). A previous study confirmed that fertilization during rainy periods can induce much higher N\u003csub\u003e2\u003c/sub\u003eO emissions (Groffman et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Pang et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn addition, the annual cumulative N\u003csub\u003e2\u003c/sub\u003eO emissions and EFs of different stand ages showed significant differences, and the old orchard had noticeably higher N\u003csub\u003e2\u003c/sub\u003eO emissions, which were 42.86% higher than those of the new orchards (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The EF in the old orchard (0.54%) was significantly higher than that in the young orchard (0.37%), suggesting that more N was lost as N\u003csub\u003e2\u003c/sub\u003eO gas in older apple orchard. Referring to the results of our previous study (Han et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), a 15-year apple orchard in this region showed that the EF was 0.34%-0.45%, and the EF was lower than that from the 25-year orchard but higher than that from the 5-year orchard in this study. This indicated that EF of apple orchards was affected by stand age, thus, estimating the EF of perennial crops should consider the different stand ages. However, compared with the EF (0.72%) of orchards in continental climate regions (Gu et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), the results of this study were relatively low. This is probably because of uncertainty in the EF, including soil properties, farmland management, and climate being key factors affecting the EF (Zhou et al., \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The EF in orchard soil related to climate types, temperature and continental climate had a lower EF (Gu et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This study region has a typical temperature continental monsoon climate, and the EF was 60%-72% lower than that of a peach orchard (EF\u0026thinsp;=\u0026thinsp;1.32%) in subtropical monsoon climate zone where had more precipitation and higher temperature (Cheng et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), despite the similar N application rate. Moreover, soil texture also significantly influenced the EF and fine-textured soils could result in larger N\u003csub\u003e2\u003c/sub\u003eO emissions (Stehfest and Bouwman, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). However, the medium-textured soil texture in this study had difficulty forming an anaerobic environment to provide conditions for denitrification (Ball, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), resulting in a low EF. At present, the evidence that orchard age affects N\u003csub\u003e2\u003c/sub\u003eO emissions is still insufficient, but in Chinese tea plantations, there is evidence that different stand ages will lead to significantly different N\u003csub\u003e2\u003c/sub\u003eO releases (Yao et al., \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The establishment of woodland can form a soil organic layer that promotes the release of N\u003csub\u003e2\u003c/sub\u003eO (Merino et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Peichl et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), and SOC and TN in the topsoil play a key role in promoting N\u003csub\u003e2\u003c/sub\u003eO emissions (Pang et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Older orchard soils with higher SOC and TN could provide more abundant substrates for nitrification and denitrification processes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eMicrobial community in orchards under different stand ages\u003c/h2\u003e \u003cp\u003eThe alpha diversity of the bacteria, fungal and \u003cem\u003enir\u003c/em\u003eS communities did not change with the stand age of the apple orchard, but the alpha diversity of AOB significantly increased with stand age (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This result disagrees with the conclusion of (Wang et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) that the soil bacterial alpha diversity increased with forest stand age. The possible reason is that agricultural management, such as weeding, has resulted in a simple and stable vegetation community since the apple orchard was established, which created a stable bacterial community (Qiao et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). A previous study reported that plant communities limited soil bacterial communities (Dang et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) due to diverse substrates from different plants being provided to bacteria (Liu et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). However, long-term weeding in our orchards led to a single vegetation species and directional bacterial community succession due to single root exudates was provided. Fungi have strong adaptability (Corneo et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), and their response to soil nutrient changes is weaker than that of bacteria (Delgado-Baquerizo et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Therefore, the alpha diversity of fungi did not change with stand age in apple orchard. The increase in AOB diversity in the old apple orchard was consistent with the findings of our meta-analysis, potentially influenced by long-term fertilization and alkaline soil conditions. Several studies have indicated that fertilizers alter the community structure of nitrogen-fixing bacteria, with higher levels of nitrification occurring in alkaline and neutral soils compared to acidic soils (Jiang et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Cui et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Meng et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSoil microbes participate in nutrient cycling and affect the development of soil plants in terrestrial ecosystems (Li et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Numerous findings indicated that soil microbial communities in apple orchards are influenced by edaphic factors (Zheng et al., \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). A report from a citrus orchard indicated that with plant growing and development, changes in the understory microenvironment and root system led to changes in microbial community composition (Qiang et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Thus, it is instructive to study soil microorganisms of different stand ages for long-term apple orchard planting management. In this study, the community structure composition of bacteria and fungi was significantly affected by stand age and the interaction between fertilization and stand age. However, fertilization had minimal impact on the composition of the microbial community (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). This finding was supported by our meta-analysis results, which contrasted with previous reports indicating significant changes in the soil microbial community due to nitrogen fertilizer in crop fields (Ullah et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Hu et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The important driving factor may be changes in soil nutrients (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea), and some studies in orchards showed a strongly positive link between soil bacteria/fungi and soil nutrients, especially TN and SOC (Qiang et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zheng et al., \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). As shown in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, there were higher TN and SOC contents in the old apple orchard, particularly in the N-amendment plots. The results agreed with previous research showing that the soil microbial community changed with stand age in orchards (Zhang et al., \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2017b\u003c/span\u003e; Wu et al., \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, different opinions also existed in orange orchards, that stand ages increased soil C and N but decreased microbial activity due to soil acidification (Wan et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). With increasing stand age, the relative abundances of some phyla of bacteria and fungi changed obviously (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), and a classification phenomenon related to tree age occurred (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Notably, the relative abundance of \u003cem\u003eFirmicutes\u003c/em\u003e obviously increased in the 25F treatment because the long-term accumulation of dead leaves and N provide substrates for bacteria in old orchards, and \u003cem\u003eFirmicutes\u003c/em\u003e is closely related to the degradation of plant residues (Tiwari et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Verzeaux et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Meanwhile, \u003cem\u003eBasidiomycota\u003c/em\u003e as saprophytic fungi (Yang et al., \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and their relative abundance was significantly increased in old orchards that had abundant plant residues (Wan et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). \u003cem\u003eBasidiomycota\u003c/em\u003e have an advantage in degrading stubborn lignin (Lundell et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) and its increase could be driven by the accumulation of soil recalcitrant C components in old stands (Wang et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Soil moisture was also an important factor limiting bacterial and fungal communities (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea), agreeing with the conclusion of (Wu et al., \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Higher vegetation coverage and more litter coverage on soil surface in older stands would better maintain soil moisture (Qiao et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Thus, the relative abundance of \u003cem\u003eGemmatimonadetes\u003c/em\u003e, which like dry environment (Fawaz, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), was lower in the old orchard.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eInfluencing factors of N\u003csub\u003e2\u003c/sub\u003eO emissions in orchards under different stand ages\u003c/h2\u003e \u003cp\u003eNumerous studies have strongly proven that stand age affects N\u003csub\u003e2\u003c/sub\u003eO emissions from the soil in tea plantations (Yao et al., \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), arbor forests (Christiansen and Gundersen, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Shrestha et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Yin et al., \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Ishizuka et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and sisal plantations (Wachiye et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) which is attributed to differences in soil properties, including carbon and nitrogen availability, pH, C/N, mineral N level, temperature and WFPS. Moreover the plant N requirement and N use efficiency are also considered to be responsible for the differences in N\u003csub\u003e2\u003c/sub\u003eO emissions (Shrestha et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). However, there is currently insufficient evidence on the effect of stand ages on N\u003csub\u003e2\u003c/sub\u003eO emissions in fruit orchards, especially in apple orchards.\u003c/p\u003e \u003cp\u003eIn apple orchards, the differences in soil abiotic properties in young and old orchards may be the more important inducing factors to N\u003csub\u003e2\u003c/sub\u003eO emissions (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea, b and c). Precipitation influences soil water and oxygen availability, which are closely linked to soil N\u003csub\u003e2\u003c/sub\u003eO emissions (Saggar et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Song et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Comprehensive analysis reveals a significant correlation between mean annual precipitation (MAP) and N\u003csub\u003e2\u003c/sub\u003eO emissions (Fig. S4), with N\u003csub\u003e2\u003c/sub\u003eO release particularly sensitive to mineral N content and WFPS (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea). Hence, the predominant role of chemical processes in N\u003csub\u003e2\u003c/sub\u003eO production is paramount and should not be overlooked (Zhang et al., \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). First, the availability of soil N was the most critical driver of N\u003csub\u003e2\u003c/sub\u003eO emissions (Levy-Booth et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; P\u0026auml;rn et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and with the growth of apple orchard stand ages, mineral N in orchard soil was strongly accumulated (Wan et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The meta-analysis results indicate that orchards aged 15\u0026ndash;25 years have NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N levels 89.74% higher than orchards aged 0\u0026ndash;5 years (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Second, WFPS also play an important role in N\u003csub\u003e2\u003c/sub\u003eO emissions, and a certain range of WFPS (50%-80%) created the optimal conditions for N\u003csub\u003e2\u003c/sub\u003eO production (P\u0026auml;rn et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The range of this region could be considered to be 35%-75% because the N\u003csub\u003e2\u003c/sub\u003eO fluxes larger than the average flux were mostly distributed in this range (Fig. S3). Soil moisture influenced the soil oxygen availability (Song et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and nitrification and denitrification were synchronized to produce higher N\u003csub\u003e2\u003c/sub\u003eO under moderate soil WFPS conditions (P\u0026auml;rn et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Above all, soil NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N contents and moisture explained 72% of N\u003csub\u003e2\u003c/sub\u003eO emissions (P\u0026auml;rn et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Meanwhile, the higher NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N contents could induce more N\u003csub\u003e2\u003c/sub\u003eO fluxes. Because NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N is involved in the nitrification process (Levy-Booth et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) to produce N\u003csub\u003e2\u003c/sub\u003eO fluxes, this process is particularly pronounced in calcareous soils with well-aerated and high pH conditions (Wolf and Russow, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, the pH had no impact on N\u003csub\u003e2\u003c/sub\u003eO emissions which was different from a previous study (Cui et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Deng et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The reason may be that the pH value of orchard soil in this study was relatively stable and had not induced a drastic effect on N\u003csub\u003e2\u003c/sub\u003eO. Moreover, this study was conducted in arid and semiarid regions, and previous researchers confirmed that mineral N contents and soil water content were the main factors affecting N\u003csub\u003e2\u003c/sub\u003eO emissions in semiarid areas (Galbally et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe RDA indicated that N\u003csub\u003e2\u003c/sub\u003eO emission was positively associated with bacteria and fungi in young and old orchards (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eb and c). Several reports (Baggs, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Chen et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) confirmed that bacterial and fungal processes were important contributions to N\u003csub\u003e2\u003c/sub\u003eO emissions; fungi in particular have been increasingly identified as major contributors to N\u003csub\u003e2\u003c/sub\u003eO emissions in recent years (Mothapo et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Wankel et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) because the final product of fungal denitrification is N\u003csub\u003e2\u003c/sub\u003eO instead of N\u003csub\u003e2\u003c/sub\u003e (Baggs, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Mothapo et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Considering that \u003cem\u003eAscomycota\u003c/em\u003e and \u003cem\u003ebasidiomycota\u003c/em\u003e contain many N\u003csub\u003e2\u003c/sub\u003eO-producing fungi and their strong N\u003csub\u003e2\u003c/sub\u003eO-producing activity (Mothapo et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), the higher relative abundance of \u003cem\u003eAscomycota\u003c/em\u003e and \u003cem\u003eBasidiomycota\u003c/em\u003e may be an important microbiological reason for the higher N\u003csub\u003e2\u003c/sub\u003eO emissions in old orchards. The results of this field study were different from laboratory experiments, which concluded that ammonia oxidizer and denitrifier abundances dominated N\u003csub\u003e2\u003c/sub\u003eO emissions (Hink et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Qiu et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This may be related to the time of sampling; the microbial activity varies with season, and the abundances over a period of time indicate the relative number of microorganisms but are not a complete representation of activity (Shrewsbury et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Another reason for the low correlation between N\u003csub\u003e2\u003c/sub\u003eO and soil microbes may be that the key role of soil properties masked the impact of ammonia oxidizers and denitrifiers (Graham et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Levy-Booth et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; P\u0026auml;rn et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In conclusion, soil abiotic properties, rather than nitrification and denitrification bacteria, caused the difference in N\u003csub\u003e2\u003c/sub\u003eO emissions in apple orchards of different stand ages. This result also agrees with the previous study showing that compared to denitrifiers, environmental attributes were more important influencers of N\u003csub\u003e2\u003c/sub\u003eO emissions in larger landscapes (Shrewsbury et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). A meta-analysis also reported similar results that N loading induced soil N\u003csub\u003e2\u003c/sub\u003eO emissions rather than ammonia oxidizer and denitrifier abundance (Zhang et al., \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2021\u003c/span\u003e); thus, abiotic factors were the main drivers of the changes in soil N\u003csub\u003e2\u003c/sub\u003eO emissions.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study showed that higher N\u003csub\u003e2\u003c/sub\u003eO emissions occurred in older apple orchards and that the main contributors were soil abiotic properties rather than soil bacteria, fungi, AOB and \u003cem\u003enir\u003c/em\u003eS genes. The richness and diversity of soil bacteria, fungi and \u003cem\u003enir\u003c/em\u003eS showed no significant differences between the young and old apple orchards, but that of AOB was higher in the old orchard. The soil microbial community composition was obviously affected by the stand age of the apple orchard. Stand age and the combination of stand age and fertilization explained most of the variation in microbial communities, but the effect of fertilizer addition was not significant. Nonetheless, microbial processes were not the key drivers of N\u003csub\u003e2\u003c/sub\u003eO release, instead, soil reactive N availability, soil moisture and enzyme activity were more direct factors. Therefore, in future research on soil N cycling and transformation of apple orchards, soil abiotic processes should not be ignored when considering soil microbial processes.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Natural Science Foundation of Shaanxi Province (grant numbers 2023-JC-QN-0356, 2022JM-154); the Open Foundation of Key Laboratory in Jiangxi Academy of Water Science and Engineering (grant numbers 2021SKTR02, 2022SKTR02); the National Natural Science Foundation of China (grant numbers 41601321, 42177327, 42007063).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data curation, and analysis were performed by Man Zhang and Cui Li. The first draft of the manuscript was written by Man Zhang and Cui Li. Weixin Wang, Xin Tong, Kaixuan Wang, Minmin Qiang, Qiong Zhang contributed to the review and editing of the manuscript. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBaggs, E. M. (2011). Soil microbial sources of nitrous oxide: recent advances in knowledge, emerging challenges and future direction. \u003cem\u003eCurrent Opinion in Environmental Sustainability\u003c/em\u003e 3, 321\u0026ndash;327. doi: 10.1016/j.cosust.2011.08.011\u003c/li\u003e\n\u003cli\u003eBall, B. C. (2013). Soil structure and greenhouse gas emissions: a synthesis of 20 years of experimentation. \u003cem\u003eEuropean Journal of Soil Science\u003c/em\u003e 64, 357\u0026ndash;373. doi: 10.1111/ejss.12013\u003c/li\u003e\n\u003cli\u003eBao, S., 2008. 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A 3-year field measurement of methane and nitrous oxide emissions from rice paddies in China: Effects of water regime, crop residue, and fertilizer application. \u003cem\u003eGlobal Biogeochemical Cycles\u003c/em\u003e 19. doi: 10.1029/2004GB002401 \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":"apple orchard, N2O emission, stand ages, microbial community, nitrification, denitrification","lastPublishedDoi":"10.21203/rs.3.rs-5201652/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5201652/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eAims\u003c/h2\u003e \u003cp\u003eThe continuously expanding apple plantation and excessive nitrogen input have made it a major source of nitrous oxide (N\u003csub\u003e2\u003c/sub\u003eO) emissions over the past 40 years in the Loess Plateau, China. However, the difference in N\u003csub\u003e2\u003c/sub\u003eO emissions from different stand ages of orchards and its key driving factors remain unclear.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA three-year field study was set up to evaluate the soil N\u003csub\u003e2\u003c/sub\u003eO emissions and the soil properties in apple orchards of two different stand ages (young orchard: 5 years and old orchard: 25 years), and soil bacteria, fungi, ammonia oxidizing bacteria (AOB) and denitrification bacteria (\u003cem\u003enir\u003c/em\u003eS) were determined via amplicon sequencing.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe higher N\u003csub\u003e2\u003c/sub\u003eO emissions and emission factors (EFs) were recorded in the old apple orchard under the conventional nitrogen (N) strategy. The microbial community composition in topsoil was obviously shifted by stand age (22.2% interpretation, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.022) and stand age and fertilization also had a combined effect (36.6% interpretation, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003). The relative abundances of \u003cem\u003eFirmicutes\u003c/em\u003e and \u003cem\u003eBasidiomycota\u003c/em\u003e involved in the decomposition of plant residues increased with stand age. Nonetheless, N\u003csub\u003e2\u003c/sub\u003eO fluxes were not significantly correlated with soil nitrifiers and denitrifiers, but were strongly correlated with NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, soil moisture and enzyme activity. In general, abiotic factors, especially mineral N availability, resulted in differences in N\u003csub\u003e2\u003c/sub\u003eO emissions between orchards of different stand ages.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe selection of future N\u003csub\u003e2\u003c/sub\u003eO emissions mitigation strategies for apple orchards should take into account both nonbiological processes and biological processes, and the assessment of N\u003csub\u003e2\u003c/sub\u003eO emissions in apple orchards should consider stand age.\u003c/p\u003e","manuscriptTitle":"Microbial Stimulation in Apple Orchards of Different Ages on the Loess Plateau: Poor Predictability of Increased Soil N2O Emissions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-29 12:11:29","doi":"10.21203/rs.3.rs-5201652/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":"014aaef1-f3cf-4b32-9ffe-fdfd2db8a956","owner":[],"postedDate":"November 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-12-21T10:36:24+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-29 12:11:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5201652","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5201652","identity":"rs-5201652","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}