Effect of elevated tropospheric ozone on soil carbon and nitrogen: a meta-analysis

All peer-reviewed literature, including journal articles, degree dissertations, and monographs related to soil C and N status with reference to elevated [O₃], were extensively searched through the ISI Web of Science (https://apps.webofknowledge.com) and the Scopus (www.scopus.com) by using the following key terms: [O₃ OR ozone] AND [soil OR rhizospher* OR terrestrial OR land] AND [carbon OR TC OR *OC OR TN OR *nitr* OR ammon* OR mineraliz*]. The relevant Chinese literatures were searched in the China National Knowledge Infrastructure Database (CNKI, www.cnki.net/). This search produced a total of 579 publications in ISI, 533 publications in Scopus, and 507 publications in CNKI. After examining the relevance, eliminating the duplications in both ISI and Scopus, and cross-checking the reference lists to avoid missing relevant studies, 75 publications were identified for further screening.

The searched publications were carefully screened according to the following criteria. (i) The studies were conducted in the field, and reported O₃ effects on at least one of the interesting variables, including total C (TC, including soil organic C), dissolved organic C (DOC), microbial biomass C (MBC), total N (TN), ammonia N (NH₄ ⁺), nitrate N (NO₃ ⁻), microbial biomass N (MBN), nitrification (NTF) rate, and denitrification (DNF) rate. (ii) The microclimate, vegetation, and initial soil conditions were the same between the control (ambient O₃) and O₃ enrichment treatments. (iii) The means, standard deviations (SDs), and replicate numbers (n) of the target variables could be directly acquired or obtained from the corresponding authors. (iv) The O₃ fumigation was performed over at least one growing season using open top chamber (OTC) or free-air O₃ concentration enrichment (FACE) technology. (v) Published articles and their measurements were excluded if the data were reported more completely in another article. (vi) For studies on O₃ and CO₂ treatments with full factorial design (i.e. control, CO₂ exposure, O₃ exposure, and combined CO₂ and O₃ exposure), the data of CO₂ exposure alone were not extracted. According to these criteria, 22 studies based on pot-plants, 2 studies on lab cultivations, and 2 studies on lysimeters were excluded. Four publications that only reported charcoal-filtered air as control were not used. Furthermore, four articles that reported data more completely in another article were eliminated. The final database consisted of 41 articles published between 2003 and 2020, including 28 English papers indexed by both ISI and Scopus and 13 Chinese literatures indexed by CNKI (supplementary material A available online at stacks.iop.org/ERL/17/043001/mmedia). The data were acquired from 17 experiments performed at 25°28' N to 60°49' N latitude and 88°14' W to 123°24' E longitude. The mean annual temperature (MAT) ranged from 1.1 °C to 26.6 °C, and the mean annual precipitation (MAP) ranged from 460 to 1280 mm. The average increment in [O₃] across all studies was 27.6 ± 18.7 nl l⁻¹. Note that studies in the Southern hemisphere were absent from the database (Hu et al 2021).

Values for each variable were extracted independently if they were obtained from different experiments, corresponded to multiple O₃ levels or additional [CO₂] exposure within a single study. To acquire as many observations as possible, values were assumed independently if they were obtained from the same experiment but measured on different dates, or when the soil samples were collected from distinct layers within the soil depth range of 0–25 cm. Similar assumptions have been widely held in previous studies (Wittig et al 2009, Li et al 2017, Feng et al 2018, Meng et al 2019). Results obtained from the same experiment were considered independent and included separately in the database if they were associated with different plant species or cultivars, N treatments, and warming or drought stresses (de Graaff et al 2006).

Values of each variable from text or tables were extracted directly. Data from figures were digitized and extracted using GetData Graph Digitizer software (Version 2.26; http://getdata-graph-digitizer.com). If the data were provided with standard errors (SEs), the SD was calculated as follows: ${\text{SD}} = {\text{SE}} \cdot \sqrt n$. If confidence intervals (CIs) were reported, they were transformed into SD according to the following equation: ${\text{SD}} = \left( {{\text{C}}{{\text{I}}_{\text{U}}} - {\text{C}}{{\text{I}}_{\text{L}}}} \right) \cdot \sqrt n \cdot {Z_{\alpha /2}}/2$, where CI_L and CI_U are the lower and upper confidence limits, respectively, and Z_{α /2} is the Z score for a given significance level, which is 1.96 when α = 0.05 and 1.645 when α = 0.10 (Luo et al 2006). Unidentified error bars were assumed to represent SE, and unidentified replicate numbers were assumed to be the replication of the plot (Treseder 2004).

The following categorical variables were included to explain the variations in soil C and N status in response to elevated [O₃]: (i) experimental O₃ level: low ([O₃] ⩽ 1.5 × ambient), moderate (1.5 × ambient < [O₃] < 2 × ambient), and high ([O₃] ⩾ 2 × ambient); (ii) experimental duration, i.e. the duration from the beginning of experiments to the time of measurements: short (duration ⩽ 1 year), medium (1 year < duration ⩽ 3 years), and long (duration > 3 years); (iii) fumigation methods: FACE and OTC; (iv) soil sampling type: rhizosphere or bulk soil; (v) soil pH: acid (pH ⩽ 6.5), neutral (6.5 < pH ⩽ 7.0), and alkaline (pH > 7.0); (vi) terrestrial ecosystem type: woodland, grassland (including meadows), and cropland; (vii) additional elevated [CO₂] treatment: no or yes. The mean [O₃] in the control and in the treatment was defined as the daylight mean [O₃] throughout the entire experimental season. In addition, information such as source of data, study site, latitude, longitude, O₃ exposure method (FACE or OTC), soil texture and sampling depth, climate, and fertilizations were acquired directly from the selected articles or their references. Considering that very few studies focused on the interaction of elevated [O₃] with drought and N additional treatments, how these factors modify soil C and N responses to elevated [O₃] could not be assessed.

The detailed information of analysis methods can be found in supplementary material B. Briefly, the meta-analyses were performed using MetaWin software (Version 2.1; Sinauer Associates, Inc., Sunderland, MA, USA) with the effect size—natural log of the response ratio (lnR) and its corresponding variance. Normal quantile plots (figure S.1) and frequency histogram of lnR (figure S.2) were plotted to check the normality of the data. A weighted random-effects model was used to assess the overall and grouped effect size. Bias-corrected 95% CIs around the effect size were generated using a bootstrapped resampling technique with 64 999 iterations. Twelve studies reported both TC and TN, representing total 64 data pairs for the calculation of C/N ratio by dividing their means. The mean effect size and CIs of C/N was estimated using an unweighted bootstrapped resampling method, since the SD of C/N could not be calculated directly. The response to elevated [O₃] were considered significant if the bias-corrected 95% bootstrapped CIs did not overlap from zero. The O₃-induced percentage change from the control was calculated as (R − 1) × 100% (de Graaff et al 2006). A negative percentage change indicates a decrease in the response to elevated [O₃] treatment, and vice versa.

The total heterogeneity of each variable was assessed using the statistic Q_T and I² index (table S.1) (Rosenberg et al 2000, Higgins and Thompson 2002, Huedo-Medina et al 2006). Categorical analysis were performed for all data by dividing Q_T into within-group heterogeneity (Q_W) and between-group heterogeneity (Q_B). The datasets were then grouped on the basis of the levels of the categorical variables with significant Q_B (table S.1) (Wittig et al 2009). The difference between the means of categories were considered to be significant if their bias-corrected 95% bootstrapped CIs did not overlap (Gurevitch and Hedges 1999, Ainsworth 2008).

Continuous models were applied to determine the relationships between lnR and environmental or forcing factors, such as O₃ fumigation levels, experimental duration, soil pH value, and latitude, MAT and MAP of the experimental site. A parametric mixed model approach was used to test whether the slopes of weighted regressions differed from zero (Rosenberg et al 2000). The regressions were considered to be significant at P < 0.05.

Multiple methods were included in the test of publication bias, including funnel plots (figure S.3), Kendall's tau (table S.2) and Spearman's rho (table S.3) rank correlation tests, as well as Rosenthal's fail-safe number (α = 0.05) (table S.4) (Rosenthal 1979). A publication bias was confirmed only if all three methods showed positive results simultaneously, namely, that the funnel plot clearly showed asymmetry (only for the overall data set), or the rank correlation test was significant (P < 0.05), and Rosenthal's fail-safe number appeared less than 5k + 10, where k is the observation number. For the data set with confirmed publication bias, the 'true' overall/grouped effect size and CIs were estimated using a 'Trim and Fill' approach (Jin et al 2015).

The TC and DOC reduced by 2.4% and 10.8%, respectively, under elevated [O₃] compared with those under control [O₃] across all studies (figure 1). The decrease in DOC content caused reduction in the MBC of 6.1%. The reduction in MBN (13.7%) was more than that in MBC, showing a generally increased C/N ratio in soil microbes under elevated [O₃]. Elevated [O₃] reduced soil TN and NH₄ ⁺ by 4.5% and 5.8%, respectively, whereas it increased soil NO₃ ⁻ by 7.0%. In addition, the changes on substrate NH₄ ⁺ and NO₃ ⁻ availability for nitrifying and denitrifying bacteria led to a decrease in soil NTF (7.5%) and an increase in soil DNF (5.0%), respectively. Elevated [O₃] significantly increased C/N by 3.1%. And there was a strong positive relationship between effect sizes of elevated [O₃] on pairwise TC and TN (P < 0.001, n = 64; figure S.4). Though the overall effects of elevated [O₃] on all response variables were significant (P < 0.05), the significant Q_T (P < 0.05) and positive I² values suggested heterogeneity among studies for presenting response variables except C/N (table S.1).

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The magnitude of O₃ effects on soil TN, MBN, NTF, and DNF was dependent on the O₃ exposure level due to their significant Q_B values (P < 0.05; table S.1). As illustrated in figure 2, elevated [O₃] generally has the most pronounced effect at high O₃ level, and it has no significant effect on these four variables at moderate O₃ levels.

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Notably, Kendall's tau and Spearman's rho rank correlation tests for publication bias of TN were significant at low O₃ level (P < 0.05), and its Rosenthal's fail-safe number was less than 295 (i.e. 5k + 10, where the observation number k = 57; tables S.2 and S.4), indicating that stronger O₃ effects on soil TN at low O₃ level were somewhat more likely to be published than were weaker O₃ effects. To make up for the publication bias, a 'true' grouped mean effect size and CIs were estimated using the 'Trim and Fill' approach. Based on the adjusted results, TN only showed a significant decrease of 5.2% at a high O₃ level (P < 0.05), while it has no significant changes at low or moderate O₃ levels. The overlapped CIs suggested that no significant difference in TN existed among the three O₃ levels (figure 2).

In terms of MBN, the O₃-induced percentage decrease was significantly larger (P < 0.05) at high O₃ levels (23.5%) than at a low O₃ level (11.8%), and both were significantly different from zero (P < 0.05). Likewise, the reduction of NTF induced by elevated [O₃] was significantly larger (P < 0.05) at high [O₃] (16.1%) than at moderate [O₃] (5.4%). As for DNF, the effect of O₃ treatment was not significant at low and moderate O₃ levels, and a significant increase was noted at high O₃ level (12.3%; P < 0.05). No between-group heterogeneity was found for other response variables when the studies were grouped by O₃ exposure level (table S.1 and figure S.5).

Weighted meta-regression analyses showed that the level of elevated [O₃] was significantly negatively related with the effect sizes of TN and MBN (P < 0.05; table S.5). Moreover, there was a significant negative correlation between the effect size of NTF and O₃ exposure level (P < 0.001; table S.5), whereas the increasing [O₃] had a significant positive effect on the effect size of DNF (P < 0.01; table S.5). As expected, the higher O₃ concentration is, the greater the impacts on these variables would be.

The temporal patterns of responses of soil NO₃ ⁻, MBN, NTF, and DNF were investigated by grouping the data by experimental duration, because of their significant Q_B values (P < 0.05). Soil MBN and NTF progressively decreased with increasing duration of exposure to elevated [O₃], whereas an inverse trend was observed for DNF (figure 3 and table S.5). Short O₃ exposure had no significant effect on MBN and DNF, whereas it marginally significantly increased soil NO₃ ⁻ and NTF by about 15.0% and 6.0%, respectively. For the medium experimental duration, MBN and NTF declined moderately with average reductions of 17.2% and 14.2% (P < 0.05), respectively, whereas the changes of NO₃ ⁻ were not significantly different from zero. Note that the DNF for medium experimental duration showed publication bias according to the results of Kendall's tau and Spearman's rho rank correlation tests, as well as Rosenthal's fail-safe number (tables S.2 and S.4). After adjustments using the 'Trim and Fill' method, the 'true' effect size of DNF for medium O₃ exposure duration was still significantly increased (10.4%, P < 0.05). The O₃-induced percentage decline of MBN for long O₃ exposures (16.0%) were similar to that at medium exposures, whereas NTF was markedly (22.9%) decreased by O₃ exposure of >3 years. The percentage increase in DNF after long O₃ exposure was approximately two times higher than that after medium exposure. As expected, the longer the duration of O₃ exposure, the more pronounced was the O₃ impact on these variables. The only exception was NO₃ ⁻, which showed a similar change (16.3%, P < 0.05) under long O₃ exposure as that under short duration.

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The heterogeneities on the responses of soil TN, NTF and DNF to different O₃ durations could be explained by the continuous model, according to the significant Q_M values (P < 0.05). As expected, the longer the duration of O₃ exposure, the more pronounced were the O₃ impacts on NTF and DNF. The duration of ozone exposure did not induce significant differences in other response variables (tables S.1 and S.5; figure S.6). Soil TC content for medium experimental duration showed publication bias (tables S.2 and S.4), indicating that stronger O₃ effects were somewhat more likely to be published than weaker O₃ effects under such circumstance. After being adjusted using the 'Trim and Fill' method, the 'true' mean effect size of TC for medium experimental duration showed a significant decrease of 10.4% (P < 0.05; figure S.6).

Of the total 41 articles, 24 and 17 reported the use of FACE and OTC, respectively. According to the results of heterogeneity analysis (table S.1), the fumigation method significantly affects the responses of MBC, MBN and NTF to elevated [O₃], but did not induce significant differences in other response variables. Of three variables with significant Q_B values, MBC and MBN showed overlapped CIs between FACE and OTC (P > 0.05; figure S.7), while only NTF showed a significant larger decrease in FACE than that in OTC (P < 0.05; figure S.7).

Soil MBC, MBN, NH₄ ⁺, NO₃ ⁻, NTF and DNF showed significant Q_B values (P < 0.05) between rhizosphere soil and bulk soil (table S.1). For rhizosphere soil samples, elevated [O₃] significantly decreased MBC, MBN and NTF by 14.9%, 18.0% and 12.9%, respectively (P < 0.05); and increased soil NO₃ ⁻ and DNF by 20.1% and 11.5%, respectively (P < 0.05); while it has no significant impact on soil NH₄ ⁺ (figure 4). On the contrary, elevated [O₃] has no significant effect on these variables in bulk soil, except it significantly reduced bulk soil NH₄ ⁺ by 10.1% (P < 0.05; figure 4). The differences on the responses of soil MBC, MBN, NTF and DNF between rhizosphere and bulk soil were significant (P < 0.05) owing to their un-overlapped CIs (figure 4). No between-group heterogeneity was found on other response variables when the studies were grouped by soil sampling type (table S.1 and figure S.8).

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The O₃-induced impacts on soil C and N were potentially related to the pre-existing soil pH (table S.1). The responses of soil TC, TN, NH₄ ⁺, and NTF to elevated [O₃], grouped by soil pH according to their significant Q_B values (P < 0.05) are shown in figure 5.

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A statistically significant decrease in TC under elevated [O₃] occurred in neutral soil (7.2%, P < 0.05), whereas a marginally significant decrease was noted in alkaline soil (1.9%), and no significant change was found in the acid soil. Soil TN content in response to elevated [O₃] was reduced by 4.3% (after being adjusted by the 'Trim and Fill' approach due to significant publication bias) and 6.1% in neutral and alkaline soils, respectively; and not affected significantly in acid soil (figure 5). Detailed meta-regression analysis showed that the O₃-induced effects on soil TN content were significantly and negatively related to soil pH (P < 0.001; table S.5), indicating that the negative O₃ effects on TN were exacerbated at higher soil pH.

In addition, lower soil pH caused stronger declines in soil NH₄ ⁺ content under elevated [O₃] (figure 5). Soil NH₄ ⁺ was remarkably decreased by 23.6% (P < 0.05) in acidic soil, but was not significantly affected in both neutral and alkaline soils. Indeed, the effect size of soil NH₄ ⁺ was positively correlated with soil pH (P < 0.001; table S.5), which indicated that higher soil pH ameliorates the negative O₃ effects on soil NH₄ ⁺ content.

Elevated [O₃] strongly decreased soil NTF by 5.7% and 20.6% in acid and neutral soils, respectively (figure 5), and this effect was significantly negatively related to soil pH (P < 0.001; table S.5).

Although statistically or marginally significant negative effects of elevated [O₃] were noted on soil MBN, no significant differences were observed among soil pH classes (figure S.9). However, the weighted meta-regression analysis showed a significant negative relationship between the effect size of MBN and soil pH (P < 0.01; table S.5).

The number of studies for DNF in neutral and alkaline groups were not sufficient to conduct quantitative comparisons. However, the weighted meta-regression analysis showed a strong significant negative relationship between the effect size of O₃ on DNF and soil pH (P < 0.01; table S.5).

Significant heterogeneities were found on TN and MBN in response to elevated [O₃] across terrestrial ecosystems (P < 0.05; table S.1). However, there were no significant differences among groups for TN owing to the overlapped CIs (figure S.10). On the contrary, soil MBN was significantly reduced by 17.9% in cropland, and was significantly different from that in grassland which tended to be increased (P < 0.05; figure S.10).

In addition, only TN, NH₄ ⁺ and NO₃ ⁻ have sufficient studies (k > 10) to conduct comparisons between solely O₃ stress and combined O₃ and CO₂ exposure. However, there was no between-group heterogeneity for either variable (table S.1). Thus, the magnitude of the responses to elevated [O₃] were independent of the additional [CO₂] exposure in the present analysis.

The results of weighted meta-regression analyses for response variables in correlation with latitude, MAT, and MAP are shown in table S.6. The effect sizes of soil TN, MBN, and DNF were significantly correlated with latitude (P < 0.05). Taking as an example the regressing results of MBN (lnR(MBN) = 1.02 × 10⁻² × Latitude−0.528), the fitted effect size of MBN varied from −0.269 to 0.090 within a latitude range of 25°28' N to 60°49' N in the present study. This means the ozone effects on MBN were negative at lower latitudes, but were positive at higher latitudes. Thus, the negative intercepts and positive slopes of the regression on TN and MBN implied stronger detrimental effects (i.e. more negative effect sizes) of O₃ on these variables at lower latitudes. In contrast, the intercept of DNF was positive, and the slope was negative. It suggested that the O₃ effect on the soil DNF process may be stimulated (i.e. more positive effect sizes) at lower latitudes.

The magnitude of response variables were significant (TC, MBN, and DNF; P < 0.05) or nearly significantly (NH₄ ⁺; P = 0.079) related with MAT. The regressions of TC and DNF had negative intercepts and positive slopes, whereas NH₄ ⁺ and MBN had positive intercepts and negative slopes. With an increase in MAT, the effect size of DNF became more positive, whereas the effect sizes of NH₄ ⁺ and MBN became more negative. It indicated that higher temperature would benefit the O₃ induced adverse effects on soil NH₄ ⁺ and MBN and the positive effects on soil DNF, which were consistent with the results on latitude, since low latitude is generally related to a higher temperature.

In addition, response variables except DOC, MBN and NTF exhibited significant (P < 0.05) or marginally significant (P = 0.061 for TC; P = 0.059 for MBC) responses to MAP. Although they had very small slopes, TC and TN showed positive intercepts and negative slopes, whereas MBC, NH₄ ⁺, NO₃ ⁻, and DNF showed negative intercepts and positive slopes. Thus, the negative responses of TC and TN, and the positive responses of NO₃ ⁻ and DNF to elevated [O₃] would be promoted at high precipitation, however, the O₃-induced inhibiting effects on MBC and NH₄ ⁺ would be mitigated.

The overall meta-analysis showed that elevated [O₃] decreased both C and N pools in soil. Ozone is a highly reactive and oxidative pollutant and is destroyed rapidly when interacting with plant surfaces, causing a vertical gradient of decreasing O₃ concentrations towards the ground and preventing any appreciable penetration of O₃ into the soil (Turner et al 1974, Blum and Tingey 1977). Therefore, the effects of O₃ on soil C and N seem unlikely to be direct, but are likely to be mediated indirectly through altering the quantity and quality of plant C inputs and resource allocations (Andersen 2003, Kasurinen et al 2005, Kanerva et al 2006).

Previous meta-analytic studies revealed that chronic O₃ exposure decreased photosynthetic rates, Rubisco activity, stomatal conductance, and chlorophyll content, and eventually altered source-sink balance in sensitive plants (Andersen 2003, Valkama et al 2007, Feng et al 2008, Feng and Kobayashi 2009, Wittig et al 2009, Li et al 2017, Feng et al 2018). Although the observed responses vary depending on the species and exposure conditions, elevated [O₃] is known to seriously influence the transport of photosynthates from leaves to roots. In many cases, decreased root C allocation occurred rapidly and even before shoot responses could be observed (Andersen 2003). Decreased C allocation below ground altered rhizodeposition, reduced the quantity of root residue and exudates and thereby decreased C flux from root exudates to the soil labile C pool (Yoshida et al 2001, Andersen 2003, Jones et al 2009). Therefore, the contents of soil TC and DOC generally decreased under elevated [O₃] (figure 1). As a labile form of soil C, DOC principally consists of low-molecular-weight sugars, fulvic acids, and amino acids (McKeague et al 1986, van Hees et al 2005). The greater reduction of DOC than that of TC may suggest a relative enrichment of recalcitrant organic C, such as high-molecular-weight phenolic compounds, in soil (Jones et al 2009). Since soil microorganisms are generally C-limited (Anderson and Domsch 1978), the decrease in soil labile C availability may explain the reduced soil microbial biomass (figure 6) (de Graaff et al 2006).

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The availability of soil TN, particularly NH₄ ⁺ contents, were also reduced under elevated [O₃] (figure 1). The inhibition of soil N availability indicated a deficiency in available N for soil microbes under elevated [O₃]. The reduced soil NH₄ ⁺ concentrations or MBN in response to elevated [O₃] could be attributed to the decreased belowground C inputs (Kanerva et al 2006, Bhatia et al 2011), or, in some cases, higher NTF and DNF rates (Pereira et al 2011). Indeed, the DNF rate was generally higher under elevated [O₃] (figure 1). Previous studies have shown that soil oxygen concentration, NO₃ ⁻ availability, as well as organic C supply are the three most important factors that determine the DNF rate (Del Grosso et al 2000, Lu et al 2011). Since soil DOC was generally decreased under elevated [O₃], the acceleration of the DNF process may be attributed to the increased NO₃ ⁻ availability (figure 6), and in some cases, decreased oxygen concentration due to the relative increases in the soil water content driven by the reduced stomatal conductance and transpiration rates under elevated [O₃] (McLaughlin et al 2007, Hu et al 2018). In contrast, the NTF rate was generally reduced by O₃ stress (figure 1). Soil NTF rates have shown to be mainly controlled by soil NH₄ ⁺ availability (figure 6) (Norton 2008). Decreased substrate quantity and quality under elevated [O₃] may exert negative effects on soil NTF. In theory, the inhibited NTF rate and accelerated DNF rate would decrease soil NO₃ ⁻ under elevated [O₃]. The marginally significant increase of soil NO₃ ⁻ may be explained by the suppressed N demand for plant shoots due to reduced biomass (Broberg et al 2017). Moreover, the increased NO₃ ⁻ may result in a high risk of soil N loss via leaching and runoff (Hu et al 2018).

The response magnitudes of most variables to elevated [O₃] were affected by O₃ levels, experimental duration, soil pH and sampling type, etc. However, these categorical factors may couple with each other. For example, [O₃] in the control and the elevated treatment for rhizosphere soil were generally higher than that for bulk soil (figure 4), which may lead to more significant responses of MBC, MBN, NH₄ ⁺, NO₃ ⁻, NTF and DNF in rhizosphere soil than that in bulk soil. In addition, MBN responded more significantly in cropland than that in grassland because of the much higher [O₃] in the former, and higher a O₃ level would exacerbate the detrimental effect of O₃ on soil MBN owing to the significant negative regression relationship (P < 0.05; table S.5).

Note that the O₃ effects on MBN and NTF were non-significant under a moderate O₃ level, while they significantly decreased at low and high O₃ levels. One possible reason is the relatively lower mean [O₃] of both control and experimental treatments for theses response variables under a moderate O₃ level (figure 2). In another case, medium O₃ exposure had no significant effect on NO₃ ⁻, while it significantly increased at short and long O₃ exposures. The relative smaller or even opposite responses of these variables at a moderate (medium) group than that at a low (short) or high (long) groups may also reflect the possibility of soil microbial acclimation to moderate doses of O₃ stress. Recently, Agathokleous et al (2019a, 2019b, 2019c) found that many determinants of plant yield followed hermetic-like biphasic dose-response relationships under elevated [O₃] stress. Since the effect of elevated [O₃] on soil processes are generally mediated indirectly through plants, it may also yield pleiotropic responses. Based on this assumption, the moderate doses of O₃ stress may upregulate adaptive responses that may enhance both microbial and biochemical resilience, while high doses may induce inhibitory responses to soil microbes (Agathokleous et al 2019a).

In general, autotrophic nitrifiers function better under neutral and/or slightly alkaline conditions, and NTF rates are often low in acidic soils (Chen et al 2013). In the present analysis, the responses of NTF in acid soil was significantly less negative than that in neutral soil according to their un-overlapped CIs (P < 0.05; figure 5). Note that the increment of [O₃] was higher in acid soils (42 nl l⁻¹) than that in neutral soils (19 nl l⁻¹). And higher O₃ level under acid condition should have exacerbated the detrimental effect of O₃ on NTF. The seemed paradoxical results may be explained by the longer experiment duration (average of 3.7 years) in neutral soil than that in acid soil (average of 1.9 years), since O₃ effect on NTF was significantly negatively related to experimental duration (P < 0.001; table S.5). In other words, the regression relationship between NTF and soil pH may be essentially related to the regression relationship between NTF and O₃ exposure duration.

The O₃-induced effect on NTF was more negative at alkaline condition (figure 5). It means the ability for oxidizing NH₄ ⁺ to NO₃ ⁻ was more restrained by elevated [O₃] at higher soil pH, which is conducive to the reserve of soil NH₄ ⁺ (i.e. the substrate for NTF). In other words, the negative O₃ effects on soil NH₄ ⁺ content was ameliorated at higher soil pH (figure 5). Moreover, the number of DNF data was not enough to conduct a categorical analysis at different soil pH levels. But its general increasing pattern (figure 1) may imply an increase in soil N₂O emissions under elevated [O₃]. ŠImek and Cooper (2002) reported that heterotrophic denitrifiers function better at higher soil pH. Consequently, the total gaseous N loss via DNF would be higher in neutral and slightly alkaline soils than acidic ones. It may explain the exacerbated TN loss due to elevated [O₃] at higher soil pH.

In addition, the increase in [O₃] used in OTC experiments (40.0 nl l⁻¹) was generally higher than that used in FACE experiments (23.1 nl l⁻¹) for NTF, and higher [O₃] was supposed to exacerbate the detrimental effect of O₃. However, NTF decreased more in FACE than that in OTC (figure S.7), which may be attributed to the longer experiment duration in FACE studies. Consequently, extended duration may counteract the limitation of lower O₃ levels on soil NTF responses to elevated [O₃] in FACE and neutral soil conditions.

Since O₃ exposure level and duration may offset one another in explaining response variances among groups, a better way would be to combine them together as a cumulative dose variable. The commonly used dose-based indices are accumulated O₃ exposure indices (such as AOT40, SUM06 and W126) and flux-based index (e.g. POD_Y). They all played significant roles in quantifying O₃ impacts on plant growth and soil properties (Zhang et al 2017, Hu et al 2019). However, the calculation of exposure indices requires hourly averaged [O₃] and effective fumigation time, and the calculation of POD_Y required further information including hourly data of photosynthetic photon flux density, temperature, water vapour pressure deficit and plant available water, etc. In the 41 papers of present meta-analysis, only 9 papers reported AOT40. So far, we can not obtain the hourly data for the calculation of dose indices for the other 32 studies. Estimating the cumulative dose by multiplying daylight mean [O₃] and plant growing season is infeasible and will import immeasurable errors. Because [O₃] has obvious daily and seasonal variations, and O₃ fumigation usually only applied during the daylight 7–9 h except for rain, dew and fog. Moreover, the effect of elevated [O₃] on soil C and N varied with plant growing stages (Hu et al 2018). Once enough data has been gathered, an updated categorical and/or continuous meta-analysis based on dose index will be more efficient in revealing the variation mechanisms of O₃ impacts on soil C and N.

Previous studies have reported contrasting effects of warming and O₃ on plant physiology, rhizosphere chemical environment, and microbial communities (Changey et al 2018, Qiu et al 2018). In addition, some studies showed that moderate drought might alleviate the O₃ effects on photosynthesis, physiology, stomata characteristics, fine-root dynamics, and soil respiration (Xu et al 2017). However, the results of weighted meta-regression analyses in the present study revealed that the responses of soil C and N dynamics to elevated [O₃] were generally enhanced at low latitudes—warmer areas with higher precipitation. Note that the lower the latitude is, the longer the annual growing season as well as the effective O₃ fumigation time would be. In the present study, the average O₃ exposure duration was estimated to be approximately 6 months per year for sites south of 40° N, while it was about 4 months for sites north of 40° N. This may partially explain the detrimental O₃ effect at low latitudes. Moreover, soil microbial activities associated with N cycling, such as NTF and DNF, were usually stimulated with increasing temperature (Saad and Conrad 1993). Moreover, increased MAT and MAP usually accelerated soil N leaching losses from terrestrial ecosystems (Jabloun et al 2015). Thus, a decrease in soil N availability due to increased temperature and precipitation may exacerbate the negative O₃ effect on soil N pool (Lu et al 2011).