No-till increases soil denitrification via its positive effects on the activity and abundance of the denitrifying community

Journal Pre-proof No-till increases soil denitrification via its positive effects on the activity and abundance of the denitrifying community Jinyang Wang, Jianwen Zou PII:

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https://doi.org/10.1016/j.soilbio.2020.107706

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Soil Biology and Biochemistry

Received Date: 20 September 2019 Revised Date:

31 December 2019

Accepted Date: 5 January 2020

Please cite this article as: Wang, J., Zou, J., No-till increases soil denitrification via its positive effects on the activity and abundance of the denitrifying community, Soil Biology and Biochemistry (2020), doi: https://doi.org/10.1016/j.soilbio.2020.107706. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

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No-till increases soil denitrification via its positive effects on the activity

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and abundance of the denitrifying community

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Jinyang Wanga,b,* and Jianwen Zoua

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a

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Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing 210095,

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Jiangsu, China

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b

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2UW, Gwynedd, UK

Jiangsu Key Laboratory of Low Carbon Agriculture and GHGs Mitigation, College of

School of Natural Sciences, Environment Centre Wales, Bangor University, Bangor LL57

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*

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Agricultural University, Nanjing 210095, Jiangsu, China

Corresponding author: Dr. J. Wang. E-mail: [email protected]. Weigang 1, Nanjing

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Abstract

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Shifting from conventional tillage to a no-till system can contribute to improving soil carbon

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sequestration and sustaining crop productivity. However, our understanding of the soil

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nitrogen (N) process through insights into the no-till effect on soil denitrification remains

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elusive. Here, we compiled data from 323 observations in 57 studies and quantified the

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responses of soil denitrification and the size and activity of the denitrifying community to no-

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till vs. conventional tillage. Across all studies, no-till significantly increased soil

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denitrification (85%) compared to conventional tillage. The no-till effect on soil

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denitrification was significantly dependent upon N fertilizer management, with a greater

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increase with N fertilization than without (101 vs. 46%). The increased soil denitrification

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under no-till was attributed to increases in the size and activity of the denitrifying community.

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On average, the potential denitrification activity, the total number of denitrifiers, and the

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abundance of denitrifying genes were increased by 66, 116, and 14–70%, respectively, in

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response to no-till. Our results demonstrate that soil denitrification under no-till leads to

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increased soil nitrous oxide (N2O) emission. This is supported by a larger response of soil

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N2O emission compared to the total denitrification, together with a significant increase (33%)

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in the (nirK+nirS)/nosZ ratio under no-till conditions. Therefore, the increased soil

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denitrification under no-till conditions may have negative impacts on soil N cycling and

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mitigation of N2O emission.

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Keywords:

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Conservation tillage; Nutrient management; Denitrifying genes; Nitrous oxide; Microbial

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diversity

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1 Introduction

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No-till is one of the three crop management principles in conservation agriculture (FAO,

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2011), which has been proposed as a component of climate-smart agriculture (Lipper et al.,

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2014). In recent decades, widespread adoption of no-till has occurred over approximately 125

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million hectares, equivalent to 9% of global arable land (Friedrich et al., 2012). A large body

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of research supports the idea that, compared to conventional tillage, no-till has greater

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potential for soil carbon sequestration, the improvement of soil functioning and quality, and

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the sustainability of crop productivity (Knapp and van der Heijden, 2018; Lal, 2015; Paustian

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et al., 2016).

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Recent evidence from both experimental and meta-analytic studies, however, suggests

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that these beneficial effects may be overstated (Pittelkow et al., 2015a; Powlson et al., 2014;

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Six et al., 2004). For example, results of comprehensive meta-analyses across the globe

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suggest that the no-till effect on crop yield is strongly dependent upon crop type, climate, and

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residue and fertilizer management (Pittelkow et al., 2015a, 2015b). In addition, Powlson et al.

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(2014) argued that the adoption of no-till agriculture has limited potential to enhance soil

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organic carbon (SOC) stock and thereby to mitigate climate change. Further, nitrous oxide

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(N2O) emissions from arable and managed grassland soils, which are believed to contribute to

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c. 60% of global N2O emissions, have mixed responses to no-till. Whereas several meta-

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analytical and experimental studies showed an overall increase in N2O emissions from no-till

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systems (Huang et al., 2018; Mangalassery et al., 2014; Mei et al., 2018; Six et al., 2004),

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others reported the no-till effect depending on environmental and management conditions

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(Rochette, 2008; van Kessel et al., 2013; Zhao et al., 2016). Although such discrepancy is, to

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some extent at least, attributed to the different data sets or different statistical methods used,

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the effects of no-till on crop productivity, soil carbon sequestration, and N2O emission remain

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contested.

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The adoption of no-till, via changes in soil biophysical characteristics, would be

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expected to affect not only the issues as mentioned above but also soil denitrification. Soil

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denitrification is a facultative anaerobic reaction and has attracted a lot of attention because

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of its contribution to fertilizer N loss and N2O emissions from agricultural soils (Bouwman et

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al., 2013; Firestone and Davidson, 1989). No-till tends to increase soil denitrification because

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of the high prevalence of anaerobic microsites under high moisture content and bulk density

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(Baggs et al., 2003; Bateman and Baggs, 2005). In poorly aerated or fine-textured soils, for

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example, no-till leads to an increase in N2O emission, which has often been attributed to

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enhanced N2O production from the denitrification process (Mei et al., 2018; Rochette, 2008).

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Although numerous experiments have investigated the effect of no-till on soil denitrification,

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results of these studies are conflicting, showing either an increase (Estavillo et al., 2002; Rice

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and Smith, 1982), decrease (Fuller et al., 2016; Menéndez et al., 2008) or no significant

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change in denitrification (Fan et al., 1997; Liu et al., 2007).

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Soil denitrification is catalyzed by a number of enzymes, including nitrate reductase,

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nitrite reductase, nitric oxide (NO) reductase, and N2O reductase, which is encoded by the

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genes narG/napA, nirS/nirK, norB, and nosZ, respectively (Philippot et al., 2007). As it is

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difficult to measure the dominant end-product (N2) of soil denitrification, the potential

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denitrification activity, in the form of the concentration of denitrifying enzymes in a soil is

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often used as a proxy (Tiedje, 1982). Some studies have shown that the response of potential

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denitrification to no-till was either positively or negatively correlated with the changes in the

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abundances of the denitrifying genes in question (Baudoin et al., 2009; Domeignoz-Horta et

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al., 2018; Melero et al., 2011). However, there is also emerging evidence that soil

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environmental conditions rather than the abundance and/or diversity of denitrifying genes are

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mainly responsible for changes in potential denitrification activities (N2O+N2) or N2O fluxes

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(Attard et al., 2011; Liu et al., 2013). Through manipulating the soil microbial community

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using a dilution approach, Philippot et al. (2013) demonstrated that the reduction in denitrifier

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diversity rather than its gene abundance resulted in the lower potential denitrification.

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Consequently, it remains unclear how soil denitrification would respond to the adoption of

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no-till and its interactions with abiotic and biotic factors under a wide range of environmental

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and experimental factors.

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In this study, we aimed to identify the general trend of soil denitrification in response

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to the adoption of no-till and its underlying mechanisms at the global scale as compared to

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conventional tillage. To address this, we conducted a meta-analysis based on a global data set.

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For this analysis, we included studies that had reported side-by-side comparisons of soil

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denitrification rate, the potential activity and the total number of denitrifiers, as well as the

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abundance of denitrifying genes for conventional tillage and no-till plots. We hypothesized (i)

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that no-till would increase soil denitrification relative to conventional tillage, because higher

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water-filled pore space (WFPS) as a result of an increase in soil moisture and bulk density

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under no-till conditions could favor the formation of anaerobic microsites (Davidson et al.,

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2000; Linn and Doran, 1984); (ii) that enhanced soil denitrification would be driven by a

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stimulation of the activity and size of denitrifier communities; and (iii) that the positive

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response of soil denitrification to no-till would lead to increased N2O emission.

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2 Materials and Methods

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2.1 Data collection

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We extracted results for the rate of soil denitrification, the potential denitrification activity,

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the total number of denitrifiers, and the abundance of denitrifying genes (i.e., napA, narG,

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nirK, nirS, and nosZ) from no-till studies conducted in the field and/or in the laboratory

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before June 2019. Where possible, we also extracted the data on soil N2O emission from

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studies reporting the rate of soil denitrification. The literature search was performed

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following the PRISMA guidelines (Fig. S1) (Moher, 2009). An extensive literature survey of

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publications was undertaken with Web of Science (Thomson Reuters, New York, USA) using

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the search terms of “denitrif* AND till*” for the rate of soil denitrification and ((denitrifier*

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OR denitrifying OR denitrification enzyme OR denitrification) AND till*)” OR “((narG OR

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napA OR nirK OR nirS OR norB OR nosZ) AND till*) for other variables. Additional

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searches were also conducted on Google Scholar (Google, Mountain View, CA, USA) and

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China National Knowledge Infrastructure (CNKI, Beijing, China). To be included in our data

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set, studies had to meet several criteria. First, to better represent the effect of no-till on soil

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denitrification under natural conditions, studies were included when their measurements were

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performed under field conditions and/or with undisturbed soil cores in the laboratory. Second,

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the plant species, soil type, and other management practices (e.g., fertilization and irrigation)

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between the no-till and conventional tillage plots had to be identical. Third, means and

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sample sizes of observations for these variables had to be available for both treatments. In

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cases where data were presented in figures, values were digitally extracted using GetData

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Graphic Digitizer (version 2.24, http://www.getdata-graph-digitizer.com/). In total, 323

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observations from 57 studies across the globe were included in this meta-analysis (Fig. 1;

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Data S1).

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To test the potential effects of other factors described below on the no-till effect, we

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extracted auxiliary information from each study, including study location, experimental

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duration, the length of time since no-till implementation, the land-use type, soil

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physicochemical properties, soil depth, and N fertilizer management when available. The

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predictor variables for the data summary included climate, land-use type, soil texture, SOC

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content, soil pH, N fertilization, and residue retention. Specifically, we used latitude and

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longitude coordinates to obtain the aridity index values from the Global Aridity Index

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Version 2 dataset (http://www.cgiar-csi.org), which is based upon the WorldClim 2.0

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(http://worldclim.org/version2). Following the generalized climate classification scheme,

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aridity index values of less than and more than 0.65 were categorized as ‘dry’ and ‘humid’,

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respectively. The land-use types included cropland and grassland. Topsoil textural

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classification was defined according to the simplified textural classification for the first 0-30

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cm in the Soil Map of the World (Nachtergaele et al., 2008). Soils were grouped for their

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SOC content into four classes as follows: <0.6%, 0.6–1.2%, 1.2–2%, and >2% (FAO, 1995).

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Soil pH classification was conducted based on the USDA criteria: acidic ≤6.5, neutral 6.6–

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7.3, and alkaline ≥7.4 (http://soils.usda.gov). Nitrogen fertilizer and residue management

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were treated as a binary variable (yes/no). The change in soil bulk density following no-till (∆

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bulk density) was calculated when available. The number of years since the initiation of no-

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till was recorded for each observation. To determine the relative response of denitrification

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end-products to no-till, the ratio of both N2O/(N2O+N2) and N2O/N2 was calculated from

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studies that reported both soil denitrification and N2O emission.

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It is worth noting here that the acetylene inhibition technique (AIT) was commonly

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used with intact soil cores for measuring the rate of soil denitrification (Mosier and

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Klemedtsson, 1994; Tiedje, 1982; Yoshinari et al., 1977), except one study that used the 15N-

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gas flux method (Liu et al., 2007). We acknowledge that while the AIT assay may

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underestimate total soil denitrification, it is still widely used to assess the relationships

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between soil denitrification and environmental and biotic factors (Groffman et al., 2006;

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Wang and Yan, 2016). Because we focused on the relative change in the rate of soil

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denitrification in no-till compared to conventional tillage, the disadvantage of the AIT assay

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is therefore unlikely to be a potential cause of bias in our analysis. The potential

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denitrification activity was measured as described previously (Tiedje, 1982). This method is

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intended to provide an estimate of the indigenous denitrifier population and associated

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enzyme activity (i.e., denitrification enzyme activity). The population size of denitrifiers was

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determined using the most probable number as described in Woomer (1994). The abundance

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of denitrifying genes encoding nitrate (napA and narG), nitrite (nirK and nirS), and N2O

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reductases (nosZ) were determined using the quantitative PCR method as described

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previously (Henry et al., 2006; Kandeler et al., 2006). Note that the functional gene nosZ in

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our meta-dataset refers to the nosZ I clade as the nosZ II clade has only recently been

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investigated in a few studies (Domeignoz-Horta et al., 2018; Kaurin et al., 2018).

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2.2 Meta-analysis and statistics

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To evaluate the effects of no-till on the rate of soil denitrification and its end-products (N2O

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and N2O+N2), the potential denitrification activity, the total number of denitrifiers, and the

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abundance of denitrifying genes, we used a natural logarithm of the response ratio (lnRR), a

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metric commonly used in meta-analyses (Hedges et al., 1999; Pittelkow et al., 2015a; Wang

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and Yan, 2016). The lnRR was calculated as: lnRR = ln(Xt/Xc), where Xt and Xc represent the

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mean of the no-till and conventional tillage plots, respectively. Since well replicated and

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long-term studies provide more reliable estimates of soil denitrification responses to no-till,

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we weighted the individual effects by replication and experimental duration (Terrer et al.,

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2016; Wang et al., 2018). To account for multiple observations originating from the same

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study, we used multilevel meta-analytic models that include a random term using the

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‘rma.mv’ function in the R package ‘metafor’ (Konstantopoulos, 2011; Viechtbauer, 2010).

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The effect of no-till was considered significant if the 95% confidence intervals (CI) did not

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overlap zero. We used a Wald test to determine whether treatment effects were statistically

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different between study categories. For easier interpretation, all results were back-

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transformed and reported as percentage change for no-till relative to conventional tillage

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practice [(elnRR – 1) × 100].

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To examine the relative effects of various predictors on the responses of soil

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denitrification and the potential denitrification activity to no-till, the best model was selected

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using maximum likelihood estimation in the R package ‘glmulti’ (Calcagno and de

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Mazancourt, 2010). The relative importance value for each predictor was expressed as the

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sum of Akaike weights for the models in which the predictor appears. These values can be

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considered as the overall support for each predictor across all models. A cutoff of 0.8 was set

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to differentiate between important and non-essential predictors. We also used a mixed-effects

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meta-regression to explore the potential relationships between the response ratios of soil

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denitrification and continuous variables when possible.

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To ensure the findings of this meta-analysis were robust, we conducted the following

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sensitivity analyses (Fig. S2). First, publication bias in the studies was evaluated by the

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funnel plot and Egger’s regression (Jennions et al., 2013). This was further adjusted by

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adding the missing studies to the analysis using the trim and fill method. Second, we used the

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leave-one-out method to identify suspicious cases with the ‘leave1out’ function in the R

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package ‘metafor’ and ran a separate meta-analysis with the subset of experiments with

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croplands only (Viechtbauer, 2010). Third, we weighted individual experiments by three

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different weighting functions, namely replication, replication and experimental duration, and

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the inverse of the pooled variance, so as to ensure that the weights of the meta-analysis did

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not affect the outcome. Given results using the different weighting functions were quite

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similar, we provided results of the meta-analysis on effect sizes that were weighted by

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replication and experimental duration. All statistical analyses described above were

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performed using R v.3.5.3 (R Development Core Team, 2016).

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3 Results

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When averaged across studies, no-till significantly increased the rate of soil denitrification by

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85% (95% CI: 48–124%, P < 0.001) relative to conventional tillage (Fig. 2). The increase in

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soil denitrification was higher but nonsignificant (P = 0.511) in the dry climates (98%, 95%

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CI: 42–175%, P < 0.001) than in the humid climates (72%, 95% CI: 31–126%, P < 0.001).

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The no-till effect did not differ between the land-use types probably because of small sample

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size in grassland (P = 0.816; n = 5). The positive response of soil denitrification to no-till

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was significant in both medium- and fine-textured soils but not in coarse-textured soils (P =

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0.247). No significant differences between subgroups of SOC and soil pH were detected in

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our analysis (both P = 0.74). The no-till effect on soil denitrification was significantly

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affected by N fertilizer management (P = 0.037), with a larger effect with N fertilization

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(101%, 95% CI: 59–154%) than without (46%, 95% CI: 9–97%). The no-till effect tended to

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be greater but nonsignificant with residue retention than without (P = 0.263). There was no

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evidence of publication bias based on the results of sensitivity analyses: The Egger’s

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regression did not support the asymmetry of the funnel plot (P = 0.097) and the trend of no-

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till effect was consistent but only with changes in their uncertainties when the data sets with

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or without grassland and different weighting functions were used for analysis.

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Changes in the no-till effect on soil denitrification (lnRR) were positively correlated

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with mean soil bulk density (n = 56, P = 0.002; Fig. 3a), but were not related to the change in

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soil bulk density (Fig. 3b). The response of soil denitrification to no-till was marginally and

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negatively correlated with the duration of no-till across all studies (n = 84, P = 0.085; Fig. 3c).

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However, when we limited our analysis to studies which provided additional details on the

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potential drivers of soil denitrification responses to no-till, model selection analysis showed

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that the effect of no-till on soil denitrification was best predicted by the change in soil bulk

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density, climate, and N fertilizer management (Fig. 3d).

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When using the data sets where soil denitrification and N2O emission were measured

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simultaneously (n = 34), we found that no-till significantly increased soil denitrification and

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N2O emission by 67% (95% CI: 33–109%) and 87% (95% CI: 49–134%), respectively (Fig.

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4). However, the changes in the ratio of denitrification end-products were positive but

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nonsignificant in response to no-till, with an increase of 2.5% (95% CI: –20–31%) for the

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N2O/(N2O+N2) ratio and 0.3% (95% CI: –30–44%) for the N2O/N2 ratio.

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No-till significantly increased both the potential denitrification activity (66%, 95% CI:

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33–108% P < 0.001) and the total number of denitrifiers (116%, 95% CI: 36–244%, P < 0.01;

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Fig. 5a) compared to conventional tillage. Overall, no-till had a positive effect on the

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abundance of denitrifying genes (Fig. 5a). The stimulating effect of no-till was significant for

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the genes nirK (23%, 95% CI: 4–46%; P < 0.05) and nosZ (20%, 95% CI: 1–43%; P < 0.05),

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but was not significant for the genes nirS (14%, 95% CI: –13–48%; P > 0.1) and narG (16%,

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95% CI: –8–47%; P > 0.1), and marginally significant for the gene napA (70%, 95% CI: –4–

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204%; P < 0.1). Although the no-till responses of the gene nirK- and nirS-to-nosZ ratio were

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either not, or only marginally, significant for studies which simultaneously measured both

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genes encoding nitrite-reductase, no-till significantly increased the ratio of (nirK+nirS)/nosZ

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(33%, 95% CI: 4–70%; P < 0.05). Model selection analysis showed that the response of the

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potential denitrification activity to no-till was best predicted by the midpoint of soil depth,

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such that the no-till effect was negatively correlated with the change in soil depth (Fig. 5b).

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4 Discussion

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The introduction of no-till can alter soil biophysical properties and thereby affect carbon

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dynamics and nutrient cycling in soils (Liebig et al., 2004; Sheehy et al., 2013). In this study,

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we provide strong empirical evidence, based on a meta-analysis of individual observations

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across the globe, that the adoption of no-till can significantly increase soil denitrification

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compared to conventional tillage from croplands and managed grasslands. This corroborates

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our first hypothesis that no-till would have a stimulating effect on soil denitrification. Our

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results clearly suggest that increased soil denitrification is attributed to the positive responses

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of the activity (i.e., potential denitrification activities) and size (i.e., denitrifiers and the gene

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abundances) of the denitrifying communities to no-till (Fig. 6). This is partially supported by

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the results of a recent meta-analysis showing higher microbial biomass and activity in no-till

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as compared to tilled soils (Fig. 6; Zuber and Villamil, 2016). Additionally, results of the

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model selection analysis suggest that the change in soil bulk density is one of the most

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important factors for predicting the no-till effect on soil denitrification, although we do not

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see a clear relationship between the responses of soil denitrification and bulk density to no-till

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(Fig. 3). Therefore, in agreement with previous studies, no-till tends to increase soil WFPS

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because of greater soil moisture and bulk density, which in turn contributes to enhanced

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heterotrophic denitrification and/or nitrifier denitrification (Angers and Eriksen-Hamel, 2008;

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Linn and Doran, 1984) (Fig. 6).

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In addition to the edaphic and biotic controls, our results suggest that the no-till effect

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on soil denitrification may be affected by climate and management practices. In line with the

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previous work reporting a greater effect of no-till on soil N2O emission in dry climates (Mei

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et al., 2018; van Kessel et al., 2013), the present analysis suggests that the no-till effect on

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denitrification appears to be higher in dry than humid climates. This is presumably due to that

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the increased soil WFPS is sufficient to enhance heterotrophic denitrification and/or nitrifier

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denitrification in dry but not in humid climates (Linn and Doran, 1984; van Kessel et al.,

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2013). As expected, we find that N fertilization can exacerbate soil denitrification under no-

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till conditions. This is supported by a significant and positive impact of N fertilization on

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agricultural soil denitrification under various climatic, edaphic, and management conditions

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(Wang et al., 2018). Also, the present analysis suggests that the positive impact of no-till on

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soil denitrification tends to gradually decline in the long run, but note that this analysis is

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based on fewer observations from the long-term experiment. Nevertheless, our finding

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supports the argument that the duration of no-till implementation is of great importance when

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evaluating no-till impacts on soil ecosystem functions (Pittelkow et al., 2015a; Powlson et al.,

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2014; Six et al., 2004; van Kessel et al., 2013).

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Several lines of evidence support the hypothesis that increased soil denitrification

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under no-till would lead to enhanced N2O emissions. First, this hypothesis is most likely

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supported by a larger positive response of N2O emission to no-till than that of soil

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denitrification. In support of this, a recent study that using 22 farm soils with no-till practiced

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for 5-10 years in the UK showed significantly higher (54%) potential N2O emissions from

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no-till relative to tilled soils (Mangalassery et al., 2014). Second, the observed positive

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response of (nirK+nirS)/nosZ ratio to no-till indicates that under no-till conditions, N2O

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production is higher than its reduction during denitrification. In agreement with this argument,

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some studies have reported a lower nosZ/16S rRNA ratio in no-till compared to conventional

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tillage plots, which means that a lower proportion of bacteria possessing the nosZ gene

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encoding the N2O reductase was often found under no-till conditions (Badagliacca et al.,

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2018; Grave et al., 2018; Kaurin et al., 2018; Krauss et al., 2017). Lastly, a recent meta-

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analysis suggested that no-till can significantly lessen soil NO emissions by 30% compared to

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conventional tillage (Fig. 6; Liu et al., 2017). This not only corroborates the ‘diffusion

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limitation’ hypothesis, especially under anaerobic condition (Firestone and Davidson, 1989;

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Russow et al., 2009), but also provides further support for our interpretation that no-till may

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increase the possibility of NO produced in soils being further reduced to N2O and/or N2 by

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the denitrifying communities (Fig. 5a). Altogether, these lines of evidence suggest that no-till

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leads to a substantial increase in soil denitrification, which in turn results in increased N2O

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emission.

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It is worth noting that our analysis has several limitations. First, although we find a strong link between the responses of soil denitrification and the size and activity of the

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denitrifying community to no-till, recent evidence suggests that variations in soil

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denitrification may be closely related to the differences in the structure of the denitrifying

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community. It has been well documented that soil denitrification activities in soils are

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strongly affected by various environmental factors (Aulakh et al., 1992; Philippot et al.,

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2007). In addition to these abiotic factors, increasing evidence suggests that soil

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denitrification is primarily governed by the denitrifier diversity but has weak or no

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relationship with the denitrifier abundance (Domeignoz-Horta et al., 2018; Enwall et al., 2010;

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Philippot et al., 2013). For example, using a dilution approach to manipulate the soil

316

microbial community, Philippot et al. (2013) point out that the decrease in denitrifier

317

diversity rather than the lower denitrifier biomass may result in a significantly lower potential

318

denitrification activity. These results collectively support the view that the importance of

319

functional redundancy is overstated, especially within more narrowly defined functional

320

groups such as denitrifiers in soil (Cavigelli and Robertson, 2000; Philippot et al., 2013;

321

Schimel and Schaeffer, 2012). As well as soil abiotic factors, the changes in the abundance

322

and structure of the denitrifying community must be addressed in the future investigation to

323

better understand the link between the response of soil denitrification and the shifts in

324

denitrifier diversity to no-till.

325

Second, the nosZ gene as a marker for N2O-reducing communities are commonly

326

used, but attention should be paid to a recently discovered clade of N2O-reducers (i.e., nosZ

327

clade II) which is diverse and abundant in soils (Jones et al., 2014, 2013). To our knowledage,

328

only two studies have investigated the effect of conservation tillage on the nosZ clade II and

329

showed contrasting results (Domeignoz-Horta et al., 2018; Kaurin et al., 2018). Some studies

330

have suggested that the nosZ clade II is not only more sensitive to environmental factors than

331

the nosZ clade I, but can also act as an important N2O sink and thereby contribute to

332

greenhouse gas mitigation (Domeignoz-Horta et al., 2018, 2015; Jones et al., 2014). The third

14

333

limitation of the present study is that the geographical spread of observations included in our

334

analysis is uneven (Fig. 1). Although c. 45% of the global area under conservation tillage is

335

located in South America, for example, only one study from Argentina is available and

336

included in our analysis. Moreover, results from our model selection analysis and previous

337

studies reveal that climate plays a vital role in determining soil ecosystem function responses

338

to no-till (Pittelkow et al., 2015a; van Kessel et al., 2013). Further research from these

339

regions is therefore strongly warranted to understand the role of climate and confirm our

340

findings. Furthermore, the available evidence indicates divergent effects of no-till on soil

341

denitrification between the top- and subsoil layers (Attard et al., 2011; Elmi et al., 2003;

342

Venterea and Stanenas, 2008). More research is needed to clarify how the no-till effect would

343

be affected by the potential interaction between N fertilization strategy (e.g., placement and

344

amount) and soil depth. Compared with the AIT assay, other advanced methods such as gas-

345

flow helium incubation and 15N-gas flux methods can more accurately quantify not only the

346

total denitrification but also its end-products (Butterbach-Bahl et al., 2013; Groffman, 2012).

347

Therefore, to better understand the response of soil denitrification to conservation tillage,

348

more research under field conditions using these advanced methods combined with molecular

349

biology techniques is needed.

350

In summary, our study provides empirical evidence that the transition from

351

conventional tillage to no-till results in an increase in soil denitrification, which is atrributed

352

to the enhanced activity and abundance of the denitrifying community. We also demonstrate

353

that the increased soil denitrification under no-till conditions may lead to increased N2O

354

emission. This is supported by both a higher response of soil N2O emission and a higher

355

(nirK+nirS)/nosZ ratio indicating a greater abundance of the N2O-producing microbial

356

communities. Both the structure of the denitrifying communities and the unaccounted N2O-

357

reducing microbial community might play essential roles in regulating soil N2O emissions

15

358

(Hallin et al., 2018; Jones et al., 2014; Philippot et al., 2013). Owing to the paucity of data

359

from regions where no-till is widely practiced, we need to be aware of the impact that might

360

have on our meta-analysis. For these reasons, further investigation is thus warranted to

361

confirm our findings. Taken together, our evidence suggests that we should be cautious when

362

shifting from conventional tillage to a no-till system, as the latter may have negative

363

consequences on fertilizer management and greenhouse gas mitigation.

364

Acknowledgments

365

This work was supported by the funding for High-Level Talent Introduction Project of

366

Nanjing Agricultural Unviersity and the European Commission under Horizon 2020 for a

367

Marie Skłodowska-Curie Actions COFUND Fellowship (663830-BU-048). We thank Prof.

368

Dave Chadwick for his comments on an early version of this manuscript and Clare M.

369

Brewster for her help with proofreading. We acknowledge the work carried out by the

370

researchers whose published data are included in this synthesis. Finally, we thank the two

371

anonymous reviewers who provided excellent feedback regarding this manuscript. Data

372

associated with this study (Data S1) is deposited in figshare:

373

https://doi.org/10.6084/m9.figshare.9772451.v1.

374

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FIGURE CAPTIONS

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Figure 1. Global distribution of study sites included in this meta-analysis. Green points

598

represent studies measuring soil denitrification rate, and red points represent studies

599

measuring the potential denitrification activity (PDA) and the size of the denitrifying

600

community.

601

Figure 2. The overall effect and comparisons for individual categorical variables of no-till on

602

soil denitrification rates. Values are means ± 95% confidence intervals. The number of

603

observations for each category is given in parentheses. The vertical dashed line is drawn at

604

zero.

605

Figure 3. Relationships between the responses (lnRR) of soil denitrification to no-till and (a)

606

mean soil bulk density (n = 56), (b) the increase in soil bulk density (∆ bulk density) since

607

no-till implementation (n = 52) and (c) the duration of no-till implementation (n = 84), and

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(d) model-averaged importance of the predictors of the no-till effect on soil denitrification (n

609

= 48). The regression line is fitted based on a mixed-effects meta-regression model with their

610

95% confidence interval. The sizes of the symbols which is grouped by soil texture are drawn

611

proportional to the weighs in the meta-regression analysis. The importance value is based on

612

the sum of Akaike weights derived from the model selection using corrected Akaike’s

613

information criteria. The cutoff is set at 0.8 to differentiate between essential and nonessential

614

predictors. Soil depth represents the midpoint of soil depth. The values of SOC, pH, bulk

615

density, and soil C:N ratio are means of no-till and conventional tillage plots.

616

Figure 4. The overall effects of no-till on soil denitrification rates, N2O emissions, and the

617

ratio of denitrification end-products (n = 34). Values are means ± 95% confidence intervals.

618

The vertical dashed line is drawn at zero.

26

619

Figure 5. No-till responses of potential denitrification activity (PDA), the total number of

620

denitrifiers, and the abundance and ratio of denitrifying genes (a), and model-averaged

621

importance of the predictors of the no-till effect on potential denitrification activity (n = 56)

622

(b). Values are means ± 95% confidence intervals. The vertical dashed line is drawn at zero

623

or 0.8. The importance value is based on the sum of Akaike weights derived from the model

624

selection using corrected Akaike’s information criteria. The cutoff is set at 0.8 to differentiate

625

between essential and nonessential predictors. Soil depth represents the midpoint of soil

626

depth. The values of SOC, pH, clay content, and soil C:N ratio are means of no-till and

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conventional tillage plots.

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Figure 6. Summary diagram of no-till effects on soil denitrification via changes in soil

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physicochemical and biological parameters. Responses of soil moisture, water-filled pore

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space (WFPS), total/dissolved organic carbon, soil temperature, microbial biomass, β-

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glucosidase, and NO emission to no-till are extracted from previous individual studies or

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meta-analyses (Angers and Eriksen-Hamel, 2008; Attard et al., 2011; Lal, 1976; Liu et al.,

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2017; Powlson et al., 2014; Zuber and Villamil, 2016). The white arrows indicate results

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based on this meta-analysis. The up, down, and two-directional arrows represent the positive,

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negative, and mixed response to no-till, respectively. CT, conventional tillage; NT, no-till.

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See text for further explanation.

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Highlights •

The effect of no-till on soil denitrification was meta-analyzed.

•

No-till significantly increased soil denitrification and N2O emissions.

•

No-till enhanced the activity and abundance of the soil denitrifying community.

•

An overall positive response of (nirK+nirS)/nosZ to no-till was found.

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: