Effects of repeated applications of urea with DMPP on ammonia oxidizers, denitrifiers, and non-targeted microbial communities of an agricultural soil in Queensland, Australia

Highlights

• Urea application reduced soil pH and increased soil total nitrogen (N), total carbon and nitrate;

• Use of DMPP increased soil pH and reduced soil nitrate at a higher application rate of 120 kg Nha⁻¹ relative to N alone;

• Addition of N did not influence soil bacterial composition at phylum level but increased AOB abundance at application rate of 80 and 120 kg N ha⁻¹.

• DMPP addition did not influence soil bacterial composition at phylum level but reduced AOB and nirK abundance at an application rate of 120 kg N ha⁻¹.

Abstract

Nitrification inhibitors have been reported to reduce nitrous oxide emission and nitrate leaching in agricultural systems. The effects of repeated applications of urea alone or in combination with nitrification inhibitors on nitrogen (N) cycling microbes involved in nitrification and denitrification together with non-targeted microbes are not well understood. Therefore, the objective of this study was to investigate the effects of repeated application of urea and DMPP on soil physio-chemistry, ammonia oxidizers and total bacteria in the soil. We collected soil samples from a 4.5-year field experiment under crop rotation with repeated application of seven treatments, namely control (CK), Urea (U), Urea + DMPP (UE) applied at 40, 80 and 120 kg N ha⁻¹, each treatment with three replicates. Ammonia-oxidizing bacteria (AOB) gene copy numbers increased as the N application rate increased (from 0 to 120 kg N ha⁻¹). The use of DMPP significantly reduced AOB and nirK gene copy numbers compared to urea alone at an application rate of 120 kg N ha⁻¹. There was no treatment effect on the abundance of ammonia-oxidizing archaea (AOA), Comammox clade A and B, nosZ and bacterial 16S rRNA genes. The community composition of AOB and AOA changed with N addition and use of DMPP but increasing N addition rate changed the composition of AOB only. Addition of N increased potential nitrification rates at 80 and 120 kg N ha⁻¹. There was no significant treatment effect on the relative abundance of bacteria at the phylum level. This experiment demonstrated that the application of N (with or without DMPP) at rates lower than 120 kg N ha⁻¹ would not result in significant impacts on soil archaeal and bacterial ecology.

Introduction

Soil microorganisms are important players in nutrient transformations and maintenance of soil functions (Aislabie et al., 2013; Brevik et al., 2015; Bei et al., 2018). However, they are highly sensitive to agricultural management practices like applications of fertilizers (Eo and Park, 2016; Shen et al., 2016; Chen et al., 2019) and can be used as indicators for soil quality (Sharma et al., 2010).

High input of nitrogen (N) fertilizers has been used to increase crop yields (Duan et al., 2014). However, there is low crop nitrogen use efficiency because applied N fertilizer can be lost through ammonia (NH₃) volatilization, nitrous oxide (N₂O) emissions, nitrate (NO₃⁻) leaching, erosion, and runoff processes (Chen et al., 2008; Castaldelli et al., 2019; Fuertes-Mendizábal et al., 2019). Nitrification has previously been thought to be a two-step process involving ammonia oxidation and nitrite (NO₂⁻) oxidation. Ammonia oxidation to NO₂⁻ is the rate-limiting step of nitrification, regulated by the amoA gene encoding the alpha subunit of the NH₃ monooxygenase (AMO) within ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) (Gao et al., 2016; Fuertes-Mendizábal et al., 2019; Miao et al., 2019). Nitrite oxidation to NO₃⁻ is the second step of nitrification and is regulated by nitrite-oxidizing bacteria (NOB). Recently, a group of bacteria within the Nitrospira genus was discovered with the capacity to completely oxidize NH₃ to NO₃⁻ in a single organism (comammox Nitrospira) (Daims et al., 2015; van Kessel et al., 2015v. Denitrification involves the conversion of NO₃- back to N₂O and dinitrogen gas (N₂) via NO₂⁻, nitric oxide (NO) (Kuypers et al., 2018; Castaldelli et al., 2019). Nitrous oxide can also be formed through nitrification by the chemical decomposition of hydroxylamine (NH₂OH) (Fuertes-Mendizábal et al., 2019). Denitrification is catalyzed by nitrate reductase encoded by the narG gene, nitrite reductase encoded by nirS / nirK gene, nitric oxide reductase encoded by norB and nitrous oxide reductase encoded by the nosZ gene (Braker and Tiedje, 2003; Shrewsbury et al., 2016).

Nitrification inhibitors are compounds that delay the oxidation of ammonium (NH₄⁺) to NO₃⁻ thereby preventing N losses through nitrification and denitrification (Suter et al., 2016; Fuertes-Mendizábal et al., 2019). 3, 4-Dimethylpyrazole phosphate (DMPP) is one of the nitrification inhibitors that has gained commercial use (Zerulla et al., 2001).

Fertilizer application has been shown to cause short and long-term effects on soil physicochemical properties which in turn influence soil microbial communities (Shen et al., 2016; Dai et al., 2018). Although several studies have investigated the effects of repeated N fertilizer application on soil microbial communities (Geisseler and Scow, 2014; Zhou et al., 2015; Shen et al., 2016), little information is available on the response of soil nitrifying, denitrifying and non-targeted microbes (i.e., who are not supposed to involve in nitrogen cycling processes) following repeated application of N fertilizers with nitrification inhibitors (Shi et al., 2017).

We measured changes in soil chemical properties, and the structure and composition of soil bacterial and N-cycling microbial functional communities, following the repeated applications of urea (U), and Urea + DMPP (UE) at different N rates for 4.5 years. The objective of this study was to investigate the effects of repeated application of urea and DMPP on soil physio-chemistry, ammonia oxidizers and total bacteria in the soil. We tested the following hypotheses to achieve our objectives: a) Repeated applications of urea and DMPP will significantly reduce soil pH and increase soil total carbon (TC) and total nitrogen (TN); and b) The levels of soil TC, TN, and pH will decrease bacterial composition and increase AOB and nirK gene copy numbers in the soil. This study will contribute to our understanding of how agricultural N management practices influence soil microbial ecosystems and their biological functions.