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The urease inhibitor N -(n -butyl) thiophosphoric triamide (NBPT) has been reported to effectively reduce nitrogen (N) losses by inhibiting urea hydrolysis. However, the effect of NBPT on soil ureolytic and ammonia-oxidizing microbes is not well understood, with inconsistent effects on crop yield and nitrogen use efficiency (NUE). Further, repeated use or overuse of chemical fertilizers can have environmental implications like soil acidification. A field experiment was conducted at Leigh Creek, Victoria at a site with a history of repeated application of urea alone (40U) or with NBPT (as Green Urea NV® (40GU)) at 40 kg N ha−1, and urea applied at 80 kg N ha−1 (80U), to perennial ryegrass (Lolium perenne L.). We aimed to investigate the temporal effects of NBPT applied with urea on ureolytic and ammonia-oxidizing microbes, pasture dry matter (DM) yield, and NUE within a season following treatment applications. The abundance of ureolytic microbes was higher in the control (CK) compared to all N treatments on all sampling days. The ureC gene copy numbers in 40 GU were significantly lower than in 40U on sampling days seven and 45. There was no significant effect of NBPT on the abundance of ammonia oxidizers, but increasing urea application rate to 80 kg N ha−1 (80U) significantly increased the abundance of ammonia-oxidizing bacteria (AOB) on days 7 and 45, and complete ammonia oxidizers (comammox Nitrospira) clade B on day 7 compared to 40U. There was no significant effect of NBPT on pasture DM yield, N-uptake, or NUE. Increasing N application rate significantly increased pasture DM yield and N- uptake but this did not influence the pasture NUE. [ABSTRACT FROM AUTHOR]

• 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. 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. [ABSTRACT FROM AUTHOR]