Abstract
Nitrification inhibitors (NIs) applied to soil reduce nitrogen fertilizer losses from agro-ecosystems. NIs that are currently registered for use in agriculture appear to selectively inhibit ammonia-oxidizing bacteria (AOB), while their impact on other nitrifiers is limited or unknown. Ethoxyquin (EQ), a fruit preservative shown to inhibit ammonia-oxidizers (AO) in soil, is rapidly transformed to 2,6-dihydro-2,2,4-trimethyl-6-quinone imine (QI), and 2,4-dimethyl-6-ethoxy-quinoline (EQNL). We compared the inhibitory potential of EQ and its derivatives with that of dicyandiamide (DCD), nitrapyrin (NP), and 3,4-dimethylpyrazole-phosphate (DMPP), NIs that have been used in agricultural settings. The effect of each compound on the growth of AOB (Nitrosomonas europaea, Nitrosospira multiformis), ammonia-oxidizing archaea (AOA; “Candidatus Nitrosocosmicus franklandus,” “Candidatus Nitrosotalea sinensis”), and a nitrite-oxidizing bacterium (NOB; Nitrobacter sp. NHB1), all being soil isolates, were determined in liquid culture over a range of concentrations by measuring nitrite production or consumption and qPCR of amoA and nxrB genes, respectively. The degradation of NIs in the liquid cultures was also determined. In all cultures, EQ was transformed to the short-lived QI (major derivative) and the persistent EQNL (minor derivative). They all showed significantly higher inhibition activity of AOA compared to AOB and NOB isolates. QI was the most potent AOA inhibitor (EC50 = 0.3–0.7 μM) compared to EQ (EC50 = 1–1.4 μM) and EQNL (EC50 = 26.6–129.5 μM). The formation and concentration of QI in EQ-amended cultures correlated with the inhibition patterns for all isolates suggesting that it was primarily responsible for inhibition after application of EQ. DCD and DMPP showed greater inhibition of AOB compared to AOA or NOB, with DMPP being more potent (EC50 = 221.9–248.7 μM vs EC50 = 0.6–2.1 μM). NP was the only NI to which both AOA and AOB were equally sensitive with EC50s of 0.8–2.1 and 1.0–6.7 μM, respectively. Overall, EQ, QI, and NP were the most potent NIs against AOA, NP, and DMPP were the most effective against AOB, while NP, EQ and its derivatives showed the highest activity against the NOB isolate. Our findings benchmark the activity range of known and novel NIs with practical implications for their use in agriculture and the development of NIs with broad or complementary activity against all AO.
Highlights
Modern agricultural systems depend heavily on large inputs of synthetic N fertilizers to maintain crop productivity and meet the increasing global food demand (Fowler et al, 2018)
This study is the first to investigate the inhibitory effect of EQ, a novel nitrification inhibitors (NIs) of potential agricultural relevance, and its oxidation derivatives, quinone imine (QI) and EQNL, on soil nitrifiers grown in pure cultures and demonstrate greater inhibition of all three compounds on ammonia-oxidizing archaea (AOA) compared to ammoniaoxidizing bacteria (AOB) isolates
Considering that (i) in all cultures, QI showed equivalent or higher inhibitory activity compared to its parent compound, and (ii) in EQ-amended cultures, QI was formed at concentrations equal or higher than those expected to induce an inhibitory effect on the AO tested, and EQNL was formed at levels substantially lower than those expected to result in an inhibitory effect on the AO tested (Figure 1 and Supplementary Table 2), we suggest that QI is the main determinant for the persistent inhibitory effect of EQ on AO and nitrite-oxidizing bacteria (NOB), in line with our previous soil studies (Papadopoulou et al, 2016)
Summary
Modern agricultural systems depend heavily on large inputs of synthetic N fertilizers to maintain crop productivity and meet the increasing global food demand (Fowler et al, 2018). Hundreds of compounds have been identified that inhibit nitrifying prokaryotes (Bédard and Knowles, 1989; McCarty, 1999) including plant-derived molecules (Coskun et al, 2017), aliphatic and aromatic n-alkynes (Taylor et al, 2015; Wright et al, 2020), statins (Zhao et al, 2020), and PTIO (2-phenyl-4,4,5,5tetramethylimidazoline-1-oxyl 3-oxide; Martens-Habbena et al, 2015) Many of these are used as selective inhibitors of ammoniaoxidizing bacteria (AOB; e.g., octyne) or archaea (AOA; e.g., PTIO) in laboratory cultures, soil microcosms or slurries, but are not suitable for use in an agricultural setting due to rapid degradation in soil or application in a gaseous state. The precise mode of action of these NIs has yet to be fully elucidated
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