Comparison of Novel and Established Nitrification Inhibitors Relevant to Agriculture on Soil Ammonia- and Nitrite-Oxidizing Isolates

Abstract

Nitrification inhibitors (NIs) applied to soil reduce nitrogen fertilizer losses from agr o -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 (EC 50 = 0.3–0.7 μM) compared to EQ (EC 50 = 1–1.4 μM) and EQNL (EC 50 = 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 (EC 50 = 221.9–248.7 μM vs EC 50 = 0.6–2.1 μM). NP was the only NI to which both AOA and AOB were equally sensitive with EC 50s 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.

The Effects of Quinone Imine, a New Potent Nitrification Inhibitor, Dicyandiamide, and Nitrapyrin on Target and Off-Target Soil Microbiota

“…Earlier studies by Papadopoulou et al ( 26 ) showed a clear and persistent inhibition of the transcription of amoA genes of both AOA and AOB by QI applied at rate of 16.1 mg kg −1 d.w. soil (i.e., approximately half of the high QI dose in the current study) in an alkaline soil (pH 7.9). The weaker effect of QI on AOB in the current study is probably a function of the lower dose rate of QI used in this study (10 mg kg −1 d.w. soil) compared to our earlier study and reflects the lower intrinsic sensitivity of AOB to QI compared to AOA ( 27 ).…”

“…Our hypothesis fits well with the theory that nonrandom associations exist between different AOM and NOB lineages that, along with edaphic factors, shape field-scale spatial patterns of nitrifying communities ( 52 ). Still a higher intrinsic tolerance of Nitrospira over Nitrobacter to NIs cannot be excluded, although data from relevant in vitro assays are only available for Nitrobacter ( 27 ).…”

“…A follow-up analysis of the spectrum of the inhibitory activity of EQ and its derivatives, compared to DCD and NP, against a range of soil AOB and AOA strains revealed a selective inhibitory activity of DCD on AOB, whereas NP showed equivalent inhibitory activity against AOB and AOA. EQ showed inhibitory activity against AOA and AOB, comparable to that of NP and DCD, respectively, with its major transformation product QI being the active NI ( 27 ). Considering the low cost of EQ ( 25 ), and its ability to be transformed in soil to a highly potent NI like QI, there is a major potential for its use in agricultural settings.…”

“…To minimize the involvement of other confounding soil factors on NIs performance, we selectively determined NI effects in two soils that largely differed in their pH (acidic versus alkaline) but had otherwise similar main physicochemical properties ( Table 1 ). We hypothesized that (i) the efficacy of the tested NIs toward nitrification would depend on the functionally dominant AOM groups/phylotypes in each soil, in accord with the pH-driven niche specialization of AOM ( 20 ), and the different sensitivity of the distinct AOM groups to the tested NIs ( 27 ); (ii) the inhibitory effects of NIs go beyond ammonia oxidation and extend to other soil microbiota components within and beyond N cycling; (iii) high NI dose rates would minimize differences in NIs efficacy and maximize potential off-target effects on soil microbiota. To test these hypotheses, we compared the impact of QI, DCD, and NP, applied at two dose rates, a low one equivalent to the agriculturally recommended application (low dose, LD ) and a high dose aiming at universal AOM inhibition (high dose, HD ), on (i) the activity of AOM by monitoring N-pools, potential nitrification rates, and changes in amoA gene and transcripts abundance; (ii) the diversity and dynamics of AOM communities and functionally linked NOB, respectively; and (iii) the diversity and dynamics of the total prokaryotic and fungal soil communities.…”

Soil Microbial Indicators within Rotations and Tillage Systems

“…C. Nitrosotalea (S) is an ammonia oxidizer found in acidic agricultural soil [107][108][109][110], which is the case in our study as the CCC rotation has the lowest pH. Lehtovirta-Morley, et al [111] and Papadopoulou, Bachtsevani, Lampronikou, Adamou, Katsaouni, Vasileiadis, Thion, Menkissoglu-Spiroudi, Nicol, and Karpouzas [110] both confirmed that C. Nitrocosmicus (S) occupy contrasting ecological niches compared to C. Nitrosotalea (S) and C. Nitrocosmicus is present in larger RAs at neutral pH. The unspecified AOA Nitrososphaeraceae (F) showing elevated RAs from the SSS rotation matches Behnke, Zabaloy, Riggins, Rodriguez-Zas, Huang, and Villamil [25].…”