Effects of the nitrification inhibitor nitrapyrin on urea-based fertilizers in a Mediterranean calcareous soil: N dynamics and microbial functional genes.

Nitrogen (N) fertilization is an essential agronomic practice, which increases crop yields and improves soil fertility. Globally, more than 110 x 10⁹ kg of chemical N fertilizers are applied each year with urea-N being one of the most affordable options. Upon urea hydrolysis, any portion not assimilated by crops is either volatilized as NH₃ or microbially nitrified (i.e., NH₄⁺ oxidized) to leachable NO₃^- and NO₂^-. Nitrification inhibitors (NI) are increasingly co-applied as a sustainable agricultural practice and block the process of nitrification, resulting in a temporal increase of NH₄⁺ in the soils. Several studies have documented the effectiveness of NIs in retaining soil NH₄⁺ and increasing crop yields, but less is known about the effects of NIs on the fate of urea–N and the overall impact of NIs on the soil microbial community.

In a 60 day soil mesocosm experiment, we investigated the effects of Nitrapyrin (NI; 2-chloro-6-(trichloromethyl)pyridine) co-applied with a selection of urea-based fertilizers: urea (U); U with urease inhibitors (U+UI); methylene-urea (MU); and zeolite-coated urea (ZU), on a typical Mediterranean soil under ambient summer conditions. We showed that NI applied with urea fertilizers resulted in a slower decay of extractable NH₄⁺ with a concurrent increase in NH₃ volatilization. Integrated measures of soil NH₄⁺ were 1.5 to 3.3-fold greater when NI was applied. At the same time, there was a 10 to 60% reduction in integrated measures of NO₃^- and NO₂^- when NI was applied with the tested fertilizer types, except MU fertilizer where the integrated measures of NO₃^- and NO₂^- doubled. Upon urea hydrolysis, the released NH₄⁺ was transformed to NO₃^- and NO₂^-, which subsequently decreased in concentration following a typical nitrification - denitrification pathway in the absence of plants. Soil N₂O emissions from urea fertilizers were reduced by 40% with UI, 50% with NI, and 66% with NI + UI.

Interestingly, 15 days after the application of NI, there was a decrease in bacterial abundance (eub genes; qPCR) in all fertilized treatments. NI dramatically reduced the abundance of ammonia-oxidizing microbes (amoA genes) and there were fewer bacteria associated with denitrification genes (nirK, nirS, nosZ) when NI was applied. 

At the end of the experiment, there was no significant difference in total N among all fertilized soils. Total N was in excess when compared to the control, and it was a considerable N pool potentially immobilized in microbial biomass in the absence of crops.

In conclusion, the use of NI doubled NH₄⁺ retention in the soil and decreased soil N₂O emission by 50%, through negatively affecting ammonia oxidizing and denitrifying microbes and subsequently reducing soil available NO₃^- and NO₂^-. The application of NIs should be carefully planned and synchronized (timing) with crop growth to reduce subsequent N transformations and N loss to the environment.

Keywords: urea, zeolite, methylene-urea, nitrification inhibitor, nitrapyrin, calcareous soil, soil nitrogen