Effects of the nitrification inhibitor nitrapyrin on urea-based fertilizers in a Mediterranean calcareous soil: N dynamics and microbial functional genes.
Authors: Giannopoulos Georgios , Elsgaard Lars , Zanakis Georgios , B. Franklin Rima , L. Brown Bonnie , Barbayannis Nikolaos 
Published: March 2020

&lt;p&gt;Nitrogen (N) fertilization is an essential agronomic practice, which increases crop yields and improves soil fertility. Globally, more than 110 x 10&lt;sup&gt;9&lt;/sup&gt; 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 NH&lt;sub&gt;3&lt;/sub&gt; or microbially nitrified (i.e., NH&lt;sub&gt;4&lt;/sub&gt;&lt;sup&gt;+&lt;/sup&gt; oxidized) to leachable NO&lt;sub&gt;3&lt;/sub&gt;&lt;sup&gt;-&lt;/sup&gt; and NO&lt;sub&gt;2&lt;/sub&gt;&lt;sup&gt;-&lt;/sup&gt;. Nitrification inhibitors (NI) are increasingly co-applied as a sustainable agricultural practice and block the process of nitrification, resulting in a temporal increase of NH&lt;sub&gt;4&lt;/sub&gt;&lt;sup&gt;+&lt;/sup&gt; in the soils. Several studies have documented the effectiveness of NIs in retaining soil NH&lt;sub&gt;4&lt;/sub&gt;&lt;sup&gt;+&lt;/sup&gt; and increasing crop yields, but less is known about the effects of NIs on the fate of urea&amp;#8211;N and the overall impact of NIs on the soil microbial community.&lt;/p&gt;&lt;p&gt;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 NH&lt;sub&gt;4&lt;/sub&gt;&lt;sup&gt;+&lt;/sup&gt; with a concurrent increase in NH&lt;sub&gt;3&lt;/sub&gt; volatilization. Integrated measures of soil NH&lt;sub&gt;4&lt;/sub&gt;&lt;sup&gt;+&lt;/sup&gt; 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 NO&lt;sub&gt;3&lt;/sub&gt;&lt;sup&gt;-&lt;/sup&gt; and NO&lt;sub&gt;2&lt;/sub&gt;&lt;sup&gt;-&lt;/sup&gt; when NI was applied with the tested fertilizer types, except MU fertilizer where the integrated measures of NO&lt;sub&gt;3&lt;/sub&gt;&lt;sup&gt;-&lt;/sup&gt; and NO&lt;sub&gt;2&lt;/sub&gt;&lt;sup&gt;-&lt;/sup&gt; doubled. Upon urea hydrolysis, the released NH&lt;sub&gt;4&lt;/sub&gt;&lt;sup&gt;+&lt;/sup&gt; was transformed to NO&lt;sub&gt;3&lt;/sub&gt;&lt;sup&gt;-&lt;/sup&gt; and NO&lt;sub&gt;2&lt;/sub&gt;&lt;sup&gt;-&lt;/sup&gt;, which subsequently decreased in concentration following a typical nitrification - denitrification pathway in the absence of plants. Soil N&lt;sub&gt;2&lt;/sub&gt;O emissions from urea fertilizers were reduced by 40% with UI, 50% with NI, and 66% with NI + UI.&lt;/p&gt;&lt;p&gt;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.&amp;#160;&lt;/p&gt;&lt;p&gt;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.&lt;/p&gt;&lt;p&gt;In conclusion, the use of NI doubled NH&lt;sub&gt;4&lt;/sub&gt;&lt;sup&gt;+&lt;/sup&gt; retention in the soil and decreased soil N&lt;sub&gt;2&lt;/sub&gt;O emission by 50%, through negatively affecting ammonia oxidizing and denitrifying microbes and subsequently reducing soil available NO&lt;sub&gt;3&lt;/sub&gt;&lt;sup&gt;-&lt;/sup&gt; and NO&lt;sub&gt;2&lt;/sub&gt;&lt;sup&gt;-&lt;/sup&gt;. 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.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Keywords&lt;/strong&gt;&lt;strong&gt;:&lt;/strong&gt; urea, zeolite, methylene-urea, nitrification inhibitor, nitrapyrin, calcareous soil, soil nitrogen&lt;/p&gt;