Responses of ammonia-oxidizers and comammox to different long-term fertilization regimes in a subtropical paddy soil
Introduction
Nitrification is the fundamental process of the global N cycle in which ammonium is oxidized by microorganisms to nitrate in a two-step process [1]. The first step, in which ammonium is converted to nitrite, involves ammonium oxidizing bacteria (AOB) and/or archaea (AOA) [2]. The second step is the oxidation of nitrite to nitrate mediated by nitrite-oxidizing bacteria (NOB) [3]. Recently, bacteria with the capacity to perform both steps was discovered in aquatic ecosystems (comammox) [4,5]. These comammox bacteria belong to the genus Nitrospira lineage II, which were traditionally considered strictly nitrite oxidizing bacteria, although recent studies demonstrated they possess considerable metabolic diversity to utilize formate and H2 at oxic-anoxic interfaces and in anoxic environments [3]. The yet identified commamox bacteria can be subdivided into two clades (A and B) based on the phylogeny of their ammonia monooxygenase [4]. Metagenomic screening of the comammox amoA gene sequence in published databases revealed the widespread presence of comammox Nitrospira in a range of terrestrial and aquatic habitats [[4], [5], [6], [7]]. Although no commamox culture has yet been obtained from agricultural soils, the comparable this limits our understanding of comammox's ecological role in soils, the comparable amoA gene abundances of comammox and those of AOA and AOB in forest and paddy soils suggest that they are functionally relevant for soil nitrification [8,9]. Comparison of ammonia oxidation kinetics for the pure comammox Nitrospira inopinata with AOA and AOB cultured indicated that AOA are not necessarily the most competitive ammonia oxidizers for ammonia substrates under oligotrophic conditions [10]. Comammox, AOA, and AOB might co-exist in agricultural soils and serve different functions in their response to fertilization management. Therefore, it is necessary to re-evaluate the niche differentiation between comammox and canonical ammonia oxidizers (AOA, AOB).
In agricultural systems, which receive 25% of the total annual global N budget [11], nitrification may reduce soil N concentrations through volatilization after ammonia fertilizer application. Measurements under standardized conditions have demonstrated that the potential nitrification rate (PNR) of soil is strongly affected by N fertilization measurement [12,13]. The abundance, activities and distribution of AOB and AOA are well defined in agricultural ecosystems, which demonstrate that a large number of NH4+ substrate inputs can selectively alter the growth of AOA and AOB in clay loam soils [14]. The AOB respond to NH4+ addition quickly in moist soils [15], while AOA are usually less sensitive in neutral agricultural soils [16]. A previous study of an aquic Inceptisol with a sandy loam texture revealed that inorganic N fertilizer application changed the abundance and community composition of AOB, but had no significant effect on the AOA community [17]. The abundance of AOA tend to be to be more abundant than AOB for most agricultural soils [18], but the response of the population of comammox to long-term fertilization in agricultural soils is still not understood.
The objective of this study was to assess the response of the three microbial groups involved in nitrification to different long-term agricultural N-fertilization regimes. We hypothesized that (1) comammox may exist in purple paddy soils, and (2) comammox may exhibit different responses to variable fertilization regimes. In this study, purple paddy soils at the Purple Soil Ecology Experimental Station of Southwest University were used to measure effects of different fertilizer regimes on soil PNR. The abundances of AOA and AOB were evaluated via real-time quantitative PCR (qPCR), and the presence of comammox Nitrospira was quantified by molecular investigation using the newly-released PCR primer sets [9].
Section snippets
Site description and sample collection
The soil samples were collected from the Purple Soil Ecology Experimental Station of Southwest University, located in Beibei, Chongqing, China (N29°48′, E106°24′). The subtropical Southwest China mean annual temperature is 18.2 °C and mean annual total rainfall is 1080 mm. Soils are Orthic Entisols (Chinese taxonomy) or Regosols (FAO taxonomy). The long-term fertilization experiment was established in 1990 and planted to winter wheat from November to the early April and summer rice from early
Impacts of long-term different fertilization regimes on potential nitrification rates (PNR)
The long term fertilization treatments affected several physicochemical factors of the soils, including pH, organic matter, TN, TP, TK and other physicochemical factors (Table 2). PNR was significantly different between the different fertilization regimes (P < 0.05) (Fig. 2). The control treatment had the lowest PNR which increased after application of N fertilizer (P < 0.05), while the highest PNR (34.3 mg N kg−1 day−1) occurred for the NPK treatment. The treatment without added phosphorus (N
Discussion
The results of this study revealed that the commamox was widely distributed in paddy soils and that these bacteria exhibited different responses to fertilization. The qPCR results for amoA gene showed that comammox Nitrospira was highly abundant in the purple paddy soil, indicating that the newly reported comammox may have functional relationships with nitrification. For the no N control, the ratio of comammox to AOA was 8.9, and the ratio of comammox to AOB was 524.4 (Table 3). Both the ratios
Acknowledgements
This work was funded by the National Basic Research Program of China, (973 Program), China (2016YFD0300901) and National Natural Science Foundation of China, China (41671232).
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