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The discovery of complete ammonia oxidizers, comammox Nitrospira, represents a breakthrough in the history of nitrification research. Nitrospira inopinata, which was obtained from an aquatic ecosystem, is the only pure comammox bacterial isolate reported so far. Ammonia oxidation kinetics of N. inopinata indicated that they prefer an oligotrophic lifestyle and may directly compete with ammonia-oxidizing archaea (AOA), but representatives of comammox Nitrospira from terrestrial ecosystems are lacking. Current studies demonstrate that the functionally dominant comammox Nitrospira in terrestrial ecosystems are separated from most sequences obtained from aquatic and engineering systems. Clades A and B of comammox Nitrospira seemingly have different ecological preferences in soils, possibly due to their different ammonium uptake systems. Analyses of the evolutionary history indicate that some genes of comammox Nitrospira involved in ammonia oxidation could be laterally transferred from β-ammonia-oxidizing bacteria (AOB). Some comammox Nitrospira species may have similar ecological preferences with AOB and be functionally active in the niches where nitrification was previously considered to be dominated by AOB. Taken together, this review summarizes the recent findings of the biogeographical distribution and ecological preference of comammox Nitrospira in large-scale soil surveys, the responses of comammox Nitrospira to nitrogen application in soil, and the putative mechanisms underpinning the ecological niche of terrestrial comammox Nitrospira. These studies from terrestrial ecosystems suggest that comammox Nitrospira are not strictly oligotrophic but both oligotrophic and copiotrophic with a broader ecological niche breadth. Comammox Nitrospira in terrestrial ecosystems are abundant and active in both oligotrophic and copiotrophic environments. These findings have profoundly expanded our knowledge of the environmental niches of comammox Nitrospira and their relative contribution to nitrification in soils. [ABSTRACT FROM AUTHOR]

Abstract The new discovery of complete ammonia oxidizers (comammox), single organisms capable of oxidizing ammonia into nitrate, redefined the traditional view of nitrification. However, little is known about the relative contributions of comammox and other nitrifiers to nitrification, particularly in agricultural soils with long-term intensive input of nutrients. Herein, we investigated the communities of ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA) and comammox Nitrospira in agricultural soils under nutrients input gradient of nitrogen (0–675 kg N ha−1 year−1), phosphorus (0–405 kg P 2 O 5 ha−1 year−1), and potassium (0–675 kg K 2 O ha−1 year−1) fertilizers for 19 years. The results showed that N and K fertilizers input significantly (P < 0.05) increased the AOB- amoA gene abundance, while AOA were not as sensitive as AOB. The comammox- amoA gene copies were increased in all fertilizer treatments and was significantly correlated (P < 0.05) with the amount of N fertilizer added. Terminal restriction fragment length polymorphism (T-RFLP) combined with clone-library assays of comammox- amoA gene showed that increasing gradient of nutrients input increased the relative abundance of 73 bp T-RF (assigned to Clade A) but decreased the relative abundance of 198 bp T-RF (representing Clade B). Correlation analyses and stepwise linear regression analyses demonstrated that AOB were the dominate contributors to soil potential nitrification, while comammox Nitrospira did not play a significant role (P > 0.05). This study provided insights into the adaptive responses of comammox Nitrospira and canonical ammonia oxidizers to long-term fertilizations and their relative contributions to potential nitrification in arable soils. Graphical abstract Unlabelled Image Highlights • Long-term fertilizers inputs increased the abundance of comammox. • Specific comammox clades responded distinctly to fertilizers inputs. • Comammox played a less important role in soil nitrification than AOB. • The abundance of AOB increased with the gradients of fertilizers inputs. [ABSTRACT FROM AUTHOR]

The recent discovery of comammox Nitrospira capable of converting ammonia to nitrate in a single organism radically challenged our century-long perception of the classic two-step nitrification performed by ammonia oxidizers and nitrite oxidizers. However, our understanding of the niche separation of comammox Nitrospira and canonical nitrifiers in forest ecosystems remains limited, especially under a global scenario of elevated nitrogen (N) deposition. Here we evaluated the impacts of six-year N deposition on the dynamics of comammox Nitrospira , ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in a subtropical forest soil. Soil inorganic N concentrations significantly increased under the six-year simulation of N deposition, while soil pH, available phosphorus, total carbon (C), C/N ratio and microbial biomass significantly decreased. Quantitative PCR showed that the amoA gene abundances of comammox Nitrospira clade B and AOA substantially increased under the increasing rates of N deposition. By contrast, the AOB amoA gene abundance significantly decreased with the higher levels of N deposition (100 and 150 kg N ha−1 yr−1). Increased 13CO 2 incorporation into the AOA communities, rather than comammox Nitrospira or AOB, was demonstrated in a DNA-stable isotope probing microcosm, indicative of the capacity of AOA to assimilate 13CO 2 through autotrophic nitrification in the investigated subtropical forest soil under long-term N deposition. Phylogenetic analysis revealed that the autotrophic AOA assemblages belonged to the Nitrosotalea cluster, and their capacity for assimilating CO 2 through autotrophic nitrification was not affected by the long-term N deposition. Taken together, we provided new evidence for the niche separation of comammox Nitrospira and canonical ammonia oxidizers in soil nitrification under the long-term N deposition in the acidic subtropical forest soil. Highlights • The abundances of comammox Nitrospira clade B and AOA increased with N input. • The abundance of AOB decreased with N input rates higher than 100 kg N ha−1 yr−1. • Increased 13CO 2 was only incorporated into soil AOA through autotrophic pathway. • N input showed no effect on autotrophic AOA within the Nitrosotalea cluster. [ABSTRACT FROM AUTHOR]

The discovery of comammox Nitrospira has expanded our knowledge of classical labor division in nitrification and opened up new avenues to unravel the nitrification process in terrestrial ecosystems. However, the effects of fertilization and rhizosphere on comammox Nitrospira remains largely limited, especially in alkaline agricultural soils. To address this knowledge gap, the community structure of comammox Nitrospira and canonical ammonia oxidizers in the bulk and rhizosphere of an alkaline agricultural soil following 8 years of fertilizer applications was investigated. The fertilization treatments included no fertilization (CON), mineral NPK fertilizer (NPK), manure + straw (MS), NPK + manure (NPKM), NPK + manure and straw (NPKMS), and NPK + manure and straw by deep ploughing (NPKMSD). Five phylogenetic clusters of comammox Nitrospira were detected in the soil including clades A.1, A.2.1, A.2.2, A.3 and B, with A.2.1 and B as the dominant clades. Fertilization had a greater effect than rhizosphere on the community structure of comammox Nitrospira , while both fertilization and rhizosphere had no significant effect on the community structure of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). Compared with CON and NPK, the relative abundance of comammox Nitrospira clade A, especially clade A.2.1, increased while clade B decreased following manure applications. Soil nitrate played a critical role in driving the variation of comammox Nitrospira community structure. These findings showed novel evidence of niche specialization of comammox Nitrospira and canonical ammonia oxidizers in agricultural soils. • Fertilization impacted more on comammox Nitrospira than canonical ammonia oxidizers. • Comammox Nitrospira clade B dominated in the alkaline agricultural soils. • Clade A.2.1 increased, but clade B decreased with manure application. • Nitrate might be an important driver shaping comammox Nitrospira community structure. [ABSTRACT FROM AUTHOR]

The newly discovered comammox Nitrospira has fundamentally overturned the perception of labor division in nitrification and attracted growing interests in the niche preferences of comammox Nitrospira in soils. Here, we investigated the abundance and community structure of comammox Nitrospira in an acidic Ultisol which received the fertilizers continuously for 32 years. Fertilization treatments included chemical nitrogen (N), phosphorus (P), and potassium (K) fertilizers (NPK), NP, NK, PK, and NPK plus peanut straw, rice straw, radish residue or pig manure. Our results revealed that comammox Nitrospira were undetectable in the NK, which was excluded from further analysis. Compared with NPK, NPK plus pig manure significantly increased the abundance of comammox Nitrospira clade A, while the other treatments did not. Available P was the most important factor positively associated with the abundance of clade A. In contrast, soil pH was the best predictor for the variation of comammox Nitrospira community structure, which was distinctly separated by three groups: PK, NPK plus pig manure and the other treatments. Four comammox Nitrospira clusters were identified: clades A.2.1, A.2.2, B.1, and B.2. Of these clusters, clade A.2.1 accounted for 100% of the comammox Nitrospira sequences in NPK, whereas PK and NPK plus pig manure increased the relative abundance of clades A.2.2, B.1, and B.2. The relative abundance of clade A.2.1 was negatively correlated to soil pH, while that of clade A.2.2 showed an opposite trend, indicating the potential niche differentiation of clade A in the soils. Moreover, clade B.1 were more abundant in NPK plus pig manure soils while clade B.2 favored growth in PK, suggesting their different niche preferences. Together, we provide novel evidence for the niche differentiation of individual clusters of comammox Nitrospira clades A and B in an acidic agricultural soil, which was fundamental to the sustainable management of microbial N cycle. • Pig manure rather than crop residues increases comammox Nitrospira clade A abundance. • Available P was the strongest factor positively associated with the abundance of clade A. • Soil pH was the key driver for the community structure of comammox Nitrospira. • Clade A.2.1 was the dominant comammox Nitrospira cluster in tested soils. • Niche differentiation of the individual clusters of clades A and B was found in the soils. [ABSTRACT FROM AUTHOR]

The urease inhibitor N -(n -butyl) thiophosphoric triamide (NBPT) has been reported to effectively reduce nitrogen (N) losses by inhibiting urea hydrolysis. However, the effect of NBPT on soil ureolytic and ammonia-oxidizing microbes is not well understood, with inconsistent effects on crop yield and nitrogen use efficiency (NUE). Further, repeated use or overuse of chemical fertilizers can have environmental implications like soil acidification. A field experiment was conducted at Leigh Creek, Victoria at a site with a history of repeated application of urea alone (40U) or with NBPT (as Green Urea NV® (40GU)) at 40 kg N ha−1, and urea applied at 80 kg N ha−1 (80U), to perennial ryegrass (Lolium perenne L.). We aimed to investigate the temporal effects of NBPT applied with urea on ureolytic and ammonia-oxidizing microbes, pasture dry matter (DM) yield, and NUE within a season following treatment applications. The abundance of ureolytic microbes was higher in the control (CK) compared to all N treatments on all sampling days. The ureC gene copy numbers in 40 GU were significantly lower than in 40U on sampling days seven and 45. There was no significant effect of NBPT on the abundance of ammonia oxidizers, but increasing urea application rate to 80 kg N ha−1 (80U) significantly increased the abundance of ammonia-oxidizing bacteria (AOB) on days 7 and 45, and complete ammonia oxidizers (comammox Nitrospira) clade B on day 7 compared to 40U. There was no significant effect of NBPT on pasture DM yield, N-uptake, or NUE. Increasing N application rate significantly increased pasture DM yield and N- uptake but this did not influence the pasture NUE. [ABSTRACT FROM AUTHOR]

Comammox Nitrospira are a newly discovered group of nitrifying prokaryotes and might be key contributors to nitrogen cycling in terrestrial ecosystems. Their large-scale distribution patterns and the dominant environmental factors shaping their ecological niches are not yet well documented. Here, we investigated the biogeographic distribution of comammox Nitrospira over 4000 km in eastern Australia and explored the niche specialization of individual comammox Nitrospira phylotypes. Our results revealed that the abundance, richness and community composition of comammox Nitrospira clade A were best predicted by mean annual precipitation (MAP) among all the determined environmental parameters. We identified four phylogenetic clusters of comammox Nitrospira : clade A.1, A.2.1, A.2.2 and A.3. MAP was consistently the strongest factor correlated with the relative abundances of the dominant clades, A.2.1 and A.3. MAP and other variables including soil nitrate, mean annual temperature and total nitrogen showed contrasting effects on the relative abundances of comammox Nitrospira clade A.2.1 and A.3, indicating their potential ecological niche differentiation in the soils. Together, we found a broad distribution of comammox Nitrospira clade A, but not clade B, in various terrestrial ecosystems across eastern Australia, and the abundance and diversity of comammox Nitrospira clade A can be mainly predicted by MAP among all the determined environmental parameters. These findings provide novel evidence for the environmental adaptation and niche specialization of comammox Nitrospira in the terrestrial ecosystems. • A broad distribution of comammox Nitrospira clade A was found in soils collected over 4000 km in eastern Australia. • Mean annual precipitation was a key driver of soil comammox Nitrospira clade A in terrestrial ecosystems. • Two comammox Nitrospira clade A clusters dominated in the terrestrial ecosystems. • These two comammox Nitrospira clade A clusters had contrasting ecological preferences. [ABSTRACT FROM AUTHOR]

• Organic fertilizer influences comammox community structure more than aggregate size. • Comammox Nitrospira clade A dominated in Ultisols. • Pig manure increased comammox Nitrospira clade B, especially in microaggregates. • Comammox Nitrospira had more co-occurrence links with AOA than with AOB. • Co-occurrence of comammox Nitrospira and AOA was more intensive in microaggregates. Complete ammonia oxidizers (comammox Nitrospira) were recently discovered to be capable of oxidizing ammonia and nitrite in a single microorganism, which refutes the long-held paradigm that nitrification required two distinct groups of microbes. However, the niche differentiation of comammox Nitrospira and their co-occurrence with ammonia-oxidizing archaea (AOA) and bacteria (AOB) remain elusive in different soil aggregates. Here, we investigated how 27-year application of mineral and/or organic fertilizers affects comammox Nitrospira , AOA and AOB in aggregates from an Ultisol. Aggregate sizes significantly influenced the community structure of AOA and AOB, rather than comammox Nitrospira. However, organic fertilizers had a greater role in mediating the community structure of comammox Nitrospira , AOA, and AOB than aggregate sizes. Comammox Nitrospira clade A dominated in the tested soils, while pig manure application significantly increased the relative abundance of clade B, especially in microaggregates. Comammox Nitrospira had more co-occurrence links with AOA than with AOB, indicating that comammox Nitrospira may share more similar ecological niches with AOA than with AOB. The co-ocurrence links between comammox Nitrospira and AOA were more intensive in microaggregates than in macroaggregates, supporting their similar oligotrophic lifestyles. However, comammox Nitrospira also co-occurred with AOB, despite the frequency was much lower than that with AOA, indicating that some comammox Nitrospira could share niches with AOB, especially in macroaggregates. Together, these results suggest that comammox Nitrospira could co-occur with both AOA and AOB, although they were more likely to co-occur with AOA than with AOB, particularly in microaggregates of the soils. These findings advance our understanding of the ecology and physiology of comammox Nitrospira in microhabitats of agricultural soils. [ABSTRACT FROM AUTHOR]

Unravelling the ecological niche differentiation between comammox Nitrospira , ammonia-oxidizing archaea (AOA) and bacteria (AOB) is essential to understand forest nitrogen dynamics. Although genomic and physiological studies have suggested that comammox Nitrospira may outcompete AOA and AOB under oligotrophic conditions, empirical evidence is lacking in terrestrial ecosystems. Here we investigated the environmental prevalence and functional importance of comammox Nitrospira and their niche differentiation with AOA and AOB in forest soils from eastern Australia. Clade A comammox Nitrospira was widely detected in these forest soils, and their abundance was positively correlated with ammonium, total nitrogen and pH. The ammonium concentration was the most important driver of clade A comammox Nitrospira abundance, based on random forest regression analysis. Clade B comammox Nitrospira was not detected in these forest soils and thus excluded from the downstream analyses. Urea application stimulated the growth of clade A comammox Nitrospira and AOB, but not AOA, during a microcosm incubation. DNA-stable isotope probing revealed that clade A comammox Nitrospira incorporated 13C–CO 2 in the incubations, suggesting that clade A comammox Nitrospira may contribute to autotrophic carbon fixation in these forest soils. Taken together, our findings indicate that clade A comammox Nitrospira is prevalent and probably contributing to nitrification in forest soils of eastern Australia. • Clade A comammox Nitrospira are widely detected in forest soils. • Abundance of clade A comammox Nitrospira was positively correlated with ammonium, total nitrogen and pH. • Clade A comammox Nitrospira incorporated 13CO 2 in SIP incubations. [ABSTRACT FROM AUTHOR]

The recent discovery of complete ammonia oxidizers (comammox Nitrospira) challenged the paradigm of the two-step nitrification mediated by two distinct groups of nitrifiers, and raised fundamental questions regarding their niche specialization and relative contribution to nitrification in agricultural soils. Previous studies suggest that comammox Nitrospira have an oligotrophic lifestyle and would outcompete canonical ammonia oxidizers (ammonia-oxidizing bacteria and ammonia-oxidizing archaea) under ammonia-limited conditions. Here, we demonstrated that comammox Nitrospira clade A were significantly more abundant than canonical ammonia oxidizers and 13CO 2 -DNA-stable isotope probing revealed that comammox Nitrospira clade A incorporated 13CO 2 into their genomes in fertilized agricultural soils during the microcosm incubation. Phylogenetic analysis of the amoA gene revealed that 13CO 2 -labelled comammox Nitrospira clade A belonged to the Nitrospira inopinata -related cluster and a new cluster that was distinct from the known comammox isolates. These results demonstrated the potential important role of comammox Nitrospira in autotrophic ammonia oxidation in agricultural soils amended with nitrogen fertilizers and their lifestyle may be not strictly restricted to oligotrophic habitats. There is a potential contribution of comammox Nitrospira to soil nitrification, which calls re-evaluation of the microbial nitrogen cycling processes and the subsequent impacts on agriculture and the environment. • Comammox Nitrospira clade A played a potential role in nitrification of fertilized agricultural soils. • The N fertilizer input significantly increased the abundances of comammox Nitrospira clade A, AOA and AOB. • Acetylene significantly impeded comammox clade A, AOA and AOB, but 1-octyne only inhibited AOB abundance. • Comammox Nitrospira clade A incorporated 13CO 2 into their genomes during the incubation. • A new cluster of comammox Nitrospira may play an important role in nitrification of fertilized soils. [ABSTRACT FROM AUTHOR]