Niche Specialization Of Comammox Nitrospira And Canonical Ammonia Oxidizers In Soils And Its Regulating Factors

Nitrification has been considered a two-step process with ammonia oxidized to nitrite followed by oxidation to nitrate by two functionally-distinct groups of organisms-ammonia oxidizers and nitrite oxidizing bacteria(NOB)for over a century.However,this long-held assumption of labor division between the two functional groups was challenged by the discovery of complete ammonia oxidizers within the Nitrospira genus that are capable of converting ammonia to nitrate in a single organisms(Comammox)at the end of 2015.To date,comammox Nitrospira have been detected in a range types of soils such as agricultural soils,grassland and forest soils,and their abundance differed greatly in soil with different land uses.The distribution patterns of comammox in soil ecosystems and the influencing environmental factors are still in its fancy.The comparable abundances of comammox and canonical ammonia oxidizers in a range types of soils and both are capable of oxidizing ammonia into nitrite and then obtain energy from the process to contribute to their metabolic growth,indicating there exist a niche specialization of comammox Nitrospira and canonical ammonia oxidizers in soils.Previous research found that soil p H、substrate(NH3)availability and autotrophic and heterotrophic nutrient differentiation were key factors influencing niche differentiation of AOA and AOB.Nevertheless,factors affecting the niche differentiation of comammox Nitrospira and canonical ammonia oxidizers are still unclear as the structure of ammonia monooxygenase gene(amo A)of comammox differs significantly from that of canonical ammonia oxidizers.Up to now,the current enrichment cultures of comammox Nitrospira were only three strains,and all of which were just isolated from aquatic ecosystems and thus,could not illustrate the physiological and ecological characteristics of comammox Nitrospira in soil ecosystems.Therefore,it is crucial to explore whether or not there exist a niche differentiation of comammox Nitrospira and canonical ammonia oxidizers in soil ecosystems and its regulating environmental factors and the relative contribution of comammox Nitrospira to nitrification.Based on the above scientific issues,we firstly collected a total of 93 soil samples located in 26 sampling sites along different climatic zones in China,including 9 soil types and 4 land uses,to explored the distribution characteristics and community structure composition of comammox and canonical ammonia oxidizers(AOA and AOB)in soils and its regulating environmental factors across large spatial scales using macro-genome sequencing.And secondly,soil samples under six different fertilization treatments and two tillage managements(ridge tillage and conventional tillage)of paddy soils of long-term field experiment station were collected,respectively,to investigated the effects of ammonia and oxygen availability on niche differentiation of comammox Nitrospira and canonical ammonia oxidizers in soil by using q-PCR,clonal sequencing,high-throughput sequencing,and terminal restriction length polymorphism.What’s more,nitrification activity of comammox Nitrospira and canonical ammonia oxidizers was determined in two neutral rice soils at the DNA and RNA levels and their seasonal changes to assessed the functional importance of comammox in soil nitrification and the relative contribution of comammox to soil nitrification.The main findings were as follows:(1)The overall distribution patterns of ammonia oxidizers in soils across China from different latitudinal zones and the influencing environmental factors.Metagenomics revealed that comammox Nitrospira was detected in all soils,and its relative abundance was abundant in most of the soil samples.The relative abundance of AOA、Comammox and AOB were 71.8%、20.8% and 7.4%,respectively.The relative abundance of Comammox and canonical ammonia oxidizers varied considerably among different soil habitats(forest soils,paddy soils,upland soils and fallow fields).In terms of comammox,the average relative abundance of comammox was the lowest in upland soils(12%),but showed to be the highest in some forest and paddy soils(~20%).The results of correlation analysis showed that the relative abundance of ammonia oxidizers in soils was significantly positive correlated to soil moisture content,indicating that soil moisture content were the main environmental factors affecting the spatial distribution of ammonia-oxidizing microorganisms.The results of correlation and RDA analysis indicated that soil moisture content、soil temperature、NH4+ content were the main factors in influencing the community structure of comammox Nitrospira in soils.(2)Effects of long-term fertilization of agricultural soils on the ecological distribution of ammonia-oxidizing microorganisms in soils.The abundance and community structure of ammonia oxidizing microorganisms in neutral purple paddy soils under different fertilization patterns were analyzed using quantitative PCR and terminal restriction fragment length polymorphism(T-RFLP)methods,respectively.In the present study,the abundance of AOA and AOB ranged from 2.24×107-3.54×107copies/g soil1 and 1.63×106-6.70×106 copies/g soil,respectively.The abundance of Comammox Clade A and clade B ranged from 1.23×106-7.63×106 copies/g soil and1.11×105-1.65×105 copies/g soil,respectively.The abundance of ammonia oxidizing bacteria(AOB)and comammox clade A differed significantly in neutral rice soils under different fertilization treatments and increased with the increase of nitrogen fertilization applied.Fertilizer application significantly increased the abundance of ammonia oxidizing archaea(AOA)and comammox clade B compared to the no-fertilizer treatment group,however,both of the two microbes showed no difference in abundance between fertilizer treatment groups.The relative abundance of comammox clade A increased and comammox clade B decreased with the increase of nitrogen fertilization,and the difference in response to fertilization reflected the difference in physiological and ecological characteristics between the two comammox clades.The results of redundancy analysis showed that organic matter,NH4+ content were the key factors affecting the abundance and structural composition of Comammox Nitrospira community in soils.The positive correlation between soil potential ammonia oxidizing rate and nitrite oxidizing rate with the abundance of AOB indicated that AOB was the main driver in the ammonia oxidizing process in the study soils.(3)Oxygen availability(redox potential)in the niche segregation of ammonia-oxidizing microorganisms.Compared to conventional tillage(CT),ridge tillage(RT)effectively improved soil aeration during the growth period of rice.The plow layer(0-20 cm)for CT was submerged for the whole growth stage of rice,however,soils at the ridges for RT,especially the topsoil(0-5 cm),maintained good aeration conditions.In-situ field measurements showed that soil redox potential for RT was higher than for CT at the surface layer.Both ammonia oxidizing archaea(AOA)and ammonia oxidizing bacteria(AOB)were more abundant for RT than CT at 0-5 cm due to the well-aerated topsoil(0-5 cm)at the ridges for RT.However,no significant(p < 0.05)difference was observed on the abundance of both the two clades of comammox Nitrospira between RT and CT at all three sampling depths.Compared to canonical ammonia-oxidizing microorganisms,comammox Nitrospira showed to be less sensitive to oxygen availability(redox potential)in the soils.The intriguing insensitivity to oxygen availability for both comammox Nitrospira clade A and B indicated that the subgroups are well adapted to lower-oxygen environments in the neutral paddy soil.(4)Activity and ecological function of comammox Nitrospira in soils.The relative importance of ammonia oxidizing archaea(AOA),ammonia oxidizing bacteria(AOB)and Commomox Nitrospira clade A and clade B on the nitrification process was evaluated by means of the Q-PCR of the amo A gene both at DNA and RNA level and the nitrification activity in winter(January)and summer(June)conditions.Nitrification activity in January and June were evaluated by measuring the concentrations of NH4+-N and NO3--N of soils incubated with(NH4)2SO4(50 mg NH4+-N kg-1 soil)for 7 days at the corresponding sampling temperatures.The results showed that soil nitrification almost stopped under low temperature conditions in winter,while was vigorous in in summer.Quantitative results showed amo A transcript abundance of AOA and AOB were 1-2 orders of magnitude higher in summer soil samples than that in winter samples.In contrast,both amo A gene abundance and transcript abundance for comammox were high in winter(January)when nitrification occurred slowly and did not increase when nitrification increased in summer(June).These results indicated that comammox Nitrospira are responsible for a small fraction of the nitrification for these two neutral paddy soils.The high transcriptional activity for comammox in winter suggested that comammox Nitrospira adapted better to the low temperature environments than canonical AOM.The highly consistent pattern between nitrification rates and amo A gene expression of AOB indicated that bacteria dominated ammonia oxidation in the paddy soils.In summery,comammox Nitrospira was widely distributed in soil ecosystems and its relative abundance was abundant in most of the soil samples.Soil moisture content were the main environmental factors affecting the spatial distribution of ammonia-oxidizing microorganisms.Soil moisture content、soil temperature、NH4+ content were the main factors in influencing the community structure compositions ofcomammox Nitrospira in soils.Ammonia availability could be a key environmental factor leading niche segregation of comammox Nitrospira and canonical ammonia oxidizers in soils.Comammox clade B and AOA would adapt better to the poor N environments with low NH4+ content than comammox clade A and AOB.Oxygen availability play an important role in niche differentiation between comammox Nitrospira and canonical ammonia oxidizers in paddy soils.Comammox Nitrospira are better adapted to the environments with low dissolved oxygen than canonical ammonia oxidizers in paddy soils.Comammox Nitrospira were responsible for a small fraction of the nitrification in neutral paddy soils and adapt better to the low temperature environments than canonical ammonia oxidizers.