Archaea Are the Major Responsive Ammonia Oxidizers in a Paddy Soil Following Fertilization and Irrigation

Because ammonia-oxidizing archaea (AOA) are ubiquitous and highly abundant in almost all terrestrial soils, they play an important role in soil nitrication. However, the changes in the structure and function of AOA communities and their specic environmental drivers in paddy soils under different fertilization and irrigation regimes remains unclear. In this study, we investigated archaeal abundance, activity and community composition in acid paddy soils by a 10-year eld experiment. Results indicated that the highest potential ammonia oxidation (PAO) (0.011 µg NO 2− -N g − 1 d.w.day − 1 ) was found in T 2 (optimal irrigation and fertilization) - treated soils, whereas the lowest PAO (0.004 µg NO 2− -N g − 1 d.w.day − 1 ) in T 0 (traditional irrigation)- treated soils. Compared with the T 0 - treated soil, the T 2 treatment signicantly (P < 0.05) increased AOA abundances. Furthermore, the abundance of AOA was signicantly (P < 0.01) positively correlated with pH, soil organic carbon (SOC), and PAO. Meanwhile, pH and SOC content were signicantly (P < 0.05) higher in the T 2 - treated soil than those in the T 1 (traditional irrigation and fertilization)- treated soil. In addition, these two edaphic factors further inuenced the AOA community composition. The archaeal phylum Crenarchaeota and genus Candidatus Nitrosotalea were mainly found in the T 2 -treated soils. Phylogenetic analysis revealed that most of the identied OTUs of AOA were mainly aliated with Crenarchaeota. Together, our ndings conrmed that T 2 might ameliorate soil chemical properties, regulate the AOA community structure, increase the AOA abundance, enhance PAO and consequently maintain optimum rice yields in a subtropical paddy eld.


Introduction
The oxidation of ammonia to nitrite is the rst step in the soil nitri cation process, which was driven by ammonia-oxidizing bacteria (AOB) and archaea (AOA) 1,2 . For example, some researches showed that AOA might make more contributions than AOB in microbial ammonia oxidation through the enzyme ammonia monooxygenase (amoA) 3,4 . Conversely, other reports found that AOB played a more critical role than AOA 5,6 . This was because that these two ammonia oxidizers had different ecological niche partitioning 7 . Thus AOA and AOB play distinct roles in ammonia oxidation process under different soil and crop management systems. In this study, electrophoretic analysis of these two ammonia-oxidizers found that AOA, but not AOB, amoA genes were detected in paddy soils. Similarly, Gao et al. (2018) reported that archaea were the predominant and responsive ammonia oxidizing prokaryotes in the same type of rice paddy soil 3 . Hence, a follow-up measurement of AOA only were done in the studied paddy soils.
The subtropical paddy soil was classi ed as typical Hapli-Stagnic Anthrosols characterized by low nutrient capital, low pH value, high P xation that severely constrained rice production 8 . Rice production must, however, increase by 1% annually due to an increase in the population 9 . High rice yields mainly depend on higher inputs of nitrogen (N) and phosphorus (P) fertilizers, which inevitably increases the risk of potential eutrophication in the surrounding water bodies. Therefore, the optimizal water and fertilizer management was commonly used to increase soil nutrient bioavailability from rice plant, to achieve high yield, and to improve soil environment, and consequently, altered AOA populations 10,11 . For example, soil organic carbon (SOC), nitrate N (NO 3 − -N) and ammonium N (NH 4 + -N) had a big impact on soil ammonia-oxidizing archaeal composition 3,12 . Meanwhile, the ammonia oxidizers-driven potential ammonia oxidation (PAO) exhibited a strong response to water supply and fertilizer input 13 . Most of the present researches focused on the archaeal community change in response to alterations in edaphic properties induced by either irrigation management or fertilizer application alone, but few reports have evaluated their responses to different water and fertilizer management, especially in subtropical paddy soils. How the irrigation-and fertilization-induced heterogeneity in soil physicochemical properties further affects archaeal community remains unclear in subtropical paddy soils. Hence, studies on the characteristics of AOA in the paddy soil under water and fertilizer coupling are urgently needed to bridge this knowledge gap. Because AOA are ubiquitously distributed and extremely diverse in soils, high-throughput sequencing (HTS) is used to reveal soil ammonia-oxidizing archaeal diversity at a high-resolution. The approach can detect soil microbial taxa at very low levels. Additionally, the functional amoA gene is detected and quanti ed by an accurate quantitative real-time PCR 14 . These technologies provide opportunities to assess the abundance, composition, and activity of AOA communities in response to environmental changes associated with fertilization and irrigation.
Thus a 10-year eld experiment was conducted to estimate the effects of water and fertilizer application on AOA communities and their environmental drivers. Here we would test the following hypotheses: (i) the AOA community would be modi ed by soil factors associated with fertilization and irrigation, and (ii) alterations in AOA communities would further in uence PAO activity.

Results
Edaphic characteristics, potential ammonia oxidation,and soil productivity.
Rice grain yields in the T 2 treatment increased by 0.92 times, stover yields increased by 1.26 times compared to those in the T 0 treatment, respectively (Fig. 1A, B). However, there were no signi cant differences in the stover and grain yields between T 2 and T 1 treatments (Fig. 1A, B). Meanwhile, soil pH values varied between 5.97 and 6.24 under different fertilization and irrigation regimes, and thus the soils were acidic (Table 2).
Meanwhile, soil pH and SOC content were signi cantly (P < 0.05) higher in the T 2 treatment than those in the other two treatments. Compared with the T 0 treatment, the T 1 and T 2 treatments signi cantly (P < 0.05) increased in soil NH 4 + -N content. Nevertheless, no apparent differences occurred in the TN concentration in paddy soils under the different fertilization and irrigation regimes. There was a signi cant (P < 0.05) increase in soil NO 3 --N content in the T 1 -treated soils compared to that in the T 0 -treated soils. In addition, the PAO in the T 0 treatment signi cantly (P < 0.05) decreased compared with that in the T 1 and T 2 treatments, but the PAO in the later two treatments was not signi cantly different (Table 2).
Archaeal abundance and alpha diversity. The AOA abundance was estimated by quantifying their amoA gene copy numbers. In this study, archaeal amoA gene copy numbers (5.74 × 10 7 amoA gene copies g -1 dry soil) were higher in the T 2 -treated soils than those in the other two treatments (Fig. 2). In addition, the rarefaction curves reached saturation, indicating that the generated sequences were enough to re ect the diversity of archaeal amoA genes (Fig. 3). The diversity indices of the AOA -related OTUs did not differ signi cantly (P 0.05) under fertilization and irrigation regimes ( Table 4). The major reasons for no treatment effects on ammonia-oxidizing archaeal diversity mainly depended on their response to natural environments and not arti cial factors, such as fertilization and irrigation.
Archaeal community composition.
A total of 154,733 archaeal sequence reads, ranging from 12,837 to 21,639 per soil sample after QIIME quality ltering, were taxonomically classi ed into ve phyla and six individual genera (Fig. 6). The Venn diagram indicated that the numbers of ammonia-oxidizing archaeal OTUs at a 97 % sequence identity were 37, 33, and 41 in the T 0 -, T 1 -and T 2 -treated soils, respectively (Fig. 4). Only 36.67% of the total archaeal OTUs were shared in three soils treated by fertilization and irrigation (Fig. 4). Additionally, the proportion of shared OTUs in AOA between T 1 -and T 2 -treatments was 64.70% in their total sequences (Fig. 4). Furthermore, a query coverage was > 99.99%, suggesting that this study captured the dominant OTUs of AOA in each soil sample ( Table 4). The dominant ammonia-oxidizing archaeal phylum in paddy soils was Crenarchaeota (the sequence number at the phylum level varied from 59.36% to 75.62% in soils), while the rare archaeal phylum was characterized by low Thaumarchaeota (Fig. 6A). Meanwhile, fertilization and irrigation altered ammoniaoxidizing archaeal community structure using PLS -DA approach (Fig. 5). A total 34.21% of the variations in the archaeal community composition could be mainly explained by soil pH, SOC, and PAO (Fig. 8). Moreover, T 2 resulted in the prevalence of the archaeal phylum Crenarchaeota, which accounted for 75.62% of the total ammonia-oxidizing archaea (Fig. 6A). However, T 1 decreased the relative abundance of the phylum Thaumarchaeota by 78.38% and 73.33%, respectively, compared to that in the T 0 and T 2 treatments (Fig. 6A).
Meanwhile, the relative abundance of the genus Candidatus Nitrosotalea in the T 2 treatment increased by 4.86 and 19.50 times, respectively, as compared with that in the T 0 and T 1 treatments. In contrast, the T 1 and T 2 treatments decreased the abundance of the genus Nitrososphaera by 77.62% and 70.15% compared with that under the T 0 treatment, respectively (Fig. 6B).
Phylogenetic tree for the amoA gene of AOA was constructed based on the values of OTUs of the top 10 most abundant species, which indicated that most of ammonia-oxidizing archaeal OTUs were a liated with Crenarchaeota cluster and unclassi ed_k__norank_d__Archaea cluster, accounting for 64.12% and 21.76% of total reads, respectively (Fig. 7). In addition, the OTUs 21, 38, 40, 50, and 51 belonged to cluster Crenarchaeota, and the OTUs 35, 39, 49, and 52 for cluster unclassi ed_k__norank_d__Archaea, respectively (Fig. 7).
Relationships between ammonia-oxidizing archaeal communities and edaphic characteristics.

Discussion
The AOA abundance was re ected by the AOA amoA gene copy number. In this study, the AOA amoA gene copy number signi cantly (P < 0.05) increased in the T 2 -treated soil, but no apparent difference occurred in the T 1 -treated soil, in comparison with that in the T 0 -treated soil (Fig. 2). This nding demonstrated that the growth of AOA communities was signi cantly stimulated by the optimized fertilizer and water management practice. However, this result did not support the hypotheses that the oligotrophic nature of AOA makes them important for nutrient-limited soil ecosystems 14,15 . Additionally, these ndings were inconsistent with those reported by Fang et al. (2019), who found that there was no obvious difference in the AOA amoA gene copy number between the NPK and CK treatments 10 . These inconsistent results mainly depended on soil types, rice cultivars, and agricultural practices 11,16 . Meanwhile, the AOA amoA gene copy number ranged from 0.28 × 10 7 to 5.74 × 10 7 per gram dry soil (Fig. 2), which was about 10-fold higher than the value obtained by Fang et al.
(2019) 10 . The difference in the amoA gene copy numbers might arise from different sampling times 17 . In summary, it is di cult to accurately predict the AOA abundance by straightforward ways under different environmental conditions. This study also indicated that different fertilization and irrigation treatments formed distinct ammoniaoxidizing archaeal community compositions based on PLS -DA (Fig. 5). This agreed with a previous report showing the proper combination of fertilization rate and irrigation frequency could regulate soil ammoniaoxidizing archaeal community composition 11 . In addition, the most predominant OTUs of AOA had a liation to Crenarchaeota (Fig. 7), which differed from the previous ndings indicating that the dominant OTUs of AOA fell into Nitrososphaera in many soil systems 10,17 . The difference in AOA community structure in different types of soils reveals a separation and selectivity of AOA induced by their own growth characteristics and habitat conditions. Moreover, these ndings indicated that AOA members were dominated by distinct ammonia-oxidizing archaeal species under different environmental conditions 17,18 . In comparison with the other two treatments, the T 2 treatment increased the abundances of both ammonia-oxidizing archaeal phylum Crenarchaeota and genus Candidatus Nitrosotalea in soils. The genus Candidatus Nitrosotalea are obligate acidophiles, and thus are abundant in acidic soils 19 . In general, these ndings indicated a possible niche differentiation for AOA populations in which different soil microsites supported different AOA species.
The changes in ammonia-oxidizing archaeal abundance and community composition, in turn could result in altered rates and/or controls of corresponding functions. This study suggested that the AOA-speci c PAO in the T 1 -and T 2 -treated soils was signi cantly (P < 0.05) greater than that in the T 0 -treated soils ( Table 2). The result is similar to that obtained by previous studies indicating that enhanced PAO in soils was observed in the NPK treatments compared to that without fertilizer application 10 . These ndings demonstrated that fertilization and irrigation enhanced soil PAO by increasing AOA abundances, particularly in the T 2 -treated soils ( Table 3). The analyses supported that the hypothesis that an increase in AOA abundances could enhance PAO in soils 20 . Furthermore, the stimulation of PAO might increase N availability for rice plants and resulted in higher paddy soil productivity 10,17 , as con rmed in this study (Fig. 1A). However, long-term fertilization and irrigation had a small impact on ammonia-oxidizing archaeal diversity in paddy soils ( Table 4). The reasons for nonsigni cant effects of fertilization and irrigation treatments on the ammoniaoxidizing archaeal diversity might depend on their response to natural conditions (e.g., soil temperature, spatial pattern, and oristic composition) 15,21,22 . Taken together, this study indicated that long-term fertilization and irrigation could signi cantly in uence the abundance, activity and community structure of AOA rather than their diversity in a paddy soil.
The variation in the AOA community structure and abundance might be due to their responses to altered edaphic properties associated with different fertilization and irrigation managements. A correlation analysis revealed that the AOA abundance was negatively related to TN and NO 3 − -N contents in the acidic paddy soil (Table 3). Conversely, positive correlations existed between the AOA abundance and TN, NH 4 + -N, and NO 3 − -N contents 10 . We found that various studies produced inconsistent ndings, perhaps because of different sampling times 17 . Meanwhile, the AOA abundance was positively correlated with SOC content in the acidic paddy soil (Table 3). Likewise, Zhang et al. (2013) reported that the AOA abundance had a signi cant positive relationship with SOC content, which supported the idea of heterotrophic growth of the archaeal community 23 . Additionally, the AOA abundance was positively related to pH (Table 3) 10 . The distinct responses of the AOA populations to soil pH were perhaps these AOA communities grew well in their own narrow pH ranges 25 . Besides directly pH-selecting for acidophilic or neutrophilic ammonia oxidizers, pH could also in uence soil nutrient availability, which indirectly mediated the AOA community composition. For instance, SOC, especially dissolved organic carbon (DOC) was another important edaphic factor driving the AOA community structure in acid soils 25 . In general, these studies further demonstrated that the abundance and community composition of AOA were shifted by altered soil properties associated with different fertilization and irrigation treatments, which might be the important factors responsible for variations in PAO.

Conclusions
Our results showed that AOA (ammonia-oxidizing archaea) played the vital role in ammonia oxidation in acid paddy soils. The T 2 (optimal irrigation and fertilization) treatment increased the AOA amoA gene copy number. Additionally, Crenarchaeota was the dominant phylum of AOA communities. Furthermore, an increase in the abundances of the phylum Crenarchaeota and genus Candidatus Nitrosotalea was observed in the T 2 treatment compared with those in the other two treatments. Meanwhile, SOC (soil organic C), pH, and PAO (potential ammonia oxidation) were the highest in the T 2 -treated soils. The T 2 -treated soils had clear differences in the AOA community composition compared with that in the T 0 (traditional irrigation) -and T 1 (traditional irrigation and fertilization) -treated soils. Moreover, the AOA abundance and community composition were mainly driven by soil pH and SOC. Meanwhile, the AOA abundance showed a signi cant positive relationship with PAO. Furthermore, the T 2 treatment increased rice yield compared to the T 0 and T 1 treatments. Taken together, these results suggest that the T 2 treatment should be utilized to mediate the archaeal community structure, promote ammonia oxidation rate, improve soil nutrient availability and thus maintain rice yield in the subtropical paddy eld.  Table 1. The chemical compound fertilizer (15% N, 15% P 2 O 5 , 15% K 2 O) was produced by China Petroleum and Chemical Co., Ltd. N, P, and K fertilizers were applied in the form of urea (46.4 % N), superphosphate (12 % P 2 O 5 ), and potassium chloride (60 % K 2 O) and used according to the amount of these fertilizers as shown in Table 1. The 100% of the total amount of P, 60% of N, and 40% of K fertilizers were used as basal fertilizers before transplanting of rice seedlings, and the 40% N and 60% K fertilizers as topdressing fertilizers after tillering, respectively (Table 1) The rice straw and grain were sampled. In addition, ve samples of 0−20 cm soil layer were collected and mixed from each plot after late rice harvest. The fresh soil samples were transported immediately on ice to the laboratory. Plant residues and stones were manually removed from soil samples. The soil samples were then mixed and sieved to < 2.0 mm. One subsample was stored at -80 ℃ for soil microbial analysis, while the other subsample was air-dried for chemical analysis.

Methods
Plant, soil physiochemical properties, and Potential ammonia oxidation (PAO). The rice straw and grain yields were measured at harvest from each plot, separately (rice grain weights were adjusted to 13.5% moisture content). In addition, the rice grain and straw materials were dried at 60 ℃ for 72 h, and weighed. Meanwhile, soil moisture was calculated as the difference between oven -dry (24 h at 105 ℃) and fresh weight. Soil pH was analyzed by a pH meter (EL20 K, Mettler -Toledo, Greifensee, Switzerland) in a 1:2.5 (m:v) soil -water suspension. The SOC content was measured by means of the oxidation-reduction titration. The TN content was analyzed using a Kjeldahl digestion. Both NH 4 + -N and NO 3 − -N in fresh soils were extracted with 2 M KCl The archaeal amoA gene was ampli ed by the primers Arch-amoAF (5′ STAATGGTCTGGCTTAGACG 3′) and Arch-amoAR (5′GCGGCCATCCATCTGTATGT 3′) 29 . Base on the preliminary experiment, the reaction systems and cycling conditions such as DNA amounts, annealling temperatures, and circular times were further optimized. The preliminary PCR ampli cation was performed for 27 cycles. All PCR products after 2.0% (w/v) agarose gel electrophoresis on a 2.0% (w/v) agarose gel is used to verify ammonia-oxidizing bacterial and archaeal size and quality. The band numbers and relative intensities of PCR products were analyzed using Quantity One analysis software (Bio-Rad). However, only the ammonia-oxidizing archaeal community was found and further analyzed under the following conditions. Each 20-μL qPCR reaction mixture contained 10 μL 2X Taq  The primers Arch-amoAF and Arch-amoAR 29 were used to amplify amoA gene fragments by a GeneAmp PCR system 9700 thermocycler (Applied Biosystems, Foster City, CA, USA Simpson's index (D) were analyzed by the mothur software package 32 . Chao1 and ACE were used to evaluate the ammonia-oxidizing archaeal community richness on the basis of the degree of sequence dissimilarity. H′ and D were used to evaluate to the diversity within each individual sample 33 . In addition, a Venn diagram showing the number of shared and unique archaeal OTUs was used to describe the similarities and differences among the archaeal communities associated with three treatments. A heatmap analysis was performed to compare the relative abundance of the top 10 archaeal genera. Moreover, a heatmap of relationship between the relative genus abundances of ammonia-oxidizing archaea and soil properties (e.g., pH, SOC, and TN) was conducted by Canoco for Windows 4.5 package. In addition, environmental factors were selected by the functions of env t (permu = 999) and vif.cca, and the environmental factors such as SOC, TN, NH 4 + -N, NO 3 --N, and PAO with P < 0.05 or vif < 10 were retained. The distancebased redundancy analysis (db-RDA) and partial least squares discriminant analysis (PLS -DA ) were processed by R software (version 3.2.1). The phylogenetic analysis on the basis of the sequences acquired from this study and reference sequences from the NCBI GenBank was made using the software MEGA version 5.0 34 to construct a phylogenetic tree by the neighbor-joining method. All bioinformatics analyses for soil ammonia-oxidizing archaeal communities were performed on online "I-Sanger" (http://www.i-sanger.com/) developed by Majorbio Bio-Pharm Technology Co. Ltd. All original nucleotide sequence reads were deposited at the NCBI Sequence Read Archive (SRA) with the accession number of SRP293735.
One-way analysis of variance (ANOVA) and Duncan's multiple range tests were used to estimate the statistical signi cance of the differences of edaphic characteristics, rice yields, archaeal amoA gene abundance and alpha-diversity under different water and fertilizer regimes by SAS Version 8.02 (SAS Institute Inc, Carey, North Carolina, USA). All data were expressed as mean ± SD (n=3). Notes: T 0 = Traditional irrigation; T 1 = Traditional irrigation and fertilization practice; T 2 = Water-saving irrigation and optimizing fertilization.  potential ammonia oxidation. * P < 0.05; ** P < 0.01.