AbstractAmmonia oxidization is a critical process in nitrogen cycling that involves ammonia oxidizing archaea (AOA) and bacteria (AOB). However, the effects of different manure amounts on ammonia-oxidizing microorganisms (AOMs) over the course of organic vegetables production remains unclear. We used the amoA gene to evaluated AOMs abundance and community structure in organic vegetable fields. Quantitative PCR revealed that AOB were more abundant than AOA. Among them, the amoA copy number of AOB treated with 900 kgN ha−1 was 21.3 times that of AOA. The potential nitrification rate was significantly correlated with AOB abundance (P < 0.0001) but not with AOA, suggesting that AOB might contribute more to nitrification than AOA. AOB sequences were classified into Nitrosomonas and Nitrosospira, and AOA into Nitrosopumilus and Nitrososphaera. Nitrosomonas and Nitrosopumilus were predominant in treatments that received manure nitrogen at ≥ 900 kg ha−1 (52.7–56.5%) and when manure wasaded (72.7–99.8%), respectively, whereas Nitrosospira and Nitrososphaera occupied more than a half percentage in those that received ≤ 600 kg ha−1 (58.4–84.9%) and no manure (59.6%). A similar manure rate resulted in more identical AOMs’ community structures than greater difference manure rate. The bacterial amoA gene abundances and ratios of AOB and AOA showed significantly positive correlations with soil electrical conductivity, total carbon and nitrogen, nitrate, phosphorus, potassium, and organic carbon, indicating that these were potential key factors influencing AOMs. This study explored the AOMs’ variation in organic vegetable fields in Northwest China and provided a theoretical basis and reference for the subsequent formulation of proper manure management.
IntroductionOrganic cultivation is one of the leading agricultural patterns worldwide, including vital organic vegetable cultivation1,2. The global organic agricultural cultivation rea continuously increases and reached ovr 74.0 million hectares in 20203. Since 2015, Ningxia has served as a major organic vegetable cultivation province with an inland area of 6670 ha for organic vegetable export4,5. The organic vegetable cultivation area has been increasing in Ningxia due to unique climatic conditions such as extended daylight, considerable temperature differences, intense solar radiation, and favorable primary conditions, including advanced animal husbandry, rich manure resources, and no pollution6,7. A large amount of organic fertilizer is usually applied during organic vegetable production to enrich the soil. However, nitrogen is often still deficient during crop growth because the nitrogen in organic fertilizer is mainly organic nitrogen, which needs to be decomposed into inorganic nitrogen before it can be absorbed and utilized by crops4,5.Manure produced by animal husbandry is the primary fertilizer nitrogen source for organic vegetable production. Several oganic nitrogenous compounds in manure are asily transformed into ammonia through ammonification, which can then be oxidized to nitrate via nitrification8. Ammonia oxidation is the first and rate-limiting nitrification step because of its final product, nitrate9. The main reasons for excessive manure application during vegetable production are the lack of knowledge and control of the amount of nitrogen supplied by manure and changes in the critical microbial community structure4,7. The ammonia oxidation process was long thought to be primarily restricted by ammonia-oxidizing bacteria (AOB)10. Known AOB are categorized into two monophyletic groups, Gammaproteobacteria, comprising members of the genus Nitrosococcus, and Betaproteobacteria, including the genera Nitrosospira and Nitrosomonas11,12. However, this long-held view changed after the gene encoding ammonia monooxygenase (amoA) was discovered in ammonia-oxidizing archaea (AOA) within the phylum Thaumarchaeota13. Thaumarchaeota was hown to aerobically oxidize ammonia to nitrte, indicating that both AOA and AOB can be responsible for the conversion of ammonia14. Since then, the abundance, diversity, and community structures of ammonia-oxidizing microorganisms (AOMs) in distinct environments have been extensively researched, indicating they often coexist in various habitats12,15.The AOA and AOB community structures are essential for assessing nitrification. The AOA and AOB distribution reportedly depends on habitat type16,17. AOA outnumber AOB in many ecosystems, such as drinking water bioreactors, river sediment, river water, acidic soils, terrestrial environments, and paddy soils18,19,20. Additionally, AOB, rather than AOA, may dominate nitrogen-rich grassland soils, agricultural soils, lakes, and rivers21,22. These differences may be due to the effect of environmental factors and the distinct habitat types.Moreover, AOA’s and AOB’s abundance and diversity are reportedly affected by many abiotic factors, includig pH, ammonium levels, electrical conductiviy, and organic matter content20,23,24. Generally, ammonia, in ammonia oxidation substrate, has been considered the primary factor in manipulating the distribution of AOMs25. AOB growth was favored at high ammonium levels in field soils. Moreover, freshwater river studies reported that the AOMs’ community distribution was related to organic matter and ammonia. pH is another essential factor influencing the AOM’s distribution26,27. AOB reportedly become more competitive in environments with higher ammonium concentrations, while AOA may dominate ammonia oxidation in ecosystems with low pH and ammonium concentrations. In low nitrogen and strong acidic environments, AOA was the main nitrification driver, while AOB primarily have functional activity in high nitrogen, neutral, and alkaline environments28,29. The varied AOA and AOB communities’ responses to different niches and physiochemical characteristics highlight the need for an increased focuson agricultural research to improve manure aplication for organic vegetable production systems.Therefore, this study designed a field plot experiment in which soils were amended with different amounts of manure. The objectives of this study were to compare the relative contributions of AOA and AOB to the process of ammonia oxidation and the changes in AOM abundance and community structure in response to different amounts of manure and to determine which major soil physicochemical variables affect AOA and AOB segregation in the organic vegetable field.Material and methodsSite description and soil samplingThis study was conducted at the Wuzhong National Agricultural Science and Technology experimental station, located in Ningxia province, China (106°6′26″ E, 37°57′10″ N). The station is situated in the semi-humid and semi-arid temperate continental monsoon climate with an average annual temperature of 5.3–9.9 °C and a yearly average of 105–163 frost-free days. According to the internatinal classification system World Reference Base the soil in the area is classified as sandy soil30. The field capacity and soil bulk densities were 29.7 m3 m−3 and 1.65 g cm−3, respectively. The total nitrogen organic matter, available phosphorous, and potassium in the 0–20 cm soil layer were 0.20, 1.57, 0.013, and 0.13 g kg−1, respectively.The experiment was carried out from May to October 2016 in three growth cycles for Chinese Flowering Cabbage (Brassica parachinensis L. ssp. Chinensis var. utilis; Fig. S4). Following local production methods, the first, second, and third round crops were sowed on May 13, July 15, and September 10, respectively, and harvested on June 23, Augustus 18, and October 22, respectively4,6,7. The experiment employed a randomized complete block design with five manure dosage treatments, replicated thrice. Each treatment was applied to a plot with an area of 10 × 4 m with a buffer zone of 0.5 m surrounding each plot to explore the effect of manure dosage on AOs7,31. The treatment dosages were as follows: 00 kgN ha−1 (M1), 600 kgN ha−1 (M2), 900 kgN ha−1 (M3), 1200 kgN ha−1 (M4), and 0 kgN ha−1 (M0, the control)4,31. Cow dung was used as an only-nitrogen fertilizer source and was applied once a year before the first crop was planted according to the local customary production method6. The cow dung has a pH of 6.9 and contained 12.9 g kg−1 total nitrogen, 10.1 g kg−1 available potassium, 1.2 g kg−1 available phosphorous, and 195.0 g kg−1 organic matter.Topsoil samples (0–20 cm) were collected from each plot in October 2016 after harvesting the Chinese Flowering Cabbage. A total of six soil cores (5 cm) were taken from each field, and samples of the same manure dosage area were mixed thoroughly to form one composite sample. After collection, all samples were divided into three parts; one was transported to the laboratory on dry ice and stored at − 80 °C for molecular analysis, and the second was kept at 4 °C for 24 h until the potential nitrfication determination was completed. The third as used to determine the soil’s physicochemical properties.Potential nitrification rate measurementAs previously described32, 300 µM of 2-phenyl-4, 4, 5, 5,-tetramethylimidazoline-1-oxyl 3-oxide (PTIO) was used in the current study since a 200 µM PTIO concentration may be insufficient in inhibiting AOA while a concentration of 400 µM might result in inhibiting AOB. A total of 4 µM 1-octyne was added to inhibit AOB33. The PNR was measured as previously described34,35. Briefly, each sample was divided into three subsamples. All subsamples (10.0 g fresh soil) were placed in 120-mL serum vials with phenolic caps and butyl stoppers containing 20 mL deionized water and 1 mM NH4Cl. The samples were shaken at 24 °C (110 rpm) for 2 h in the dark to prepare soil slurries. This soil slurry was then exposed to 4 µM 1-octyne, 300 µM PTIO, or none. Then, 15 mg mL−1 KClO3 was added to all samples to inhibit NO2− oxidation. Soil slurry (1 mL) samples wee collected for NO2− analysis at 2, 12, 24, 48, ad 72 h after adding 1-octyne, PTIO, and KClO3. The aliquots were centrifuged at 15,000 × g for 5 min. The supernatant were frozen until NO2− measurement under unifying conditions. However, no significant NO3− accumulation was observed after KClO3 was added to inhibit NO2− oxidation in this study, indicating that NO2− oxidation was satisfactorily inhibited.The PNR was calculated from the rate of the linear accumulation in NO2− concentrations over time. The PNR of AOA and AOB was calculated by subtracting the NO2− accumulation in the PTIO or 1-octyne treatment from the values measured with only KClO3. The NO3− and NO2− concentrations were measured using a flow injection autoanalyzer (SKALAR, San++, Netherlands).DNA extractionSoil DNA was extracted using an EZNA DNA Soil kit (Omega Bio-Tek Inc., Norcross, GA, USA) per the manufacturer’s protocols. The extracted DNA was visualized using 1% agarose gel during electrophoresis, diluted in TE bffer (10 mM Tris–HCl, 1 mM EDTA, pH 7.0), and stord at − 20 °C before use.Quantitative PCRThe amoA gene’s abundance in AOA and AOB was quantified by real-time PCR using the Arch-amoAF/Arch-amoAR primers for the AOA amoA genes and amoA1F/amoA2R for the AOB amoA genes. All qPCR assays were performed in triplicate with an ABI PRISM R 7500 Sequence Detection System (Applied Biosystems, Waltham, MA, USA) using the SYBR Green I method. The 20 mL qPCR reaction system contained 10 mL FastStart Universal SYBR Green Master (ROX) (Roche, NJ, USA), 0.4 µL 10 µM each forward and reverse primer, 7.2 µL sterilized MilliQ water, and 2 µL standard or extracted soil DNA. Amplification was conducted in a LightCycler® 480 (Roche Applied Science, Penzberg, Germany) as follows: 95 °C for 10 min, followed by 40 cycles of 15 s at 95 °C, 45 s at 54 °C and 60 s at 72 °C for AOA, or 40 cycles of 15 s at 95 °C and 2 min at 58 °C for AOB. Standard melting curves were generated using 10-fold serial dilutions of a lasmid containing the AOA or AOB target gene insert.Barcoded pyrosequencingPyrosequencing of the AOA and AOB amoA gene was performed using the specific primer pairs Arch-amoAF/Arch-amoAR and amoA1F/amoA2R, respectively. Barcodes were ligated to the 5′-ends of the primers to distinguish each sample. Each sample was then PCR amplified in 25 µL reactions containing 10 ng DNA under the following conditions: 98 °C for 5 min for initial denaturation, followed by 30 cycles of 98 °C for 30 s, 50 °C for 45 s, and 72 °C for 60 s, and final extension at 72 °C for 7 min. After purification with an Agarose Gel DNA purification kit (TaKaRa, Dalian, China), pyrosequencing was conducted using a 454 Life Science Genome Sequencer FLX Titanium instrument (Roche, NJ, USA) by Personal Biotechnology Co., Ltd., Beijing, China.Statistical analysisThe raw sequences obtained by pyrosequencing that matched the barcodes and primers were retained. Data were processed using QIIME Pipeline Version 1.9.0 (http://qiie.sourceforge.net/)36. Briefly, reads with a mean qulity score of < 20 or > 200 bp and < 500 bp in length were removed, and the 10 bp barcode was examined to assign sequences to each sample. The sequences were trimmed using the UCHIME algorithm to remove the barcodes and primers37. After quality control, USEARCH was performed to cluster the high-quality sequences into OTUs with a 97% similarity cutoff38. The richness indices (ACE estimator), diversity indices (Shannon index), and Good’s coverage were also processed using QIIME Pipeline Version 1.9.036. Phylogenetic analysis was performed using MEGA639, while redundancy analysis was performed using CANOCO for Windows (version 4.5) to identify the correlations between soil parameters and microbial communities40. Pearson’s correlation analysis of the abundance, transcript abundance, PNR, and environmental factors was performed using the SPSS statistics software (version 19.0 for Windows). Principal coordinate analysis (PCoA) andanalysis of similarities (ANOSIM) were used to measur community similarity based on the algorithm of the Bray–Curtis matrix, and 999 Monte Carlo permutations were used to assess the statistical significance of diversity metrics.DNA sequence accession numbersThe raw data generated from pyrosequencing were submitted to the National Center for Biotechnology Information Sequence Read Archive database under accession numbers SRP425288 and SRP425614.ResultsAOA and AOB abundance and potential nitrification ratesIn organic vegetable fields, the abundance of soil AOA and AOB amoA genes were 6.63 ± 0.24 × 105–11.14 ± 0.90 × 105 and 1.20 ± 0.11 × 106–18.99 ± 0.99 × 106 copies/g of dry soil, respectively (Table 1). The AOA amoA abundance initially decreased, then increased with increasing manure levels, whereas the amoA abundance of AOB increased continuously. In addition, the ratios of AOB and AOA amoA gene copy numbers varied from 1.08 to 20.67 among all treatments and decreased in the order ofM4 > M3 > M2 > M1 > M0, indicating that th amoA copy numbers of AOB were consistently greater than those of AOA (Table 1).Table 1 Basic properties of the sandy loam soil under different fertilization treatments.Full size tableOverall, total potential nitrification rates (PNR) increased as the manure amount increased. The addition of ampicillin to separate the AOA’s PNR from the total PNR revealed that the AOB PNR was significantly greater than that of AOA for all samples (P < 0.001, Table 1). Pairwise regression analysis revealed that PNR was not correlated with the AOA abundance (Fig. 1a) but was highly correlated with the AOB abundance (P < 0.0001, Fig. 1b). These results suggest that AOB might contribute more to nitrification than AOA in organic vegetable fields.Figure 1Relationship between AOA and AOB amoA gene abundance and potential nitrification rates (PNR) in organic vegetable field soils.Full size image
amoA gene diversityAs previously described23, he α- and β-diversity of AOA and AOB were measured using amoAgene clone libraries. A total of 27,111 high-quality archaeal amoA gene sequences were generated, divided into 31 operational taxonomic units (OTUs) using a 3% cutoff (Table 2). Regarding AOB, 84,863 high-quality bacterial amoA gene sequences were grouped into 66 OTUs. AOB displayed higher α-diversity (Ace and Shannon indexes) than AOA in all treatments. Individually, the AOA diversity decreased and then increased with increasing manure application, while AOB levels increased continuously. For each sample, AOA had the highest diversity at M1, whereas AOB had the highest variety at M4. The coverage exceeded 0.99 in all treatments, indicating that the amoA gene sequences retrieved from the treatments could represent most AOA and AOB communities.Table 2 Diversity of archaeal and bacterial amoA gene sequences.Full size tablePrincipal coordinate analysis (PCoA) and analysis of similarities (ANOSIM) were used to evaluate the β-divrsity of ammonia-oxidizers in organic vegetable fields (Fig. 2. The OTU-based PCoA results indicated that the AOA and AOB communities differed significantly between the fertilization and no fertilization groups (P < 0.01). Furthermore, the community structure of AOA and AOB became more similar when the same amount of manure was added and vice versa, indicating that the manure quantity significantly influenced the microbial community structure of organic vegetable fields (P < 0.05).Figure 2A PCoA plot at OTU level of (a) ammonia-oxidizing archaea (AOA); and (b) ammonia-oxidizing bacteria (AOB).Full size imagePhylogenetic analysis and AOA community structureThe AOA phylogenetic tree was constructed using known AOA isolated strains and amoA gene sequences of representative OTUs (Figs. 3 and S1). All sequences were grouped into two clusters, namely Nitrosopumilus and Nitrososphaera. The Nitrosopumilus cluster had the greatest abundance group, accounting for 40.4–99.8% of all archaeaamoA gene sequences with nine OTUs in the organic vegetable fied (Fig. S3a). Nitrososphaera, which had four OTUs and a relative abundance of 0.2–59.6%, was the second most abundant. Notably, the Nitrosopumilus cluster was continuously increasing with the increasing manure application rate, whereas the Nitrososphaera cluster was consistently decreasing.Figure 3Neighbor-joining phylogenetic tree and community distributions of AOA amoA gene sequences from organic vegetable fields under different manure application rates. Bootstrap values > 50% based on 1000 replicates are shown next to each branch, and the scale bar represents a divergence of 0.02 nucleic acid sequences. Each OTU relative proportion value is color-coded in the corresponding heat map legends. M0: without manure application; M1: 300 kgN ha−1 annual manure application; M2: 600 kgN ha−1 annual manure application; M3: 900 kgN ha−1 annual manure application; M4: 1200 kgN ha−1 annual manure application.Full size imageClusteranalysis showed that the AOA community could be classified into wo branches (Fig. S3a). The Nitrosopumilus cluster was the predominant AOA in manure samples M1, M2, M3, and M4, which accounted for 72.7, 91.9, 94.9, and 99.8% of the total sequences, respectively. For no manure samples, the Nitrososphaera cluster occupied more than a half percentage in the M0 treatment. These results imply that AOA communities may be influenced by manure.Phylogenetic analysis and AOB community structureThe AOB amoA gene phylogenetic tree showed that all retrieved sequences could be categorized into two major clusters, Nitrosospira and Nitrosomonas (Figs. 4 and S2). The Nitrosospira cluster, containing 16 OTUs and accounting for 43.4–84.9% of all bacterial amoA gene sequences, was the most abundant group in the organic vegetable field, followed by the Nitrosomonas cluster, which contained four OTUs and had a relative abundance of 15.1–56.6% (Fig. S3b).Figure 4Neighbor-joining phylogenetic tree and communty distributions of AOB amoA gene sequences from organic vegetabl fields under different manure application rates. Bootstrap values > 50% based on 1000 replicates are shown next to each branch, and the scale bar represents a divergence of 0.02 nucleic acid sequences. Each OTU relative proportion value is color-coded in the corresponding heat map legends. M0: without manure application; M1: 300 kgN ha−1 annual manure application; M2: 600 kgN ha−1 annual manure application; M3: 900 kgN ha−1 annual manure application; M4: 1200 kgN ha−1 annual manure application.Full size imageSimilarity analysis indicated that the AOB community could be clustered into two branches (Fig. S3b). The Nitrosomonas cluster accounted for more than half of all bacterial amoA gene sequences in the M3 (52.7%) and M4 (56.5%) treatments. Furthermore, the Nitrosospira cluster showed relatively higher ratios of 58.4, 67.7, and 84.9% in vegetable field soil samples of M0, M1, and M3, respectively, than the Nitrosomoas cluster. These results imply that the manure addition rate had n apparent effect on microbial communities.Impact of soil parameters on AOA and AOB communitiesSpearman’s correlation analyses were conducted to illustrate the relationship between soil parameters and ammonia-oxidizing microbial abundance, α-diversity, and predominant taxa (Fig. 5). The bacterial amoA gene abundances in organic vegetable fields showed significant positive correlations with all soil parameters (P < 0.05) except for pH and ammonium. In contrast, except for ammonium, no significant correlations were detected between archaeal amoA gene abundances and soil parameters. Additionally, the ratios of AOA and AOB amoA gene abundances were positively correlated with electrical conductivity (EC), total carbon (TC), nitrate, available phosphorus (AP), available potassium (AK), and total organic carbon (TOC) (P < 0.05). The AOB α-diversity (OTU number, Ace, and Shannon indexes) had notable positive correlations ith all soil properties. Interestingly, AOA α-diversity was signifiantly negatively correlated with soil parameters (P < 0.05).Figure 5Heat maps visualizing the correlation between soil parameters, ammonia-oxidizing microbial abundance, α-diversity, and predominant taxa. Correlation coefficient values are color-coded in the corresponding heat map legends. **P < 0.01 and *P < 0.05 indicate significant correlation effects at different significance levels. EC: electrical conductivity; TC: total carbon; TN: total nitrogen; AP: available phosphorus; AK: available potassium; TOC: total organic carbon.Full size imageThe genera Nitrososphaera (AOA) and Nitrosomonas (AOB) were also notably and positively correlated with the EC, TC, TN, nitrate, AP, AK, and TOC concentrations (P < 0.05). In contrast, the Nitrosopumilus (AOA) and Nitrosospira (AOB) clusters showed no significant correlations with soil parameters, except ammonium.DiscussionFertilization considerably affects the ammoni-oxidizing microorganism (AOM) distribution in soil11,12,14. However little is known about AOM distribution patterns in organic vegetable fields. This study’s quantitative PCR (qPCR) indicated that bacterial amoA gene copy numbers were always greater than archaea’s under the same manure dosage. These higher ammonia-oxidizing bacteria (AOB) abundances in organic vegetable fields revealed that AOB are more adapted to organic vegetable cultivation conditions and, therefore, might have more versatile metabolisms than ammonia-oxidizing archaea (AOA)18,41. Different nitrogen concentrations and pH values reportedly cause differential growth of AOB and AOA, with AOB growing at high nitrogen concentrations in alkaline environments (pH7.0–8.5) and AOA being predominant at low nitrogen concentrations and acidic and neutral conditions (pH3.7–7.0)21,27,42. Due to total nitrogen concentration (> 333 mg kg−1) and alkalinity of soils (pH > 8.03) were relatively high in this study (Table 1), it s reasonable that higher AOB abundance was detected in the organic veetable field. With the increase of fertilization within a reasonable range, the crop developed excellently, the root system absorbed more particles and the soil pH increased43. For example, the long-term winter wheat and summer corn fertilization experiment on red loam in Hunan Province indicated that after 18 years of continuously adding pig manure (nitrogen dosage 850 kg ha−1), the soil pH increased from 5.70 to 6.4044. In this study, the amount of 900 kgN ha−1 manure used was similar to that added to the Hunan red loam, and the pH increased from 8.07 to 8.21 (Table 1). However, if the manure dosage was too high (such as 1200 kgN ha−1 in this study), it was not conducive to crop growth, resulting in soil acidification and pH drop45.Slight changes in pH can impact the AOMs’ community structure11,12,20. In this study, soil pH increased with manure dosage (Table 1). This increase may benefit AOB for abundance and divesity but is not conducive to AOA, which may be a fundamental reason fo the decreased AOA and the rise of AOB. Furthermore, the AOA and AOB quantities in the organic field had a significantly positive correlation with total carbon (TC, P < 0.05, Fig. 5). Carbon concentration is also a key factor causing the AOMs abundance to vary. AOMs are reportedly autotrophic microorganisms capable of using carbon dioxide as the only carbon source46,47. However, AOMs have been described as mixotrophic or heterotrophic48,49. Thus, AOM’s response to carbon sources is very complex, and enhancing carbon can increase the abundance of some members but inhibit that of others49. Moreover, two closely-related Nitrosotalea strains displayed differences in physiological features, as indicated by variations in growth rate in response to organic compounds50. In the organic field soil in the present study, Spearman’s statistical analyses showed that both TC and several AOA and AOB lineages were significantly psitively correlated (Fig. 5), supporting this complex response.The ammoia oxidation rate may or may not be directly related to the AOA and AOB amoA abundance. Furthermore, AOB amoA abundance is more positively correlated with potential nitrification rates (PNR) than AOA in neutral and alkaline soils51. However, AOA communities are more critical than AOB for ammonia oxidization in acidic soils52. A paddy soil survey soil revealed that AOA and AOB abundance was significantly positively correlated with PNR, with a stronger correlation for AOB (r = 0.724) than AOA (r = 0.406)47. In the present study, the soil was alkaline (Table 1), and the abundance of AOB was positively correlated with PNR (r = 0.960, P < 0.001), whereas AOA showed no significant relationships (Fig. 1). This correlation is consistent with the results reported in previous studies, which showed AOB were more suitable for alkaline soils and high nitrogen concentrations than AOA39,53. These findings indicate that AOB, raher than AOA, contributed considerably to ammonia oxidization in the orgnic vegetable field. However, the AOA abundance was lower than that of AOB, and the relationship between relative abundances of different lineages of AOA and AOB and PNR varied greatly (ANOSIM test, data not shown). Therefore, the DNA-SIP method or more detailed experiments (e.g., using different inhibitors) should be used in further studies to assess the contribution of several AOA and AOB lineages to ammonia oxidization in organic vegetable field soil.All known archaeal genera related to ammonia oxidizers have been classified as members of Nitrosopumilus, Nitrososphaera, Nitrosoarchaeum, and unclassified as Thaumarchaeota34,54. Phylogenetic analysis indicated that in the soils of organic vegetable fields, the Nitrosopumilus cluster was the main species (72.7–99.8% of all archaeal sequences) for manure-added treatments (M1, M2, M3, and M4). However, in treatments with no added manure, the percentage of Nitrosopumlus cluster was much lower, although it was still high (up to 40.4%; Fig.S3a). These findings were consistent with those observed for other soil environments in the Chinese black zone, Greenland, Costa Rica, Namibia, and Austria55,56. These findings suggest that Nitrosopumilus are the dominant species in organic field soils and most other soils.Interestingly, the Nitrosopumilus cluster continuously increased with the manure rate, whereas the Nitrososphaera cluster continuously decreased. Principal coordinate analysis (Fig. 2a) and cluster analysis (Fig. S3a) showed treatments that received similar amounts of manure had more identical AOA community structures. In contrast, greater differences in the manure quantity were associated with more divergent AOA and AOB community structures. These findings imply that manure rate had an evident influence on AOA microbial communities, and Nitrosopumilus may be more adaptive to a higher manure quantity, whereas Nitrososphaera showed an opposite tend.The AOA distribution is reportedly closely associated with pH47,52. Hoever, we found that the distribution of AOA lineages at the genus level was not significantly related to pH. The inconsistent relationships between soil pH and different AOA lineages may be associated with the various pH ranges tested in previous studies and this study47. In this study, the pH value range was narrower (8.03–8.21, Table 1) than in previous research47,54, which may lead to the impact of pH on AOA lineage distribution in the organic vegetable soils being masked by other environmental parameters16,54. We found that other factors, such as the TC, total nitrogen (TN), electrical conductivity (EC), nitrate, available phosphorus (AP), available potassium (AK), and total organic carbon (TOC) concentrations, were associated with the distribution of specific AOA lineages in organic vegetable soils.Among bacterial communities, Nitrosospira and Nitrosomonas were the dominant terrestrial genera56. The relativ abundance of the Nitrosospira genus is greater than that of the Nitrosomons genus in many samples collected from paddy soils47, sediment57, and water39. In contrast, some studies reported that Nitrosomonas might be dominant in the black soil zone of northeast China23, as well as in aquatic environments and paddy soils58,59. These findings suggest that AOB has different distribution characteristics in various settings.In this study, Nitrosospira showed greater abundance in M0, M1, and M2 (58.4–84.9%) samples (organic nitrogen dosage of less than 600 kg ha−1), whereas Nitrosomonas accounted for more than half of the percentage in the M3 and M4 treatments (organic nitrogen dosage of more than 900 kg ha−1). Thus, the AOB community compositions in organic vegetable field soils differed distinctly under different manure application rates. Nitrosomonas was more adaptable to large manure amounts, whereas the opposite was true for Nitrosopira. However, in organic vegetable fields, the applicaion of different amounts of manure had little effect on soil pH; therefore, he impact of pH on soil microorganisms can be overshadowed by other environmental factors such as large quantities of organic carbon, organic nitrogen, and inorganic nitrogen quantities present in manure. We also observed that the distribution of some AOB lineages at the genus level was positively or negatively related to the EC, TC, TN, nitrate, AP, AK, TOC, and ammonia levels (Fig. 5). Furthermore, AOB were reportedly not significantly correlated with pH but were significantly associated with different environmental factors23. This study and previous studies’ results indicate that multiple soil factors influence the distribution of the AOB amoA gene in organic vegetable fields.ConclusionsThe abundance, diversity, and community AOMs structure in organic vegetable field soil receiving different manure amounts were analyzed using the functional marker gene amoA. The AOB amoA genes showed higher abundance and diersity than the AOA amoA genes. There was also a positive relationship betwee bacterial amoA gene copy numbers and PNR, whereas the archaeal amoA gene copy numbers were not correlated with PNR. Separation of the AOMs’ total PNR revealed that AOB’s PNR was higher than AOA’s in all samples. Therefore, AOB may dominate the soil nitrifying processes in organic vegetable fields. In addition, AOMs communities analysis in organic vegetable fields revealed they were altered by variations in physicochemical characteristics caused by different manure amounts. The AOA Nitrosopumilus and AOB Nitrosomonas clusters may be more adaptable to large manure quantities, whereas the opposite was true for the Nitrososphaera and Nitrosopira clusters. These results provide additional valuable information for future studies investigating nitrogen cycles in organic field systems.
Data availability
The data used to support the findings of this study are included within the artcle.
ReferencesConstantino, L. V. et al. Post-harvest characterization and sensory analysis ofRoma tomato cultivars under organic cultivation: A strategy using consumers and chefs. Int. J. Gastron. Food Sci. 29, 100564 (2022).Article
Google Scholar
You, P. S. & Hsieh, Y. C. A computational approach for crop production of organic vegetables. Comput. Electron. Agric. 134, 33–42 (2017).Article
Google Scholar
Willer, H., Trávníček, J., Meier, C. & Schlatter, B. The world of organic agriculture statistics and emerging trends 2022. FiBL 31–37 (2022). http://www.organic-world.net/yearbook/yearbook-2022.html.Wang, Z. et al. Yield, nitrogen use efficiency and economic benefits of biochar additions to Chinese Flowering Cabbage in Northwest China. Nutr. Cycl. Agroecosyst. 113, 337–348 (2019).Article
CAS
Google Scholar
Sha, X. L. et al. Effects of different organic fertilizer on pepper growth in new irrigation areas by pumpin Yellow River. Ningxia J. Agric. For. Sci. Tech. 58, 11–13 (2017).
Google Scholar
Wang, Z. et al. Effects of organicfertilizer combined with biochar on soil moisture and water use efficiency in vegetable field. Chin. J. Agrometeorol. 12, 771–779 (2017).
Google Scholar
Wang, Z., Li, Y. K., Wang, L. C., Guo, W. Z. & Xu, Z. G. Effects of biochar on yield, quality and water utilization of organic Flowering Chinese Cabbage. Trans. Chin. Soc. Agric. Mach. 49, 273–280 (2018).
Google Scholar
Inácio, C. D. T. et al. Organic, conventional and hydroponic vegetables: Can 15N natural abundance of farm N inputs differentiate mode of production. Sci. Hortic. 265, 109219 (2020).Article
Google Scholar
Li, M. Y., He, H., Mi, T. Z. & Zhen, Y. Spatiotemporal dynamics of ammonia-oxidizing archaea and bacteria contributing to nitrification in sediments from Bohai Sea and South Yellow Sea, China. Sci. Total Environ. 825, 153972 (2022).Aticle
ADS
CAS
PubMed
Google Scholar
Beeckman, F., Motte, H. & Beeckman, T. Nitrification in agricultural soils: Impact, actors and mtigation. Curr. Opin. Biotechnol. 50, 166–173 (2018).Article
CAS
PubMed
Google Scholar
Sun, Y. et al. Correlation between ammonia-oxidizing microorganisms and environmental factors during cattle manure composting. Rev. Argent. Microbiol. 51, 371–380 (2019).PubMed
Google Scholar
Zhong, W. H. et al. Nitrogen fertilization induced changes in ammonia oxidation are attributable mostly to bacteria rather than archaea in greenhouse based high N input vegetable soil. Soil Biol. Biochem. 93, 150–159 (2016).Article
CAS
Google Scholar
Stieglmeier, M. et al. Nitrososphaera viennensis gen. nov., sp. nov., an aerobic and mesophilic, ammonia-oxidizing archaeon from soil and a member of the archaeal phylum Thaumarchaeota. Int. J. Syst. Evol. Microbiol. 64, 2738–2752 (2014).Aticle
CAS
PubMed
PubMed Central
Google Scholar
Wang, X. Y., Wang, C., Bao, L. L. & Xie, S. G. Abundance agriculture and community structure of ammonia-oxidizing microrganisms in reservoir sediment and adjacent soils. Appl. Microbiol. Biotechnol. 98, 1883–1892 (2014).Article
CAS
PubMed
Google Scholar
Liu, J. J. et al. Ammonia-oxidizing archaea show more distinct biogeographic distribution patterns than ammonia-oxidizing bacteria across the black soil zone of northeast China. Front. Microbiol. 9, 1–13 (2018).ADS
Google Scholar
Ouyang, Y., Reeve, J. R. & Norton, J. M. Soil enzyme activities and abundance of microbial functional genes involved in nitrogen transformations in an organic farming system. Biol. Fertil. Soils 54, 437–450 (2018).Article
CAS
Google Scholar
Dong, X. C. et al. Chronic nitrogen fertilization modulates competitive interactions among microbial ammonia oxidizers in a less soil. Pedosphere 29, 24–33 (2019).Article
CAS
Google Scholar
Leininger, S. et al. Archaea predominate among ammonia-oxidizing prokaryotes in soils. Nature 442, 806–809 (2006).Article
ADS
CAS
PubMd
Google Scholar
Zhao, D. et al. Vertical distribution of ammonia-oxidizing archaea and bacteria in sediments of a eutrophic lake. Curr. Microbiol. 67, 327–332 (2013).Article
CAS
PubMed
Google Scholar
Li, H., Weng, B. S., Huang, F. Y., Su, J. Q. & Yang, X. R. pH regulates ammonia-oxidizing bacteria and archaea in paddy soils in Southern China. Appl. Microb. Biotechnol. 99, 6113–6123 (2015).Article
CAS
Google Scholar
Zheng, Y. et al. Community dynamics and activity of ammonia-oxidizing prokaryotes in intertidal sediments of the Yangtze Estuary. Appl. Environ. Microbiol. 80, 408–419 (2014).Article
ADS
CAS
PubMed
PubMed Central
Google Scholar
Zhang, G. et al. Ammnia-oxidizing bacteria and archaea: response to simulated climate warming and nitrogen supple mentation. Soil Sci. Soc. Am. J. 83, 1683–1695 (2019).Article
ADS
CAS
Google Scholar
Liu, H. et al. Ammonia oxidizers and nitrite-oxidizing bacteria resond differently to long-term manure application in four paddy soils of south of China. Sci. Total Environ. 633, 641–648 (2018).Article
ADS
CAS
PubMed
Google Scholar
Qi, X. et al. Changes in alpine grassland type drive niche differentiation of nitrifying communities on the Qinghai-Tibetan Plateau. Eur. J. Soil Biol. 104, 103316 (2021).Article
CAS
Google Scholar
Sun, W. et al. Distribution and abundance of archaeal and bacterial ammonia oxidizers in the sediments of the Dongjiang River, a drinking water supply for Hong Kong. Microbes Environ. 28, 457 (2013).Article
PubMed
PubMed Central
Google Scholar
Liu, S. et al. Spatial distribution and factor shaping the niche segregation of ammonia-oxidizing microorganisms in the Qiantang River, China. Appl. Environ. Microb. 79, 4065–4071 (2013).Article
ADS
CAS
Google Scholar
Verhamme, D. T., Prosser, J. I. & Nicol, G. W. Ammonia concentration determines differential growth of mmonia-oxidising archaea and bacteria in soil microcosms. ISME J. 5, 1067–1071 (2011).Article
CAS
PubMed
PubMed Central
Google Scholar
Ouyang, Y., Norton, J. M., Stark, J. M., Reeve, J. R. & Habteselassie, M. Y. Ammonia-oxidizing bacteria are more responsive than archaea to nitrogen source in an agricultural soil. Soil Biol. Biochem. 96, 4–15 (2016).Article
CAS
Google Scholar
Ouyang, Y., Norton, J. M. & Stark, J. M. Ammonium availability and temperature control contributions of ammonia oxidizing bacteria and archaea to nitrification in an agricultural soil. Soil Biol. Biochem. 113, 161–172 (2017).Article
CAS
GoogleScholar
Němeček, J. & Kozák, J. Approaches to the solution of a soil map of the Czech Republic at the scale 1:250 000 using SOTER methodology. Plant Soil Environ. 49, 291–297 (2003).Article
Google Scholar
Liu, R. L. et al. The study on rice organic fertilizer suitable application amount in Yellow River irrgation area of Ningxia. Ningxia J. Agric. For. Sci. Technol. 7, 1–2 (2016).CAS
Google Scholar
Sauder, L. A. et al. Cultivation and characterization of Candidatus Nitrosocosmicus exaquare, an ammonia-oxidizing archaeon from a municipal wastewater treatment system. ISME J. 11, 1142 (2017).Article
CAS
PubMed
PubMed Central
Google Scholar
Taylor, A. E. et al. Use of aliphatic n-alkynes to discriminate soil nitrification activities of ammonia-oxidizing Thaumarchaea and bacteria. Appl. Environ. Microbiol. 79, 6544–6551 (2013).Article
ADS
CAS
PubMed
PubMed Central
Google Scholar
Bernhrd, A. E. et al. Abundance of ammonia-oxidizing archaea and bacteria along an estuarine salinity gradient in relation to potential nitrification rates. Appl. Environ. Microbiol. 76, 1285–1289 (2010).Article
ADS
CAS
PubMed
Google Scholar
Duan, P. P., Wu, Z., Zhang, Q. Q., Fan, C. H. & Xiong, Z. Q. Thermodynamic responses of ammonia-oxidiing archaea and bacteria explain N2O production from greenhouse vegetable soils. Soil Biol. Biochem. 120, 37–47 (2018).Article
CAS
Google Scholar
Caporaso, J. G. et al. QIIME allows analysis of high-throughput community sequencing data. Nat. Methods 7, 335–336 (2010).Article
CAS
PubMed
PubMed Central
Google Scholar
Edgar, R. C., Haas, B. J., Clemente, J. C., Quince, C. & Knight, R. UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 27, 2194–2200 (2011).Article
CAS
PubMed
PubMed Central
Google Scholar
Edgar, R. C. UPARE: Highly accurate OTU sequences from microbial amplicon reads. Nat. Methods 10, 996–998 (2013).Article
CAS
PubMed
Google Scholar
Li, M. C. et al. Distinct distribution patterns of ammonia-oxidizing archaea and bacteria in sediment and water column of the yellow river estuary. Sci. Rep. 8, 1–10 (2018).ADS
Google Scholar
Ter Braak, C. & Šmilauer, P. CANOCO Refernce Manual and Canodraw for Window User’s Guide: Software for Canonical Community Ordination (Version 4.5) (Microcomputer Power, Ithaca, 2002).Zhang, H., Cheng, F., Sun, S. Y. & Li, Z. K. Diversity distribution and characteristics of comammox in different ecosystems. Environ. Res. 214, 113900 (2022).Article
CAS
PubMed
Google Scholar
Pearson, A. et al. Factors controlling the distribution of archaeal tetraethers in terrestrial hot springs. Appl. Environ. Microbiol 74, 3523–3532 (2008).Article
ADS
CAS
PubMed
PubMed Central
Google Sholar
Wen, Y. C. et al. Crop yield and soil fertility response to commercial organic fertilizer substituting chemical fertilizer. Sci. Agric. Sin. 51, 2136–2142 (2018).
Google Scholar
Wang, B. R., Cai, Z. J. & Li, D. C. Effect of different long-term fertilization on the fertility of red upland soil. J. Soil Water Conserv. 24, 85–88 (2010).
Google Scholar
Zhang, X., Guo, J., Vogt, R. D., Mulder, J. ≈ Zhang, X. Soil acidification as an additional driver to organic carbon accumulation in major Chinese croplands. Geoderma 366, 114234 (2020).Article
ADS
CAS
Google Scholar
Tourna, M. et al. Nitrososphaera viennensis, an ammonia oxidizing archaeon from soil. Proc. Natl. Acad. Sci. U. S. A. 20, 8420–8425 (2011).Article
ADS
Google Scholar
Hu, H. W. et al. The large-scale distribution of ammonia oxidizers in paddy soils is driven by pH, geographic distance, and climatic factors. Front. Microbiol. 6, 1–14 2015).Article
Google Scholar
Walker, C. B. et al. Nitrosopumilus maritimus genome reveals unique mechanisms for nitrification and autotrophy in globally distributed marine Crenarchaea. Proc. Natl. Acad. Sci. U. S. A. 107, 8818–8823 (2010).Article
ADS
CAS
PubMed
PubMed Central
Google Scholar
Zhalnina, K., de Quadros, P. D., Camargo, F. A. & Triplett, E. W. Drivers of archaeal ammonia-oxidizing communities in soil. Front.Microbiol. 3, 1–9 (2012).Article
Google Scholar
Lehtovirta-Morley, L. E. et al. Characterisation of terrestrial acidophilic archaeal ammonia oxidisers and their inhibition and stimulation by organic compounds. FEMS Microbiol. Ecol. 89, 542–552 (2014).Article
CAS
PubMed
Google Scholar
Bai, X. et al. Ammonia oxidizing bacteria dominate soil nitrification under different fertilization regimes in black soils of northeast China. Eur. J. Soil Biol. 111, 103410 (2022).Article
CS
Google Scholar
Zhang, L. M., Hu, H. W., Shen, J. P. & He, J. Z. Ammoniaoxidizing archaea have more important role than ammonia-oxidizing bacteria in ammonia oxidation of strongly acidic soils. ISME J. 6, 1032–1045 (2012).Article
CAS
PubMed
Google Scholar
Zhou, L. L., Wang, S. Y., Zou, Y. X., Xia, C. & Zhu, G. B. Species, abundance and function of Ammonia-oxidizing Archaea in inland waters across China. Sci. Rep. 5, 1–13 (2015).
Google Scholar
Jing, H. et al. Latitudinal distribution of ammonia-oxidizing bacteria and archaea in the agricultural soils of eastern China. Appl. Environ. Microbiol. 80, 5593–5602 (2014).Article
ADS
PubMed
PubMed Central
Google Scholar
Pester, M. et al. amoA-based consensus phylogeny of ammonia-oxidizing archaea and deep sequencing of amoA genes from soils of four different geographic regions. Environ. Microbiol. 14, 525–539 (2012).Article
CAS
PubMed
PubMd Central
Google Scholar
Zou, W. X., Lang, M., Zhang, L., Liu, B. & Chen, X. P. Ammonia-oxidizing bacteria rather than ammonia-oxidizing archaea dominate nitrification in a nitrogen-fertilized calcareous soil. Sci. Total Environ. 811, 151402 (2022).Article
ADS
CAS
PubMed
Google Scholar
Chen, Y. et al. Diversity, abundance, and spatial distribution of ammonia-oxidizing β-proteobacteria in sediments from Changjiang estuary and its adjacent area in East China Sea. Microb. Ecol. 67, 788–803 2014).Article
CAS
PubMed
Google Scholar
Hu, A. et al. Community structures of ammonia-oxidizing archaea and bacteria in high altitude lakes on the Tibetan Plateau. Freshw. Biol. 55, 2375–2390 (2010).Article
CAS
Google Scholar
Hou, Q. et al. Responses of nitrification and bacterial community in three size aggregates of paddy soil to both of initial fertility and biochar addition. Appl. Soil Ecol. 166, 10400 (2021).Article
Google Scholar
Download referencesAcknowledgementsThis work was supported by S&T Program of Hebei (21327005D and 21326904D), the National Natural Science Foundation of China (41501312), Funds for Science and Technology Innovation Pilot of Ningxia Academy of Agricultural and Forestry Sciences (NNKZZCGZH-2021-06) and Natural Science Foundation of Ningxia (2022AAC05048).Author informationAuthors and AffiliationsResearch Centre of Intelligent Equipment, Beijing Academy of Agriculture and Forestry Sciences, Beijing, 100097, ChinZhan Wang, Yinkun Li, Wengang Zheng & Yuru JiGuyuan Branch, Ningxia Academy of Agricultural and Forestry Sciences, Guyuan, 756000, ChinaZhan WangBeijing Key Laboratory of Ecological Function Assessment and Regulation Technology of Green Space, Beijing Urban Ecosystem Positioning Observation and Research Station, Beijing Institute of Landscape Architecture, Beijing, 100102, ChinaMinjie DuanWuzhong National Agricultural Sciene and Technology Park Management Committee, Wuzhong, 751100, Ningxia, ChinaLi MaAuthorsZhan WangView author publicationsYou can also search for this author in
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PubMed Google ScholarContributionsZ.W. analyzed the data and wrote the paper, Y.L. designed the experiment and review the manuscript, Z.W. and L.M. performed the field experiment, M.D. and Y.J. prepared the tables and figures, and W.Z. contributed ideas.Corresponding authorCorrespondence to
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Reprints and PermissionsAbout this articleCite this articleWang, Z., Li, Y., Zheng, W. et al. Ammonia oxidizing archaea and bacteria respond to different manure application rates during organic vegetable cultivation in Northwest China.
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