长期施肥对水稻土典型氨氧化菌和全程氨氧化菌种群活性和丰度的影响

doi:10.3864/j.issn.0578-1752.2024.14.009

摘要/Abstract

摘要：

【目的】通过研究长期施用有机肥和无机肥对硝化菌种群活性和丰度的影响，揭示其种群丰度变化与硝化活性之间的关系，为农业生产中减少温室气体排放和实现可持续发展提供科学依据。【方法】使用特异性抑制剂乙炔、1-辛炔和3,4-二甲基吡唑磷酸盐（DMPP）等，结合实时荧光定量PCR技术（qPCR），分析水稻土长期不施肥（CK）、单施化肥（NPK）、单施有机肥（M），以及化肥与有机肥配合施用（MNPK）4个处理的全程氨氧化菌（Comammox）、氨氧化细菌（AOB）、氨氧化古菌（AOA）种群活性和丰度，以及硝化螺菌（Nitrospira）和硝化杆菌（Nitrobacter）种群丰度差异。【结果】长期施用有机肥显著提高了Comammox和AOA的活性（双因素方差分析，P<0.001），在单施有机肥处理中，Comammox活性贡献比例高达64.2%，而施用无机肥仅显著促进了AOB的活性（双因素方差分析，P<0.001）。qPCR分析结果表明，有机肥处理显著提高了AOA、Nitrospira、Comammox分枝A和分枝B种群的丰度（双因素方差分析，P<0.001），而无机肥处理则显著提高了AOB和Nitrobacter的种群丰度（双因素方差分析，P<0.001）。相关性分析表明，AOA和Comammox的活性与土壤含水量、有机质、全氮、全磷、速效磷、碱解氮含量以及AOA和Nitrospira种群丰度显著正相关，Comammox活性还与Comammox分枝A丰度显著正相关；而AOB活性则与AOB和Nitrobacter种群丰度、硝态氮和速效钾含量呈显著正相关。【结论】 Comammox对供试水稻土的硝化作用有着重要贡献，其活性和种群丰度主要受土壤含水量、有机质、全氮、全磷、速效磷和碱解氮等因子变化的影响。

关键词: 全程氨氧化菌, 硝化活性, 种群丰度, 有机肥, 化肥, 实时荧光定量PCR技术

Abstract:

【Objective】 This work aimed to investigate the impact of long-term applying organic and inorganic fertilizers on the abundance and activities of complete ammonia oxidizers (Comammox), ammonia-oxidizing archaea (AOA) and bacteria (AOB), as well as the abundances of Nitrospira and Nitrobacter in paddy soil, providing a scientific basis for mitigating greenhouse gas emissions and promoting sustainable agricultural production.【Method】 By utilizing multiple specific inhibitors (i.e., acetylene, 1-octyne, and DMPP) in conjunction with real-time quantitative PCR (qPCR), this work examined the differences in activities and abundances of Comammox, AOA, and AOB as well as the abundances of Nitrobacter and Nitrospira in a paddy soil under four fertilization regimes: plots without fertilizer (CK), with manure (M), with chemical fertilizer (NPK), and with combination of manure and chemical fertilizer (MNPK).【Result】 The long-term application of organic fertilizer significantly stimulated the activities of Comammox and AOA (Two-way ANOVA, P<0.001). In those plots solely receiving organic manure, Comammox accounted for as high as 64.2% of total ammonia oxidizing activity, while the inorganic fertilizer application only enhanced the activity of AOB (Two-way ANOVA, P<0.001). qPCR demonstrated that chronic organic amendment significantly increased the abundances of AOA, Nitrospira, Comammox clade A and Clade B (Two-way ANOVA, P<0.001); whereas inorganic amendment increased the abundances of AOB and Nitrobacter (Two-way ANOVA, P<0.001). The correlation analysis revealed there were positive correlations between activities of AOA and Comammox with moisture content, organic matter (OM), total nitrogen (TN), total phosphorus (TP), available phosphorus (AP), alkaline hydrolysable nitrogen (AN), as well as AOA and Nitrospira abundances, while the activity of Comammox was positively correlated with the abundance of Comammox clade A as well. Additionally, the activity of AOB showed positive correlations with AOB and Nitrobacter abundances, nitrate content, and available potassium (AK).【Conclusion】 Comammox played an important role in nitrification of the tested paddy soil, with its abundance and activity primarily influenced by the changes in moisture content, OM, TN, TP, AP, and AN etc..

Key words: Comammox, nitrification activity, population abundance, organic fertilizers, chemical fertilizers, qPCR

张小琴, 尹昌, 李政, 唐旭, 李艳, 吴春艳. 长期施肥对水稻土典型氨氧化菌和全程氨氧化菌种群活性和丰度的影响[J]. 中国农业科学, 2024, 57(14): 2803-2814.

ZHANG XiaoQin, YIN Chang, LI Zheng, TANG Xu, LI Yan, WU ChunYan. Influences of Long-Term Appling Different Fertilizers on the Activities and Abundances of Canocial Ammonia Oxidizers and Comammox in Paddy Soil[J]. Scientia Agricultura Sinica, 2024, 57(14): 2803-2814.

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参考文献 45

[1]	GRUBER N, GALLOWAY J N. An Earth-system perspective of the global nitrogen cycle. Nature, 2008, 451: 293-296.
[2]	PROSSER J I, HINK L, GUBRY-RANGIN C, NICOL G W. Nitrous oxide production by ammonia oxidizers: Physiological diversity, niche differentiation and potential mitigation strategies. Global Change Biology, 2020, 26(1): 103-118. doi: 10.1111/gcb.14877 pmid: 31638306
[3]	DAIMS H, LÜCKER S, WAGNER M. A new perspective on microbes formerly known as nitrite-oxidizing bacteria. Trends in Microbiology, 2016, 24(9): 699-712. doi: S0966-842X(16)30045-2 pmid: 27283264
[4]	PROSSER J I, NICOL G W. Archaeal and bacterial ammonia- oxidisers in soil: The quest for niche specialisation and differentiation. Trends in Microbiology, 2012, 20(11): 523-531.
[5]	DAIMS H, LEBEDEVA E V, PJEVAC P, HAN P, HERBOLD C, ALBERTSEN M, JEHMLICH N, PALATINSZKY M, VIERHEILIG J, BULAEV A, KIRKEGAARD R H, VON BERGEN M, RATTEI T, BENDINGER B, NIELSEN P H, WAGNER M. Complete nitrification by Nitrospira bacteria. Nature, 2015, 528: 504-509.
[6]	HU H W, HE J Z. Comammox-a newly discovered nitrification process in the terrestrial nitrogen cycle. Journal of Soils and Sediments, 2017, 17(12): 2709-2717.
[7]	HU J J, ZHAO Y X, YAO X W, WANG J Q, ZHENG P, XI C W, HU B L. Dominance of comammox Nitrospira in soil nitrification. Science of the Total Environment, 2021, 780: 146558.
[8]	WANG X M, WANG S Y, JIANG Y Y, ZHOU J M, HAN C, ZHU G B. Comammox bacterial abundance, activity, and contribution in agricultural rhizosphere soils. Science of the Total Environment, 2020, 727: 138563.
[9]	WANG S Y, WANG X M, JIANG Y Y, HAN C, JETTEN M S M, SCHWARK L, ZHU G B. Abundance and functional importance of complete ammonia oxidizers and other nitrifiers in a riparian ecosystem. Environmental Science and Technology, 2021, 55(8): 4573-4584. doi: 10.1021/acs.est.0c00915 pmid: 33733744
[10]	TAYLOR A E, VAJRALA N, GIGUERE A T, GITELMAN A I, ARP D J, MYROLD D D, SAYAVEDRA-SOTO L, BOTTOMLEY P J. Use of aliphatic n-alkynes to discriminate soil nitrification activities of ammonia-oxidizing thaumarchaea and bacteria. Applied and Environmental Microbiology, 2013, 79(21): 6544-6551. doi: 10.1128/AEM.01928-13 pmid: 23956393
[11]	YIN C, FAN X P, CHEN H, JIANG Y S, YE M J, YAN G C, PENG H Y, WAKELIN S A, LIANG Y C. 3, 4-Dimethylpyrazole phosphate is an effective and specific inhibitor of soil ammonia-oxidizing bacteria. Biology and Fertility of Soils, 2021, 57(6): 753-766.
[12]	ZHAO J, BELLO M O, MENG Y Y, PROSSER J I, GUBRY- RANGIN C. Selective inhibition of ammonia oxidising archaea by simvastatin stimulates growth of ammonia oxidising bacteria. Soil Biology and Biochemistry, 2020, 141: 107673.
[13]	SUN D Y, TANG X F, LI J, LIU M, HOU L J, YIN G Y, CHEN C, ZHAO Q, KLÜMPER U, HAN P. Chlorate as a comammox Nitrospira specific inhibitor reveals nitrification and N₂O production activity in coastal wetland. Soil Biology and Biochemistry, 2022, 173: 108782.
[14]	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 Biology and Biochemistry, 2016, 96: 4-15.
[15]	TAN C, YIN C, LI W J, FAN X P, JIANG Y S, LIANG Y C. Comammox Nitrospira play a minor role in N₂O emissions from an alkaline arable soil. Soil Biology and Biochemistry, 2022, 171: 108720.
[16]	JIANG L P, YU J, WANG S Y, WANG X M, SCHWARK L, ZHU G B. Complete ammonia oxidization in agricultural soils: High ammonia fertilizer loss but low N₂O production. Global Change Biology, 2023, 29(7): 1984-1997.
[17]	SHEN J P, ZHANG L M, DI H J, HE J Z. A review of ammonia- oxidizing bacteria and archaea in Chinese soils. Frontiers in Microbiology, 2012, 3: 296.
[18]	TOURNA M, FREITAG T E, NICOL G W, PROSSER J I. Growth, activity and temperature responses of ammonia-oxidizing archaea and bacteria in soil microcosms. Environmental Microbiology, 2008, 10(5): 1357-1364. doi: 10.1111/j.1462-2920.2007.01563.x pmid: 18325029
[19]	ROTTHAUWE J H, WITZEL K P, LIESACK W. The ammonia monooxygenase structural gene amoA as a functional marker: molecular fine-scale analysis of natural ammonia-oxidizing populations. Applied and Environmental Microbiology, 1997, 63(12): 4704-4712. doi: 10.1128/aem.63.12.4704-4712.1997 pmid: 9406389
[20]	JIANG R, WANG J G, ZHU T, ZOU B, WANG D Q, RHEE S K, AN D, JI Z Y, QUAN Z X. Use of newly designed primers for quantification of complete ammonia-oxidizing (Comammox) bacterial clades and strict nitrite oxidizers in the genus Nitrospira. Applied and Environmental Microbiology, 2020, 86(20): e01775-20.
[21]	WERTZ S, POLY F, LE ROUX X, DEGRANGE V. Development and application of a PCR-denaturing gradient gel electrophoresis tool to study the diversity of Nitrobacter-like nxrA sequences in soil. FEMS Microbiology Ecology, 2008, 63(2): 261-271.
[22]	HONG Y G, JIAO L J, WU J P. New primers, taxonomic database and cut-off value for processing nxrB gene high-throughput sequencing data by MOTHUR. Journal of Microbiological Methods, 2020, 173: 105939.
[23]	LI C Y, HU H W, CHEN Q L, CHEN D L, HE J Z. Comammox Nitrospira play an active role in nitrification of agricultural soils amended with nitrogen fertilizers. Soil Biology and Biochemistry, 2019, 138: 107609.
[24]	王智慧. 土壤中全程和半程硝化微生物的生态位分化及功能重要性研究[D]. 重庆: 西南大学, 2021.
	WANG Z H. Niche differentiation and functional importance of complete and incomplete nitrifiers in the soil[D]. Chongqing: Southwest University, 2021. (in Chinese)
[25]	YIN C, FAN X P, CHEN H, YE M J, YAN G C, LI T Q, PENG H Y, E S Z, CHE Z X, WAKELIN S A, LIANG Y C. Inhibition of ammonia- oxidizing bacteria promotes the growth of ammonia-oxidizing archaea in ammonium-rich alkaline soils. Pedosphere, 2022, 32(4): 532-542.
[26]	HE J Z, HU H W, ZHANG L M. Current insights into the autotrophic thaumarchaeal ammonia oxidation in acidic soils. Soil Biology and Biochemistry, 2012, 55: 146-154.
[27]	XIA W W, ZHANG C X, ZENG X W, FENG Y Z, WENG J H, LIN X G, ZHU J G, XIONG Z Q, XU J, CAI Z C, JIA Z J. Autotrophic growth of nitrifying community in an agricultural soil. The ISME Journal, 2011, 5(7): 1226-1236.
[28]	TOURNA M, STIEGLMEIER M, SPANG A, KÖNNEKE M, SCHINTLMEISTER A, URICH T, ENGEL M, SCHLOTER M, WAGNER M, RICHTER A, SCHLEPER C. Nitrososphaera viennensis, an ammonia oxidizing archaeon from soil. Proceedings of the National Academy of Sciences of the United States of America, 2011, 108(20): 8420-8425.
[29]	DONG W L, LI X, WANG E Z, LIU X D, WANG M, SONG A L, YIN H Q, FAN F L. Linking microbial taxa and the effect of mineral nitrogen forms on residue decomposition at the early stage in arable soil by DNA-qSIP. Geoderma, 2021, 400: 115127.
[30]	HINK L, GUBRY-RANGIN C, NICOL G W, PROSSER J I. The consequences of niche and physiological differentiation of archaeal and bacterial ammonia oxidisers for nitrous oxide emissions. The ISME Journal, 2018, 12(4): 1084-1093.
[31]	LEVIČNIK-HÖFFERLE Š, NICOL G W, AUSEC L, MANDIĆ-MULEC I, PROSSER J I. Stimulation of thaumarchaeal ammonia oxidation by ammonia derived from organic nitrogen but not added inorganic nitrogen. FEMS Microbiology Ecology, 2012, 80(1): 114-123.
[32]	LIU H Y, QIN S Y, LI Y, ZHAO P, NIE Z J, LIU H E. Comammox Nitrospira and AOB communities are more sensitive than AOA community to different fertilization strategies in a fluvo-aquic soil. Agriculture, Ecosystems and Environment, 2023, 342: 108224.
[33]	SUN P, ZHANG S X, WU Q H, ZHU P, RUAN Y Z, WANG Q. pH and ammonium concentration are dominant predictors of the abundance and community composition of comammox bacteria in long-term fertilized Mollisol. Applied Soil Ecology, 2021, 168: 104139.
[34]	PALOMO A, PEDERSEN A G, FOWLER S J, DECHESNE A, SICHERITZ-PONTÉN T, SMETS B F. Comparative genomics sheds light on niche differentiation and the evolutionary history of comammox Nitrospira. The ISME Journal, 2018, 12(7): 1779-1793.
[35]	JONES C M, HALLIN S. Geospatial variation in co-occurrence networks of nitrifying microbial guilds. Molecular Ecology, 2019, 28(2): 293-306. doi: 10.1111/mec.14893 pmid: 30307658
[36]	DAEBELER A, BODELIER P L E, YAN Z, HEFTING M M, JIA Z J, LAANBROEK H J. Interactions between thaumarchaea, Nitrospira and methanotrophs modulate autotrophic nitrification in volcanic grassland soil. The ISME Journal, 2014, 8(12): 2397-2410.
[37]	GAO W L, FU Y J, FAN C H, ZHANG W, WANG Y S, LI N, LIU H R, CHEN X, LIU Y Q, WU X L, LI Q F, CHEN M. Factors predictive of the biogeographic distribution of comammox Nitrospira in terrestrial ecosystems. Soil Biology and Biochemistry, 2023, 184: 109079.
[38]	LIN Y X, HU H W, YE G P, FAN J B, DING W X, HE Z Y, ZHENG Y, HE J Z. Ammonia-oxidizing bacteria play an important role in nitrification of acidic soils: A meta-analysis. Geoderma, 2021, 404: 115395.
[39]	HUANG X R, ZHAO J, SU J, JIA Z, SHI X L, WRIGHT A, ZHU-BARKER X, JIANG X J. Neutrophilic bacteria are responsible for autotrophic ammonia oxidation in an acidic forest soil. Soil Biology and Biochemistry, 2018, 119: 83-89.
[40]	ZHANG Q, LI Y, HE Y, LIU H Y, DUMONT M, BROOKES P, XU J M. Nitrosospira cluster 3-like bacterial ammonia oxidizers and Nitrospira-like nitrite oxidizers dominate nitrification activity in acidic terrace paddy soils. Soil Biology and Biochemistry, 2019, 131: 229-237.
[41]	PICONE N, POL A, MESMAN R, VAN KESSEL M A H J, CREMERS G, VAN GELDER A H, VAN ALEN T A, JETTEN M S M, LÜCKER S, OP DEN CAMP H J M. Ammonia oxidation at pH 2.5 by a new gammaproteobacterial ammonia-oxidizing bacterium. The ISME Journal, 2021, 15: 1150-1164.
[42]	HAYATSU M, TAGO K, UCHIYAMA I, TOYODA A, WANG Y, SHIMOMURA Y, OKUBO T, KURISU F, HIRONO Y, NONAKA K, AKIYAMA H, ITOH T, TAKAMI H. An acid-tolerant ammonia- oxidizing γ-proteobacterium from soil. The ISME Journal, 2017, 11(5): 1130-1141.
[43]	LI C Y, HE Z Y, HU H W, HE J Z. Niche specialization of comammox Nitrospira in terrestrial ecosystems: Oligotrophic or copiotrophic? Critical Reviews in Environmental Science and Technology, 2023, 53(2): 161-176.
[44]	LIU T L, WANG Z H, WANG S L, ZHAO Y P, WRIGHT A L, JIANG X J. Responses of ammonia-oxidizers and comammox to different long-term fertilization regimes in a subtropical paddy soil. European Journal of Soil Biology, 2019, 93: 103087.
[45]	LIN Y, YE G, DING W, HU H W, ZHENG Y, FAN J B, WAN S, DUAN C J, HE J Z. Niche differentiation of comammox Nitrospira and canonical ammonia oxidizers in soil aggregate fractions following 27-year fertilizations. Agriculture, Ecosystems and Environment, 2020, 304: 107147.

途径 Pathway	N	CH	DP	OC
硝酸盐和亚硝酸消耗 Consumption of nitrate and nitrite	√	√	√	√
异养硝化 Heterotrophic nitrification	√	√	√	√
AOA	√	－	√	√
AOB	√	－	－	－
Comammox	√	－	－	√

处理 Treatment	pH	含水量Water content (%)	硝态氮含量 NO₃^--N (mg·kg^-1)	铵态氮含量 NH₄⁺-N (mg·kg^-1)	有机质含量 Organic matter (g·kg^-1)	全氮含量Total nitrogen (g·kg^-1)	全磷含量Total phosphorus (g·kg^-1)	全钾含量Total potassium (g·kg^-1)	有效磷含量Available phosphorus (mg·kg^-1)	速效钾含量Available potassium (mg·kg^-1)	碱解氮含量Alkaline hydrolyzable nitrogen (mg·kg^-1)
CK	6.09b	25.35a	17.61a	18.12a	19.74a	1.24a	0.79a	23.29a	55.39a	64.60a	102.16a
NPK	5.49a	25.71a	63.77b	18.62a	19.47a	1.30a	1.11ac	24.19a	135.71d	88.92b	112.24a
M	6.39c	29.88b	25.08a	26.13b	29.73b	1.88b	1.42bc	24.50a	180.29b	66.65a	149.62b
MNPK	5.84b	29.51b	61.50b	18.17a	34.32c	2.29c	1.86b	24.39a	248.61c	94.55b	172.28c

项目 Item	有机肥 Organic fertilization		无机肥 Inorganic fertilization		无机肥×有机肥 Organic fertilization× Inorganic fertilization
项目 Item	F_1,8	P	F_1,8	P	F_1,8	P
AOA活性AOA activity	82.83	<0.001	3.02	0.120	5.48	0.047
AOB活性AOB activity	0.77	0.134	24.89	<0.001	2.78	0.134
Comammox活性Comammox activity	112.82	<0.001	0.56	0.477	0.10	0.760
AOA丰度AOA abundance	85.64	<0.001	14.84	<0.001	10.85	0.002
AOB丰度AOB abundance	23.68	<0.001	1179.28	<0.001	47.10	<0.001
Comammox分枝A丰度ComA abundance	31.62	<0.001	14.15	<0.001	5.65	0.024
Comammox分枝B丰度ComB abundance	36.64	<0.001	11.04	0.002	35.65	<0.001
Nitrobacter丰度Nitrobacter abundance	59.57	<0.001	470.66	<0.001	62.40	<0.001
Nitrospira丰度Nitrospira abundance	187.22	<0.001	0.042	0.840	0.75	0.394