2022, Vol. 43
硝化作用是氮素循环的重要过程[1, 2], 也是稻田土壤氮素转化的关键环节[3].硝化作用的底物(铵态氮)和最终产物(硝态氮)都是微生物和农作物生长的重要来源[4], 但是硝态氮又可以通过反硝化作用或淋洗而损失[5, 6].因此, 硝化作用的研究对于提高土壤氮肥利用率和减少氮素损失具有重要的意义.传统上, 硝化作用是由氨氧化[由微生物ammonia-oxidizing archaea(AOA)和ammonia-oxidizing bacteria(AOB)驱动]和亚硝酸盐氧化(细菌nitrite-oxidizing bacteria, NOB)合作完成的两步催化过程[7].然而, 在2015年新发现的完全氨氧化微生物——Comammox Nitrospira能够通过亚硝酸直接将氨氧化为硝酸盐[8].已经发现了Comammox Nitrospira属于硝化螺菌属谱系Ⅱ(Nitrospira Linage Ⅱ), 并且有一套完整的催化硝化反应的酶, 具有氨单加氧酶(ammonia monooxygenase, AMO)、羟胺脱氢酶(hydroxylamine dehydrogenase, HAO)和亚硝酸盐氧化还原酶(nitrite oxidoreductase, NXR)[8, 9], 这表明Comammox Nitrospira既能行使如AOA和AOB的功能, 也能行使如NOB的功能, 这也改变了对硝化作用过程的认识[8].基于Comammox Nitrospira amoA的系统发育分析, Comammox Nitrospira可以分为两个进化枝, 即Comammox Nitrospira进化枝A和B(clade A和clade B)[8].
Comammox Nitrospira在水产养殖系统、稻田土壤、淡水和废水处理厂等广泛分布[10~14], Comammox Nitrospira菌株都是在水生生态系统分离培养的, 包括Nitrospira inopinata、Nitrospira nitrosa和Nitrospira nitrificans, 均属于clade A[9].目前从土壤环境中尚未获得纯菌, 这限制了人们对土壤Comammox Nitrospira的了解, 但通过宏基因组分析和定量PCR发现Comammox Nitrospira在土壤中分布广泛[15], 尤其在水稻土中检测到了较高丰度的Comammox Nitrospira, 甚至高于AOB[10].因此有必要对Comammox Nitrospira在水稻土中的分布进行深入研究.
土壤pH是影响AOA和AOB生态位分化的重要因子[16~20], 例如专性嗜酸性Nitrosotalea devanaterra的培养证明AOA在低pH土壤中生长[21], 酸性森林土壤中高硝化作用的发生与AOA密切相关[22], 而在高pH的农业土壤或稻田土壤中, 硝化作用是由AOB而非AOA驱动的[23].此外, NOB的群落结构组成也受土壤pH的影响[24].基于以上分析, 本文推测土壤pH可能也是导致稻田土壤中Comammox Nitrospira的丰度和群落结构组成变化的主要因子.因此, 本文选取红壤发育的水稻土(酸性水稻土)和紫色土发育的水稻土(中性水稻土)为研究对象, 利用荧光定量PCR和克隆测序技术分析两种不同pH的水稻土中Comammox clade A和clade B amoA基因的丰度和群落结构组成, 以期为进一步了解土壤全程氨氧化细菌的分布提供依据.
1 材料与方法 1.1 供试土壤酸性红壤发育的水稻土采自湖南省农业科学院(113°04′47″E, 28°11′53″N), 该区域为亚热带季风气候, 年均气温18.2℃, 年均降雨量1 339.2 mm, 供试土壤类型为红壤, 成土母质为第四纪发育红色黏土.中性紫色土发育的水稻土采自重庆市北碚区西南大学国家紫色土土壤肥力与肥料效益检测基地(106°26′33″E, 30°26′36″N), 该区域为亚热带季风气候, 年均气温18.2℃, 年均降雨量1 105 mm.供试土壤为中性紫色土, 由侏罗系沙溪庙组紫色泥岩风化的残积、坡积物发育而成的.酸性水稻土的基本理化性质：pH为6.2, ω(有机质)为13.60 g·kg-1, ω(全氮)为1.70 g·kg-1, ω(NH4+-N)为6.10 mg·kg-1, ω(NO3--N)为13.60 mg·kg-1.中性水稻土的基本理化性质：pH为7.6, ω(有机质)为17.55 g·kg-1, ω(全氮)为1.76 g·kg-1, ω(NH4+-N)为17.77 mg·kg-1, ω(NO3--N)为13.86 mg·kg-1.
1.2 土壤取样和准备土壤样品采集于2019年5月, 使用直径为6.5 cm的土钻按5点取样法从0~20 cm的深度采集了土壤样品.剔除土壤中可见植物枯枝落叶及土壤动物等杂物, 混匀.将鲜土分成2份, 第1份储存在-20℃冰箱中用于提取土壤DNA以及荧光定量PCR分析; 第2份是将土壤样品风干后研磨并过2 mm筛子, 用来测定土壤的基本理化性质, 每个土壤样品3次重复.
1.3 土壤基本理化性质的测定使用1 ∶2.5的土壤/蒸馏水悬浮液在pH计上测量土壤的pH值.土壤有机质用重铬酸钾氧化法测量[25].凯氏定氮法[26]测定土壤总氮.将5 g新鲜土壤悬浮在50 mL 2 mol·L-1 KCl中来提取NH4+-N和NO3--N, 并使用紫外分光光度计进行浓度比色测定[27].
1.4 提取土壤DNA按照Power Soil DNA Isolation Kit(MoBio Laboratories, 美国)试剂盒的说明方法从0.25 g土壤样品中提取土壤总DNA.简而言之, 将土壤样品加到PowerBead管中, 使用MO BIO涡旋仪器以最大速度涡旋10 min去裂解微生物细胞, 然后洗涤DNA并从提供的二氧化硅旋转滤膜上洗脱以获得提取物.在开始后续实验之前, 分别通过NanoDrop 2000分光光度法和1%琼脂糖凝胶电泳法检测土壤DNA的纯度和完整性.
1.5 实时荧光定量PCR及克隆测序使用实时荧光定量PCR仪(Quant StudioTM 6 Flex)测定全程氨氧化细菌分支A(clade A)和分支B(clade B)amoA基因丰度.Comammox clade A和clade B amoA功能基因的引物序列如Pjevac等[10]所述.实时荧光定量PCR的总反应体系为20 μL, 具体为DNA模板1 μL, 上引物、下引物以及ROX Reference DyeII(50×)各0.4 μL, Taq DNA聚合酶10 μL, 无菌水7.8 μL, qPCR反应扩增条件与笔者先前的描述相同[15], 每个样品进行3个生物学重复.本研究qPCR分析的扩增效率为96% ~105%, R2值在0.996~0.999之间.全程氨氧化细菌clade A和clade B amoA基因克隆测序由上海凌恩生物科技有限公司完成, 每个样品挑选50个阳性克隆.
1.6 数据分析利用SPSS 21软件对Comammox clade A和clade B amoA基因拷贝数进行统计分析. 所得OTU序列在NCBI的GenBank数据库进行Blast, 获得相近的同源基因序列以及Comammox clade A纯菌序列; 然后使用MEGA7软件进行分析, 利用NJ法构建系统发育树.
2 结果与讨论荧光定量PCR分析比较酸性水稻土和中性水稻土中Comammox clade A和clade B amoA基因的丰度, 研究结果显示酸性水稻土和中性水稻土Comammox clade A amoA基因的丰度(以土计)分别为2.48×108 copies·g-1和3.45×106 copies·g-1, 酸性水稻土Comammox clade A amoA基因的丰度比中性水稻土Comammox clade A amoA基因丰度高了2个数量级, 差异显著[P＜0.05, 图 1(a)].酸性水稻土的Comammox clade B amoA基因的丰度(以土计)为4.06×106 copies·g-1, 其丰度显著高于中性水稻土的Comammox clade B amoA基因的丰度[1.52×106 copies·g-1, P＜0.05, 图 1(b)].进一步分析发现酸性水稻土中Comammox clade A amoA基因的丰度比clade B高60倍, 而中性水稻土中Comammox clade A和clade B amoA基因的丰度比约为2.Hu等[14]报道了300种森林土壤样品(pH 4.0~8.6)中Comammox clade A和clade B的丰度, 发现Comammox clade A是酸性土壤(pH < 6.0)中最丰富的硝化细菌, 其次是Comammox clade B.以上研究结果说明土壤pH会影响水稻土中的Comammox Nitrospira不同类群的丰度.
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误差线表示标准误, n=3; 不同小写字母表示P＜0.05水平上差异显著 图 1 不同土壤类型的水稻土的Comammox clade A和clade B amoA基因丰度 Fig. 1 Comammox clade A and clade B amoA gene abundances under different soil types |
笔者前期的研究结果发现, 在中性水稻土中Comammox的丰度(1.2×107~7.2×108 copies·g-1)相较于AOA和AOB达到最高, 而且水稻土中的Comammox主要以clade A为主[15]. Wang等[28]的研究利用qPCR法对Comammox amoA基因进行分析发现, 农业土壤中的Comammox的丰度范围为(6.45±4.31)×106~(5.37±0.44)×107 copies·g-1, 其丰度低于AOA和AOB, 夏季的Comammox对氨氧化的贡献为3.9% ~19.2%, 冬季为0.8% ~22.1%其数值明显低于AOA和AOB对氨氧化的贡献(77.8% ~99.2%), 这表明Comammox在农业土壤中广泛分布, 并且Comammox Nitrospira的丰度和生态位可能受季节的影响[29, 30].Li等[31]的研究调查了澳大利亚东部130种森林土壤中Comammox Nitrospira的丰度, 结果表明AOA的平均丰度[(8.97×106±1.31×106) copies·g-1]显著高于Comammox clade A[(3.73×106±9.62×105) copies·g-1]和AOB[(3.58×106±1.14×106) copies·g-1], 并且AOA是中等酸性土壤(pH 5~7)中最丰富的硝化细菌, 其次是Comammox clade A和AOB, 此外通过稳定性同位素核酸探针技术研究发现普遍存在的Comammox clade A可能有助于森林土壤中的自养碳固定, 认为Comammox clade A在森林土壤中普遍存在, 可能对硝化作用有贡献, 这表明Comammox Nitrospira对较低土壤pH环境可能表现出较高的耐受性[32].
同时克隆测序结果表明Comammox群落结构组成在酸性水稻土和中性水稻土中不完全相同(图 2).酸性水稻土的Comammox clade A系统发育分析的结果得到12个OTU(图 2), 其中OTU1占比最高, 为40%, 与OTU12, OTU14、OTU8、OTU3和OTU18占比的总和为61%, 这些OTU均属于Nitrospira inopinata cluster, 其余的6个OTU均属于Nitrospira nitrosa cluster.这表明酸性水稻土中的Comammox主要是由属于clade A的Nitrospira inopinata和Nitrospira nitrosa cluster构成的.中性水稻土的Comammox clade A系统发育分析的结果得到7个OTU(图 2), 并且全部落在Nitrospira inopinata cluster中, 其中OTU4的占比最高(30%), 其次是OTU1(28%), OTU5(26%).中性水稻土中未检测到Nitrospira nitrosa cluster. 只检测到了Nitrospira inopinata cluster.笔者前期的研究利用宏基因组技术分析不同pH紫色土发育的旱地土壤的硝化微生物丰度和群落[33], 结果发现Nitrospira inopinata在中性紫色土发育的旱地土壤的相对丰度达到最高, Lu等[29]的研究发现Nitrospira inopinata的丰度在pH 7.53~9.17之间与pH呈显著正相关关系, 这表明Nitrospira inopinata更适应在中性环境生长.克隆测序结果发现在酸性水稻土中检测到了Nitrospira nitrosa; 然而另有研究发现Nitrospira nitrosa在中性或者碱性的活性污泥样品中检测到较高的丰度[34].因此, Nitrospira nitrosa类群可能同时具有高和低pH的生态位.Comammox clade B系统发育分析如图 2所示, 对酸性水稻土的测序结果分析得到5个OTU, 其中OTU19占比最高, 为85%, 与荷兰的河漫滩土壤中获得的River Floodplain soil clone(FN395328)序列有密切的亲缘关系[35].Wang等[36]的研究也发现我国农业土壤中Comammox Nitrospira广泛分布, 其中Comammox clade B的主要序列与River Floodplain soil clone(FN395328)有密切的亲缘关系.本研究中酸性水稻土中有2%的Comammox Nitrospira 群落与Rice paddy soil, Vercelli, Italy(MF347179)有密切亲缘关系, 该菌株曾在意大利水稻土中发现[8].此外, 在本研究发现中性水稻土中未检测到Comammox clade B.以上结果说明, 土壤pH是影响全程氨氧化细菌群落结构及生态位分化的重要因子.此外, 在酸性水稻土中检测到两种Comammox, 包括Nitrospira inopinata和Nitrospira nitrosa, 中性水稻土中只检测到了Nitrospira inopinata, 说明全程氨氧化细菌与AOB相似, 种群比较单一, 意味着这些物种可能是推动土壤氨氧化过程的核心微生物.
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图 2 不同土壤类型的水稻土的Comammox Nitrospira的群落结构组成 Fig. 2 Community compositions of Comammox Nitrospira under different soil types |
在酸性红壤和中性紫色土发育的水稻土中均检测到了高丰度的全程氨氧化细菌(Comammox), 这表明全程氨氧化细菌在水稻土中广泛分布.酸性水稻土的Comammox clade A和clade B amoA的丰度均显著高于中性水稻土, 这意味着土壤pH会影响Comammox的丰度, 而且clade A比clade B更加适应酸性环境.中性水稻土中的Comammox主要隶属于Nitrospira inopinata类群, 而Nitrospira nitrosa则可能具有低pH的生态位.pH对土壤全程氨氧化细菌的丰度及群落结构组成有显著影响, 是影响其生态位分化的重要环境因子之一.
| [1] | Kuypers M M M, Marchant H K, Kartal B. The microbial nitrogen-cycling network[J]. Nature Reviews Microbiology, 2018, 16(5): 263-276. DOI:10.1038/nrmicro.2018.9 |
| [2] | Hink L, Gubry-Rangin C, Nicol G W, et al. The consequences of niche and physiological differentiation of archaeal and bacterial ammonia oxidisers for nitrous oxide emissions[J]. The ISME Journal, 2018, 12(4): 1084-1093. DOI:10.1038/s41396-017-0025-5 |
| [3] | Chen X, Zhang L M, Shen J P, et al. Abundance and community structure of ammonia-oxidizing archaea and bacteria in an acid paddy soil[J]. Biology and Fertility of Soils, 2011, 47(3): 323-331. DOI:10.1007/s00374-011-0542-8 |
| [4] | Gruber N, Galloway J N. An earth-system perspective of the global nitrogen cycle[J]. Nature, 2008, 451(7176): 293-296. DOI:10.1038/nature06592 |
| [5] | Santoro A E, Buchwald C, McIlvin M R, et al. Isotopic signature of N2O produced by marine ammonia-oxidizing archaea[J]. Science, 2011, 333(6047): 1282-1285. DOI:10.1126/science.1208239 |
| [6] | Kowalchuk G A, Stephen J R. Ammonia-oxidizing bacteria: a model for molecular microbial ecology[J]. Annual Review of Microbiology, 2001, 55: 485-529. DOI:10.1146/annurev.micro.55.1.485 |
| [7] | Daims H, Lücker S, Wagner M. A new perspective on microbes formerly known as nitrite-oxidizing bacteria[J]. Trends in Microbiology, 2016, 24(9): 699-712. DOI:10.1016/j.tim.2016.05.004 |
| [8] | Daims H, Lebedeva E V, Pjevac P, et al. Complete nitrification by Nitrospira bacteria[J]. Nature, 2015, 528(7583): 504-509. DOI:10.1038/nature16461 |
| [9] | van Kessel M A H J, Speth D R, Albertsen M, et al. Complete nitrification by a single microorganism[J]. Nature, 2015, 528(7583): 555-559. DOI:10.1038/nature16459 |
| [10] | Pjevac P, Schauberger C, Poghosyan L, et al. AmoA-targeted polymerase chain reaction primers for the specific detection and quantification of comammox Nitrospira in the environment[J]. Frontiers in Microbiology, 2017, 8. DOI:10.3389/fmicb.2017.01508 |
| [11] | Palomo A, Pedersen A G, Fowler S J, et al. Comparative genomics sheds light on niche differentiation and the evolutionary history of comammox Nitrospira[J]. The ISME Journal, 2018, 12(1): 1779. |
| [12] | Gonzalez-Martinez A, Rodriguez-Sanchez A, van Loosdrecht M C M, et al. Detection of comammox bacteria in full-scale wastewater treatment bioreactors using tag-454-pyrosequencing[J]. Environmental Science and Pollution Research, 2016, 23(24): 25501-25511. DOI:10.1007/s11356-016-7914-4 |
| [13] | Orellana L H, Chee-Sanford J C, Sanford R A, et al. Year-round shotgun metagenomes reveal stable microbial communities in agricultural soils and novel ammonia oxidizers responding to fertilization[J]. Applied and Environmental Microbiology, 2018, 84(2). DOI:10.1128/AEM.01646-17 |
| [14] | Hu H W, He J Z. Comammox-a newly discovered nitrification process in the terrestrial nitrogen cycle[J]. Journal of Soils and Sediments, 2017, 17(12): 2709-2717. DOI:10.1007/s11368-017-1851-9 |
| [15] |
王梅, 王智慧, 石孝均, 等. 长期不同施肥量对全程氨氧化细菌丰度的影响[J]. 环境科学, 2018, 39(10): 4727-4734. Wang M, Wang Z H, Shi X J, et al. Long-term fertilization effects on the abundance of complete ammonia oxidizing bacteria (comammox Nitrospira) in a neutral paddy soil[J]. Environmental Science, 2018, 39(10): 4727-4734. |
| [16] | Di H J, Cameron K C, Shen J P, et al. Nitrification driven by bacteria and not archaea in nitrogen-rich grassland soils[J]. Nature Geoscience, 2009, 2(9): 621-624. DOI:10.1038/ngeo613 |
| [17] | Hu H W, Zhang L M, Dai Y, et al. pH-dependent distribution of soil ammonia oxidizers across a large geographical scale as revealed by high-throughput pyrosequencing[J]. Journal of Soils and Sediments, 2013, 13(8): 1439-1449. DOI:10.1007/s11368-013-0726-y |
| [18] | Nicol G W, Leininger S, Schleper C, et al. The influence of soil pH on the diversity, abundance and transcriptional activity of ammonia oxidizing archaea and bacteria[J]. Environmental Microbiology, 2008, 10(11): 2966-2978. DOI:10.1111/j.1462-2920.2008.01701.x |
| [19] | Shen J P, Zhang L M, Di H J, et al. A review of ammonia-oxidizing bacteria and archaea in Chinese soils[J]. Frontiers in Microbiology, 2012, 3. DOI:10.3389/fmicb.2012.00296 |
| [20] | Wang Z H, Meng Y, Zhu-Barker X, et al. Responses of nitrification and ammonia oxidizers to a range of background and adjusted pH in purple soils[J]. Geoderma, 2019, 334: 9-14. DOI:10.1016/j.geoderma.2018.07.038 |
| [21] | Lehtovirta-Morley L E, Stoecker K, Vilcinskas A, et al. Cultivation of an obligate acidophilic ammonia oxidizer from a nitrifying acid soil[J]. Proceedings of the National Academy of Sciences of the United States of America, 2011, 108(38): 15892-15897. DOI:10.1073/pnas.1107196108 |
| [22] | Stopnisek N, Gubry-Rangin C, Höfferle S, et al. Thaumarchaeal ammonia oxidation in an acidic forest peat soil is not influenced by ammonium amendment[J]. Applied and Environmental Microbiology, 2010, 76(22): 7626-7634. DOI:10.1128/AEM.00595-10 |
| [23] | Jia Z J, Conrad R. Bacteria rather than Archaea dominate microbial ammonia oxidation in an agricultural soil[J]. Environmental Microbiology, 2009, 11(7): 1658-1671. DOI:10.1111/j.1462-2920.2009.01891.x |
| [24] | Han S, Zeng L Y, Luo X S, et al. Shifts in Nitrobacter-and Nitrospira-like nitrite-oxidizing bacterial communities under long-term fertilization practices[J]. Soil Biology and Biochemistry, 2018, 124: 118-125. DOI:10.1016/j.soilbio.2018.05.033 |
| [25] | Nelson D W, Sommers L E. Total carbon, organic carbon, and organic matter[A]. In: Sparks D L, Page A L, Helmke P A, et al(Eds. ). Methods of Soil Analysis: Part 3 Chemical Methods, 5.3[M]. Madison, America: Soil Science Society of America, 1996. 961-1010. |
| [26] | Bremner J M, Mulvaney C S. Nitrogen-total[A]. In: Page A L, Miller R H, Keeney D R (Eds. ). Methods of Soil Analysis. Part 2. Chemical and Microbiological Properties[M]. Madison, America: Soil Science Society of America, 1982. 595-624. |
| [27] | Keeney D R, Nelson D W. Nitrogen-Inorganic Forms[A]. In: Page A L, Miller R H, Keeney D R, (Eds). Methods of Soil Analysis. Part 2: Chemical and Microbiological Properties[M]. Madison, America: Soil Science Society of America, 1982. 643-698. |
| [28] | Wang X M, Wang S Y, Jiang Y Y, et al. Comammox bacterial abundance, activity, and contribution in agricultural rhizosphere soils[J]. Science of the Total Environment, 2020, 727. DOI:10.1016/j.scitotenv.2020.138563 |
| [29] | Lu S D, Sun Y J, Lu B Y, et al. Change of abundance and correlation of Nitrospira inopinata-like comammox and populations in nitrogen cycle during different seasons[J]. Chemosphere, 2019, 241. DOI:10.1016/j.chemosphere.2019.125098 |
| [30] | Shi Y, Jiang Y Y, Wang S Y, et al. Biogeographic distribution of comammox bacteria in diverse terrestrial habitats[J]. Science of the Total Environment, 2020, 717. DOI:10.1016/j.scitotenv.2020.137257 |
| [31] | Li C Y, Hu H W, Chen Q L, et al. Niche differentiation of clade A comammox Nitrospira and canonical ammonia oxidizers in selected forest soils[J]. Soil Biology and Biochemistry, 2020, 149. DOI:10.1016/j.soilbio.2020.107925 |
| [32] | Xu S Y, Wang B Z, Li Y, et al. Ubiquity, diversity, and activity of comammox Nitrospira in agricultural soils[J]. Science of the Total Environment, 2020, 706. DOI:10.1016/j.scitotenv.2019.135684 |
| [33] |
王智慧, 蒋先军. 宏基因组技术研究泥岩母质发育的三种不同pH紫色土硝化微生物群落演变规律[J]. 微生物学报, 2021, 61(7): 1933-1944. Wang Z H, Jiang X J. Metagenomics-centric assembly of nitrifying communities in three pH-distinct soils with the same origin of parent material[J]. Acta Microbiologica Sinica, 2021, 61(7): 1933-1944. |
| [34] | Zhao Z R, Huang G H, He S S, et al. Abundance and community composition of comammox bacteria in different ecosystems by a universal primer set[J]. Science of the Total Environment, 2019, 691. DOI:10.1016/j.scitotenv.2019.07.131 |
| [35] | Steenbergh A K, Meima M M, Kamst M, et al. Biphasic kinetics of a methanotrophic community is a combination of growth and increased activity per cell[J]. FEMS Microbiology Ecology, 2010, 71(1): 12-22. DOI:10.1111/j.1574-6941.2009.00782.x |
| [36] | Wang X X, Lu L, Zhou X, et al. Niche differentiation of Comammox Nitrospira in the mudflat and reclaimed agricultural soils along the north branch of yangtze river estuary[J]. Frontiers in Microbiology, 2021, 11. DOI:10.3389/fmicb.2020.618287 |