夏围围(1986—),女,安徽淮南人,博士,副教授,主要从事土壤微生物分子生态学研究。E-mail:
硝化微生物在农田土壤氮转化过程中发挥重要作用,深入开展团聚体中硝化微生物分布研究,有助于揭示土壤结构-微生物-土壤营养元素循环间的相互影响机制。选取旱地黄棕壤为研究对象,比较玉米连作(M-M)和玉米/花生轮作(M-P)两种种植方式下土壤团聚体的性质和硝化潜势(NP)的变化,并通过荧光定量PCR和高通量测序研究团聚体中不同类型硝化微生物功能基因的丰度和群落组成差异。结果表明,与M-M相比,M-P能够显著提高团聚体pH、NH4+和全碳(TC)含量。M-P使NP显著提高,但团聚体粒径对NP无显著影响。氨氧化细菌(AOB)
Nitrifiers play an important role in the process of farmland soil nitrogen transformation. A study on the distribution of nitrifying microorganisms in aggregates will help to reveal the interaction mechanism between soil structure, microorganisms and soil nutrient cycling.
The changes in soil aggregate properties and nitrification potential (NP) in maize monoculture (M-M) and maize peanut rotation (M-P) were compared, and the abundance and community composition of different nitrifying functional genes in soil aggregates were evaluated by quantitative PCR and high-throughput sequencing.
Compared with M-M, M-P significantly increased pH, NH4+ and total carbon (TC) in soil aggregates. M-P also significantly enhanced NP, but the aggregate size had no significant effect on NP. The abundance of the AOB
Soil aggregate size and cropping system can greatly affect the distribution of nitrifying microorganisms in soil aggregates. However, nitrifying microorganisms have different adaptation mechanisms among aggregates. This study provides a theoretical support for improving the ecological adaptation mechanism of soil nitrifying microorganisms in the micro-environments under Gramineae-Legume rotation.
团聚体是土壤结构的基本组成单位和土壤生态系统的功能单元,为微生物提供了空间异质的栖息场所。不同粒级团聚体上营养水平、湿度、氧气等微域环境的差异,必然会引起土壤微生物群落的空间分异,进而影响土壤养分转化和物质循环,重塑土壤团聚体的生物学性质。已有较多研究证明不同农田管理措施下,团聚体的粒级组成[
硝化作用是土壤氮素循环的关键过程,不仅降低农田土壤氮肥利用率,而且增加硝酸盐淋溶和温室气体N2O排放风险。硝化作用的两个阶段分别由氨氧化菌和亚硝酸盐氧化细菌(NOB)催化完成。氨氧化菌将铵根离子(NH4+)氧化为亚硝酸盐(NO2–),此反应亦为硝化作用的限速步骤,已发现的氨氧化菌包括氨氧化古菌(AOA)、氨氧化细菌(AOB)和新发现的全程氨氧化细菌(Comammox);继而,NOB将NO2–氧化为硝酸盐(NO3–)。国内外研究表明AOA和AOB丰度和群落结构受土壤pH、NH4+含量、有机碳、土壤质地等因素影响[
近年来,土壤团聚体和硝化菌群之间的研究取得一定进展。Zhang等[
本研究以旱地黄棕壤为研究对象,通过定量PCR和高通量测序等分子生物学手段揭示了硝化微生物群落在不同粒级团聚体上的分异特征,并比较两种不同种植方式(玉米连作和玉米/花生轮作)对土壤团聚体性质和硝化微生物群落的影响。研究结果有助于理解土壤团聚体形成过程中微生物的环境适应机制,为土壤肥力评价和农田管理提供理论依据。
试验区位于安徽省巢湖市栏杆集镇新桥村(31°58'N,117°50'E),地处江淮丘陵南部,属北亚热带湿润季风气候区,光照充分,热量条件较好,无霜期长,季风气候显著,冬寒夏热,四季分明。年平均气温15~16℃,年平均降水量约1 100 mm,主要集中在5—8月,占年总降水量的55%。典型土壤类型为下蜀黄土母质发育的黄棕壤。
田间定位试验于2018—2019年进行。根据试验区内典型农业生产模式,在试验地设置2种处理,分别为玉米连作(M-M)和玉米/花生轮作(M-P)。试验开始前,选取一块地面平整的、土壤性质均匀的传统旱作农田,平均划分为2个小区。试验小区第1年均种植玉米,第2年分别种植玉米和花生。作物每年4月播种,当年9月下旬收获,秋冬季抛荒,在试验第2年作物收获后采集土壤进行分析。玉米和花生种植过程中,基肥施用量相同,为复混肥料(N:P2O5:K2O = 26:10:15)900 kg·hm–2,玉米在大喇叭口期追施尿素150 kg·hm–2(N含量为46.4%),花生不追肥。其他管理措施如培土、除草等均按常规模式进行。
采集试验小区多个采样点0~20 cm土壤。取样时尽量避免挤压,以保持原状土壤结构。将采集的多点土壤样品混合组成代表土样。将稍大土块沿土壤自然结构脆弱面剥离成直径约5 mm小块,仔细挑除土样中的石块、植物根系。取其中一部分土壤用于团聚体粒径分级。M-M全土理化性质为:pH 6.16,全碳(TC)7.63 g∙kg–1,全氮(TN)0.82 g∙kg–1,NH4+-N 1.20 mg∙kg–1,NO3–-N 8.47 mg∙kg–1。M-P全土理化性质为:pH 6.65,TC 8.15 g∙kg–1,TN 0.91g∙kg–1,NH4+-N 4.69 mg∙kg–1,NO3–-N 9.53 mg∙kg–1。
采用干筛法[
两种种植方式下土壤团聚体的理化性质
Soil aggregate properties under different cropping systems
种植方式 |
粒级 |
土壤团聚体Soil aggregate | |||||
百分比 |
pH | 全碳 |
全氮 |
铵态氮 |
硝态氮 |
||
注:表中值为平均值±标准差( |
|||||||
M-M | > 2 | 16.5% | 6.05±0.04bB | 6.62±0.03dB | 0.80±0.02bB | 0.77±0.06cB | 8.89±0.04bA |
2~1 | 31.1% | 5.99±0.06bB | 7.41±0.36cB | 0.75±0.09abB | 1.18±0.09bB | 9.17±0.06aB | |
1~0.25 | 40.5% | 6.07±0.02bB | 8.50±0.06aA | 0.86±0.06abA | 1.57±0.02aB | 8.50±0.01cB | |
< 0.25 | 11.9% | 6.14±0.03aB | 8.27±0.03bA | 0.88±0.04aA | 1.56±0.11aB | 7.25±0.57dA | |
M-P | > 2 | 13.0% | 6.77±0.03aA | 8.21±0.06aA | 0.91±0.01aA | 2.91±0.23cA | 7.33±0.04bB |
2~1 | 22.3% | 6.65±0.07bA | 8.01±0.11bA | 0.90±0.03aA | 5.44±0.03aA | 9.84±0.15aA | |
1~0.25 | 52.0% | 6.45±0.01cA | 8.14±0.02aB | 0.90±0.01aA | 5.26±0.30aA | 9.27±0.48aA | |
< 0.25 | 12.7% | 6.47±0.02cA | 8.23±0.05aA | 0.79±0.01bB | 4.29±0.18bA | 7.42±0.11bA |
土壤pH采用电位计法测定,首先对pH计进行校准,按水土比2.5:1向土壤加入无CO2去离子水,之后将玻璃电极和饱和甘汞电极插入土壤悬液中进行读数。土壤无机氮采用氯化钾浸提,按水土比5:1向土壤加入2 mol·L–1 KCl溶液,以200 r·min–1振荡1 h,将振荡后的溶液静置后过滤,滤液采用连续流动化学分析仪(Skalar ++,Breda,Netherlands)对铵态氮(NH4+-N)和硝态氮(NO3–-N)含量进行测定。土壤TC和TN含量的测定,将土壤冷冻干燥并过100目筛,利用碳氮元素分析仪(Vario MAX CN,Germany)进行测定。以上指标测定时,均进行3次重复。
土壤硝化潜势(Nitrification Potential)通过悬浮液培养法测定[
式中,NP代表硝化潜势(mg∙ kg–1∙d–1),
采用Fast DNA® Spin Kit for Soil(MP Biomedicals)试剂盒,按照试剂盒内说明书上的步骤提取土壤微生物DNA。最终将提取到的土壤DNA溶解于100 μL DES(无核酸酶超纯水)。利用微量紫外分光光度计(Nano Drop® ND-2000 UV-Vis)在260 nm处测定DNA浓度和纯度,确保OD260/ OD280在1.8~2.0范围内,避免腐殖质对后续PCR扩增的影响。最后,用DES将土壤DNA进行10倍稀释,保存于–20℃冰箱。各粒级团聚体DNA提取均进行三次重复。
通过实时定量PCR技术对土壤不同类型硝化微生物功能基因进行定量分析。使用引物amoA-1F/ amoA-2R[
为探究土壤硝化微生物群落组成变化,开展16S rRNA基因高通量测序分析。由上海美吉生物医药科技有限公司(Shanghai Majorbio Bio-pharm Technology Co.,Ltd)对四个土壤团聚体粒级DNA样品中微生物16S rRNA基因V4区进行扩增(引物515F/907R)和建库,最后在Illumina MiSeq测序系统上进行双端测序,继而在美吉生信云平台(www.majorbio.com)上进行生物信息分析。大致分析流程如下:下机数据先进行拼接和质控,根据barcode区分样本,将所测序列在97%相似度水平划分OTU(操作分类单元),并以silva138数据库为参考进行物种注释。在属分类水平,检索已知的AOB[
通过SPSS 19.0统计软件,采用单因素方差(One-way ANOVA)分析Tukey法对不同粒径和不同种植方式下理化因素、进行统计显著性检验;采用双因素方差(Two-way ANOVA)分析检验种植方式和团聚体粒径之间是否存在交互效应;并采用Pearson双尾
土壤团聚体的理化性质见
四个粒级间的土壤团聚体硝化潜势(NP)如
不同种植方式下土壤团聚体的硝化潜势差异
Nitrification potentials within soil aggregates under different cropping systems
不同粒径土壤团聚体上硝化微生物功能基因丰度的差异
Abundance changes of nitrifying functional genes in different soil aggregate sizes
团聚体中AOB
不同粒径土壤团聚体上氨氧化微生物
以
硝化微生物
土壤团聚体理化性质、硝化微生物功能基因丰度以及硝化潜势之间的相关分析
Correlation analysis between soil properties, nitrifying functional gene abundances and nitrification potential
指标Index | 硝化菌Nitrifier | 土壤团聚体Soil aggregate | |||||
pH | TC | TN | NH4+ | NO3– | 土壤硝化潜势(NP) | ||
注:Pearson双尾显著性检验, |
|||||||
基因丰度 |
AOB |
0.548** | 0.480* | 0.150 | 0.513* | –0.425* | 0.588** |
AOA |
–0.576** | –0.742** | –0.324 | –0.754** | 0.086 | –0.686** | |
Comammox |
–0.037 | –0.730** | –0.263 | –0.301 | –0.018 | –0.330 | |
NOB |
–0.558** | 0.020 | –0.325 | –0.660** | –0.697** | –0.687** | |
AOA/AOB | –0.693** | –0.777** | –0.451* | –0.779** | 0.250 | –0.794** | |
Comammox/AOB | –0.354 | –0.824** | –0.335 | –0.583** | 0.177 | –0.690** | |
Comammox/AOA | 0.677** | 0.157 | 0.108 | 0.558** | –0.258 | 0.441 | |
土壤硝化潜势(NP) | 0.800** | 0.412 | 0.485* | 0.889** | 0.207 | 1.000 |
对24个团聚体DNA样品进行高通量测序,共获得16S rRNA基因序列约111.8万条,平均每个样品约4.66万条,硝化微生物的平均相对丰度约为1.02%(473条/样),其中,AOA、AOB和NOB的平均相对丰度分别为0.51%(239条/样)、0.08%(36条/样)和0.43%(199条/样)。
通过16S rRNA基因高通量测序分析,揭示了不同种植方式下土壤团聚体中硝化微生物群落组成(
基于16S rRNA基因的硝化微生物群落组成(a)及与土壤性质和硝化潜势的相关系数热图分析(b)
Nitrifying community structures based on 16S rRNA genes(a)and their correlation with soil properties and nitrification potential by heatmap analysis(b)
双因素方差分析种植方式和团聚体粒径对氨氧化古菌、氨氧化细菌和亚硝酸盐氧化细菌组成的影响(
Results (
硝化菌Nitrifier | 属水平分类 |
C | S | C×S |
*, | ||||
AOA | 0.001* | 0.055 | 0.319 | |
0.481 | 0.947 | 0.374 | ||
Norank_f_ Nitrososphaeraceae | 0.359 | 0.323 | 0.263 | |
AOB | 0.067 | 0.042* | 0.237 | |
0.053 | 0.261 | 0.261 | ||
NOB | 0.027* | 0.150 | 0.425 | |
0.000* | 0.543 | 0.833 |
NH4+含量和pH是影响土壤团聚体硝化微生物群落组成变化的最主要因子(
合理轮作能够协调养分供应,均衡利用土壤养分,防止连作障碍,具有很高的生态和经济效益[
同时,轮作能够通过根系向土壤输送更加多样的营养物质,提高土壤微生物群落多样性[
地理大尺度数据整合分析显示,陆地生态系统中AOB和AOA
Zhang等[
全球范围内土壤硝化速率与土壤pH、SOC、TN、NH4+、AP等显著正相关,与C/N显著负相关[
目前团聚体水平硝化活性与硝化微生物的关联报道十分有限,硝化潜势与不同类型硝化微生物(如AOB/AOA)的相关性并不一致[
值得注意的是,本研究中禾豆轮作只进行了一轮,相关土壤结构和微生物变化是在不同作物茬口下得到的,虽然能够最大程度体现种植不同作物产生的直接影响,但无法评估这些土壤结构及微生物变化的可持续性和生态效益。因此,未来可实施多年轮作,并在相同茬口和不同茬口下进行土壤和作物的多维分析,以便获取连续、详实而全面的轮作“茬口”效应研究结果。
玉米连作和玉米/花生轮作方式下,种植方式和团聚体粒径对土壤团聚体的多种理化性质均产生较大影响,且种植方式能够强烈改变土壤硝化潜势。土壤团聚体粒径和种植方式还能较大程度影响硝化微生物在土壤团聚体中的分布,不同氨氧化微生物在团聚体上的分异规律具有明显差异。AOB
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