Characteristics of structure and abundance of soil nitrogen-fixing bacterial community in alfalfa with different growing ages in the Loess Plateau
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摘要: 生物固氮是紫花苜蓿(Medicago sativa)土壤氮素的重要来源, 固氮微生物数量及其群落结构变化对土壤氮素供应和肥力维持起着重要作用。本研究采用Illumina MiSeq测序和荧光定量PCR技术, 探究了黄绵土区玉米农田和不同种植年限[2019年(2年)、2012年(9年)、2003年(18年)]紫花苜蓿地土壤nifH固氮基因丰度、nifH固氮微生物群落结构和多样性, 通过共现网络分析丰富和稀有固氮微生物的生态地位, 耦合土壤理化性质明确影响固氮微生物群落结构的主导因子。结果表明, 黄绵土固氮微生物nifH基因丰度为2.97×106~5.93×106 copies∙g−1(干土), 且表现为苜蓿地显著高于玉米农田。土壤样品经测序共获得有效序列176 367条, 主要分布在5门、8纲、11目、15科、17属。门水平上, 变形菌门和蓝藻门为主要优势类群; 属水平以斯克尔曼氏菌属和固氮弧菌属为优势属。与玉米农田相比, 多年持续种植紫花苜蓿显著提高了斯克尔曼氏菌属的相对丰度, 但其随种植年限延长呈降低趋势。长期种植紫花苜蓿促生了固氮菌属、伯克氏菌属、弗兰克氏菌属、中慢生根瘤菌属、地杆菌属和慢生根瘤菌属等生理类群, 同时也使得梭状杆菌属、红假单胞菌属和三离藻属消亡。RDA分析发现, 固氮微生物不同种群对环境因子的响应并不一致, 具有各自的生态位, 但土壤全磷是影响土壤固氮微生物群落结构的主导环境因子, 其次是有机碳和硝态氮。分子生态网络分析表明固氮菌生态网络中丰富类群占据生态系统核心地位, 且物种间均为协同合作关系, 群落结构相对稳定, 对环境变化具有较强的适应能力。综上, 黄土高原半干旱区种植紫花苜蓿显著提高了土壤nifH基因丰度, 改变了固氮微生物nifH群落结构, 该结果可为黄绵土固氮微生物多样性研究和紫花苜蓿适宜种植年限的确定提供基础数据和理论依据。Abstract: Biological nitrogen fixation is a major nitrogen source in alfalfa fields, and the nitrogen supply and soil fertility can be largely affected by the composition and quantity of the nitrogen-fixing bacterial community. In this study, a field experiment was conducted to explore the soil nitrogen-fixing bacterial community structure and abundance characteristics in loessal soil with different alfalfa growing ages (2, 9 and 18 years planted in 2019, 2012, and 2003, respectively), using farmland (maize field) as the control. The fluorogenic quantitative real-time PCR technique was adopted in the experiment, using the high-throughput sequencing platform Illumina MiSeq to target the nifH gene. We analyzed the ecological status of abundant and rare nitrogen-fixing microorganisms through co-occurrence networks and identified the dominant factors affecting the community structure of nitrogen-fixing microorganisms by soil coupling the physical and chemical properties. The results showed that long-term planting of alfalfa increased the organic carbon, total nitrogen, and soluble carbon contents of the soil. The nifH gene abundance ranged from 2.97×106 copies∙g−1 to 5.93×106 copies∙g−1 in dry soil and was significantly higher in alfalfa fields than in farmland. The correlation analysis between the abundance of nifH gene of nitrogen-fixing microorganisms and soil physicochemical factors showed that nifH gene abundance in the soil was positively correlated with bulk density (P=0.009) and soluble carbon content (P=0.005), positively correlated with total nitrogen (P=0.044) and available potassium (P=0.013) contents, and negatively correlated with total phosphorus content (P=0.000) and nitrate content (P=0.023). A total of 176 367 valid sequences were obtained, belonging to five phyla, eight classes, 11 orders, 15 families, and 17 genera. Proteobacteria and Cyanobacteria were the dominant phyla, accounting for 95.9%−98.9% and 0.2%−1.8% of the total sequences of the samples, whereas Skermanella and Azohydromonas were the dominant genera, accounting for 82.2%–87.6% and 1.6%–4.6%, respectively. Compared with farmland, continuous alfalfa planting significantly increased the relative abundance of Skermanella, but its’ relative abundance decreased with increasing alfalfa planting years. Long-term cultivation of alfalfa propagated microbial taxa, including Azotobacter, Burkholderia, Frankia, Mesorhizobium, Geobacter, and Bradyrhizobium; whereas Clostridium, Rhodopseudomonas, and Trichormus were sterilized. Redundancy analysis (RDA) showed niche differentiation for the nitrogen-fixing bacterial community in response to environmental factors, but total phosphorus, organic carbon, and nitrate-nitrogen in the soil were the dominant environmental factors for the nitrogen-fixing bacterial community structure. Analysis of the molecular ecological network showed that there were 520 nodes and 4170 edges in the network of nitrogen-fixing microorganisms in maize fields and alfalfa soil, among which 24 nodes belonged to the abundant group, 93 nodes belonged to the rare group, and 403 nodes belonged to the transitional group. There was one internal connection of abundant taxa, 2187 internal connections of transitional taxa, and 358 internal connections of rare taxa. Nitrogen-fixing bacteria have a cooperative relationship in their ecological network, with a relatively stable community structure and strong adaptability to environmental changes. This study provides basic data and a theoretical basis for the diversity of nitrogen-fixing microorganisms in loess soil and the determination of a suitable planting period for alfalfa.
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Key words:
- Loessal soil /
- Medicago sativa /
- nifH gene /
- Abundant taxa /
- Rare taxa /
- Ecological network
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图 1 不同处理土壤固氮微生物Alpha多样性
Farmland、L2019、L2012和L2003分别表示农田、苜蓿种植时间为2019年、2012年和2003年。Farmland, L2019, L2012, and L2003 denote farmland and Medicago sativa lands planting in 2019, 2012, and 2003, respectively.
Figure 1. Alpha diversity of soil nitrogen-fixing microbial communities under different treatments
图 2 不同处理土壤固氮菌非度量多维尺度分析
Farmland、L2019、L2012、L2003分别表示农田、苜蓿种植时间为2019年、2012年和2003年。Farmland, L2019, L2012, and L2003 denote farmland and Medicago sativa lands planting in 2019, 2012, and 2003, respectively.
Figure 2. Nonmetric multidimensional scale analysis of soil nitrogen-fixing microbial communities under different treatments
图 3 不同处理土壤固氮微生物群落组成(门和属水平)
“*”表示处理间在P<0.05水平差异显著; Farmland、L2019、L2012和L2003 分别表示农田、苜蓿种植时间为2019年、2012年和2003年。In the figure, “*” means significant difference at P<0.05 among treatments. Farmland, L2019, L2012, and L2003 denote farmland and Medicago sativa land planting in 2019, 2012, and 2003, respectively.
Figure 3. Soil nitrogen-fixing microbial community composition at the phylum and genus levels under different treatments
图 4 土壤固氮微生物共生网络分析
A: 丰富和稀有类群; B: 核心和非核心类群; C: 核心类群; D: 共现网络。图A、B和D中节点大小代表 OTUs 的度, 边的粗细为权重; 图C中节点大小为OTUs的平均相对丰度。群落内部和外部边的连接数用黑色字体显示, 节点数根据不同分类显示; 图D中不同颜色的节点表示不同优势属。图中蓝色连接线为正连接, 红色连接线为负连接。A: abundant and rare taxa; B: core and non-core taxa; C: core taxa; D: co-occurrence network. Node size in figures A, B, and D represents the degree of OTUs, and edge thickness is weight; node size in Figure C is the average relative abundance of OTUs. The number of connections between the inner and outer edges of the community is shown in black font, and the number of nodes is shown according to different classifications; nodes with different colors in Figure D represent different dominant genera. The blue connection line in the figures is the positive connection, and the red connection line is the negative connection.
Figure 4. Symbiosis network analysis of nitrogen-fixing microorganisms
图 5 土壤固氮微生物群落与土壤理化因子冗余分析
AK、SBD、OC、TN、DOC、NO3−-N、NH4+-N、DON、TP、 pH分别表示土壤速效钾、容重、有机碳、全氮、可溶性碳、硝态氮、铵态氮、可溶性氮、全磷和pH, Farmland、L2019、L2012和L2003分别表示农田、苜蓿种植时间为2019年、2012年和2003年。Skermanella、Azohydromonas、Nostoc、Methylobacter、Sinorhizobium、Anabaena、Paenibacillus、Azotobacter、Burkholderia、Frankia、Clostridium、Mesorhizobium、Rhodopseudomonas、Trichormus、Geobacter、Bradyrhizobium、Rhodomicrobium分别表示斯克尔曼氏菌属、固氮弧菌属、念珠藻属、甲基杆菌属、中华根瘤菌属、鱼腥藻属、类芽孢杆菌属、 固氮菌属、伯克氏菌属、弗兰克氏菌属、梭状杆菌属、中慢生根瘤菌属、红假单胞菌属、三离藻属、地杆菌属、慢生根瘤菌属和红微菌属。AK, SBD, OC, TN, DOC, NO3−-N, NH4+-N, DON, TP, pH represent soil available potassium, bulk density, organic carbon, total nitrogen, soluble carbon, nitrate nitrogen, ammonium nitrogen, soluble nitrogen, total phosphorus and pH, respectively. Farmland, L2019, L2012 and L2003 denote farmland and Medicago sativa land planting in 2019, 2012, and 2003, respectively.
Figure 5. Redundancy analysis of soil nitrogen-fixing microbial community structure at the genus level and soil physicochemical properties
表 1 目标基因的引物名称及引物序列
Table 1. Primer names and primer sequences of target genes
目标基因 Target gene 引物 Prime 引物序列 Sequence PCR反应条件 PCR condition nifH gene nifH-F 5′-AAA GGYGGW ATC GGY AAR TCC ACC AC-3′ 95 ℃ 预变性 3 min 1个循环; 95 ℃ 变性 30 s; 56 ℃ 退火 30 s;
72 ℃ 延伸 40 s, 35 个循环。
95 ℃ for 3 min × 1 cycle, 95 ℃ for 30 s, 56 ℃ for 30 s,
72 ℃ for 40 s × 35 cycles.nifH-R 5′-TTG TTS GCS GCR TACATS GCC ATC AT-3′ 
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表 2 不同处理土壤理化性状和nifH基因丰度
Table 2. Soil physicochemical properties and nifH gene abundance under different treatments
项目 Item Farmland L2019 L2012 L2003 容重 Bulk density (SBD, g∙cm−3) 1.18±0.01a 1.21±0.01a 1.23±0.02a 1.25±0.03a 有机碳 Organic carbon (OC, g∙kg−1) 9.86±0.11b 9.79±0.14b 10.48±0.26b 11.65±0.30a 全氮 Total nitrogen (TN, g∙kg−1) 0.98±0.04c 0.96±0.05c 1.13±0.01b 1.28±0.02a 全磷 Total phosphorus (TP, g∙kg−1) 0.82±0.01a 0.74±0.01bc 0.76±0.01b 0.72±0.01c 硝态氮 Nitrate nitrogen (mg∙kg−1) 17.41±0.19a 11.55±0.46c 11.42±0.14c 13.94±0.71b 铵态氮 Ammonium nitrogen (mg∙kg−1) 1.68±0.05b 2.67±0.07a 2.92±0.05a 1.99±0.20b 可溶性碳 Dissolved organic carbon (DOC, mg∙kg−1) 109.59±5.14b 118.96±3.84b 119.55±2.36b 134.65±2.63a 可溶性氮 Dissolved organic nitrogen (DON, mg∙kg−1) 56.17±4.70a 41.66±3.45a 47.92±0.54a 53.55±3.49a 速效钾 Available potassium (AK, mg∙kg−1) 155.50±9.02a 183.33±9.44a 177.00±2.26a 184.83±3.24a pH 8.42±0.06a 8.58±0.07a 8.65±0.06a 8.55±0.07a nifH基因丰度 Abundance of nifH gene [nifH, ×106copys∙g−1(dry soil)] 2.97±0.30c 5.21±0.29ab 4.54±0.32b 5.93±0.26a 数据为平均值±标准误(n=3), 同行不同小写字母表示不同处理间差异显著(P<0.05), Farmland、L2019、L2012和L2003分别表示农田、苜蓿种植时间为2019年、2012年和2003年。Data in table are mean ± standard error (n=3). Different lowercase letters in the same line indicate significant differences among different treatments (P<0.05). Farmland, L2019, L2012, and L2003 denote farmland and Medicago sativa lands planting in 2019, 2012, and 2003, respectively.
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表 3 土壤固氮菌nifH基因丰度与土壤理化性状的相关系数
Table 3. Correlation coefficients between nitrogen-fixing bacteria nifH gene abundance and soil physicochemical properties
nifH SBD OC TN TP NO3−-N NH4+-N DOC DON AK pH nifH 1.000 SBD 0.714** 1.000 OC 0.511 0.420 1.000 TN 0.589* 0.529 0.936** 1.000 TP −0.865** −0.618* −0.522 −0.498 1.000 NO3−−N −0.646* −0.462 −0.027 −0.141 0.651* 1.000 NH4+−N 0.305 0.275 −0.085 −0.026 −0.355 −0.818** 1.000 DOC 0.750** 0.451 0.795** 0.815** −0.725** −0.351 0.022 1.000 DON −0.326 −0.283 0.235 0.264 0.281 0.590* −0.686* 0.217 1.000 AK 0.688* 0.267 0.341 0.371 −0.604* −0.585* 0.398 0.509 −0.233 1.000 pH 0.531 0.664* 0.109 0.281 −0.308 −0.612* 0.619* 0.165 −0.435 0.492 1.000 **: P<0.01; *: P<0.05; 项目名称见表2。The names of items are shown in the table 2.
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