生物多样性 ›› 2015, Vol. 23 ›› Issue (1): 68-78. DOI: 10.17520/biods.2014148
所属专题: 土壤生物与土壤健康
关大伟1,2, 李力1,2, 姜昕1,2, 马鸣超1,2, 曹凤明1,2, 周宝库3, 李俊1,2,*()
收稿日期:
2014-07-10
接受日期:
2014-12-30
出版日期:
2015-01-20
发布日期:
2015-05-04
通讯作者:
李俊
作者简介:
E-mail: jli@caas.ac.cn基金资助:
Dawei Guan1,2, Li Li1,2, Xin Jiang1,2, Mingchao Ma1,2, Fengming Cao1,2, Baoku Zhou3, Jun Li1,2,*()
Received:
2014-07-10
Accepted:
2014-12-30
Online:
2015-01-20
Published:
2015-05-04
Contact:
Jun Li
摘要:
为揭示长期施肥对黑土大豆根瘤菌群体结构和多样性的影响, 采用BOX-PCR、IGS-PCR-RFLP和16S rDNA基因序列分析法, 对分离自黑龙江省7种长期不同施肥处理的254株大豆根瘤菌进行了遗传多样性和系统发育分析, 结合土壤理化性质分析了大豆根瘤菌群体结构和多样性与土壤因子间的关系。7种处理分别为不施肥(CK)、有机肥(OM)、单施氮肥(N1)、单施2倍氮肥(N2)、氮肥+有机肥(N1+OM)、氮肥磷肥混施(N1P1)和2倍氮肥磷肥混施(N2P2)。系统发育分析结果表明, 所有供试菌株均为慢生根瘤菌属(Bradyrhizobium), 其中大部分菌株与日本慢生大豆根瘤菌(Bradyrhizobium japonicum)相似性最高, 少部分菌株与辽宁慢生大豆根瘤菌(Bradyrhizobium liaoningense)相似性最高。BOX-PCR聚类分析结果表明, 供试菌株在70%相似性水平上分为15个群, 在与施肥处理相关性分析中分为3个群体, 分别对应于不施化肥处理(CK和OM)、化学氮肥处理(N1、N2、N1+OM)、氮肥磷肥处理(N1P1和N2P2)。典范对应分析结果表明, 土壤pH、速效氮和速效磷与根瘤菌群体结构相关性极显著(P=0.002, 0.004, 0.002)。不同施肥措施下大豆根瘤菌的多样性有明显差异: N2P2处理的丰富度指数和Shannon-Wiener指数显著高于其他处理; OM处理的Simpson指数最高; N1和N2处理的3种多样性指数都显著低于其他处理。通径分析结果表明, pH、速效磷对多样性指数有较高的直接正效应; 速效氮通过pH的间接负效应影响多样性指数。本研究表明, 长期施用化肥改变了根瘤菌群体结构, 单施氮肥减少大豆根瘤菌多样性, 而氮肥磷肥混施则有助于提高大豆根瘤菌多样性。
关大伟, 李力, 姜昕, 马鸣超, 曹凤明, 周宝库, 李俊 (2015) 长期施肥对黑土大豆根瘤菌群体结构和多样性的影响. 生物多样性, 23, 68-78. DOI: 10.17520/biods.2014148.
Dawei Guan, Li Li, Xin Jiang, Mingchao Ma, Fengming Cao, Baoku Zhou, Jun Li (2015) Influence of long-term fertilization on the community structure and diversity of soybean rhizobia in black soil. Biodiversity Science, 23, 68-78. DOI: 10.17520/biods.2014148.
处理 Treatment | 施肥种类 Fertilizer type | 施用量 Dose of fertilization (kg/ha) | 菌株数量 Number of strains |
---|---|---|---|
CK | 不施肥 No fertilizer | 0 | 32 |
OM | 马粪 Horse manure | 18,600 | 32 |
N1+OM | 尿素+马粪 Urea + Horse manure | 75+18,600 | 45 |
N1 | 尿素 Urea | 75 | 48 |
N2 | 尿素 Urea | 150 | 41 |
N1P1 | 尿素+过磷酸钙 Urea & Calcium superphosphate | 75+150 | 28 |
N2P2 | 尿素+过磷酸钙 Urea & Calcium superphosphate | 150 +300 | 28 |
表1 各处理施肥种类、施用量及分离菌株数量
Table 1 The dose of different fertilization and number of isolated rhizobium strains
处理 Treatment | 施肥种类 Fertilizer type | 施用量 Dose of fertilization (kg/ha) | 菌株数量 Number of strains |
---|---|---|---|
CK | 不施肥 No fertilizer | 0 | 32 |
OM | 马粪 Horse manure | 18,600 | 32 |
N1+OM | 尿素+马粪 Urea + Horse manure | 75+18,600 | 45 |
N1 | 尿素 Urea | 75 | 48 |
N2 | 尿素 Urea | 150 | 41 |
N1P1 | 尿素+过磷酸钙 Urea & Calcium superphosphate | 75+150 | 28 |
N2P2 | 尿素+过磷酸钙 Urea & Calcium superphosphate | 150 +300 | 28 |
图1 BOX-PCR图谱聚类分析树状图。括号内的处理编号代表菌株分离地点, 括号内的数字代表具有相同BOX-PCR图谱的菌株数量。
Fig. 1 The dendrogram of BOX-PCR cluster analysis. The codes of the treatments in parenthesis refer to sampling sites, and the numbers in parenthesis refer to the strain numbers with same BOX-PCR type.
图2 IGS PCR-RFLP指纹图谱聚类分析树状图。A1和A2为群A的亚群, B1、B2、B3和B4为群B的亚群, C1和C2为群C的亚群。
Fig. 2 The dendrogram of IGS PCR-RFLP cluster analysis. A1 and A2 are subgroups of A group, B1, B2, B3and B4 are subgroups of B group, and C1 and C2 are subgroups of C group.
图3 代表菌株与参比菌株16S rDNA序列的Neighbor-joining聚类图。图中分支上的数字表示可信度, 括号内为GenBank登录号, 0.01表示遗传距离。
Fig. 3 Neighbor-joining clustering phylogenetic tree of 16S rDNA sequence between representatives of isolated rhizobia and the reference strains. The values on branch stand for reliabilities and the numbers in the parenthesis are GenBank accession numbers. 0.01 denoted genetic distances.
图4 大豆根瘤菌的BOX-PCR群与施肥处理进行相关性分析。图中?代表处理; ρ代表BOX-PCR群。
Fig. 4 Correlation analysis between fertilization treatments and BOX-PCR genomic groups of soybean rhizobia. ? Treatments; ρ Groups of BOX-PCR in the figure.
施肥处理 Treatments | pH | 有机质含量 Organic manure (g/kg) | 速效氮含量 Available N (mg/kg) | 速效磷含量 Available P (mg/kg) | 速效钾含量 Available K (mg/kg) |
---|---|---|---|---|---|
CK | 6.91b | 22.3d | 54.10e | 2.32f | 175.27b |
OM | 7.01a | 25.0b | 76.96c | 13.02d | 201.85a |
N1 | 5.94d | 22.9cd | 75.85c | 2.92f | 158.40d |
N2 | 5.00e | 23.0c | 95.39a | 5.48e | 143.78e |
N1P1 | 5.94d | 24.5b | 69.12d | 58.10b | 167.00c |
N2P2 | 4.94e | 23.6c | 95.65a | 124.93a | 154.12d |
N1+OM | 6.32c | 28.0a | 85.72b | 14.35c | 199.58a |
表2 不同施肥处理土壤的理化性质
Table 2 Soil properties in different fertilization treatments
施肥处理 Treatments | pH | 有机质含量 Organic manure (g/kg) | 速效氮含量 Available N (mg/kg) | 速效磷含量 Available P (mg/kg) | 速效钾含量 Available K (mg/kg) |
---|---|---|---|---|---|
CK | 6.91b | 22.3d | 54.10e | 2.32f | 175.27b |
OM | 7.01a | 25.0b | 76.96c | 13.02d | 201.85a |
N1 | 5.94d | 22.9cd | 75.85c | 2.92f | 158.40d |
N2 | 5.00e | 23.0c | 95.39a | 5.48e | 143.78e |
N1P1 | 5.94d | 24.5b | 69.12d | 58.10b | 167.00c |
N2P2 | 4.94e | 23.6c | 95.65a | 124.93a | 154.12d |
N1+OM | 6.32c | 28.0a | 85.72b | 14.35c | 199.58a |
图5 大豆根瘤菌的BOX-PCR群与土壤理化性质典范对应分析。图中ρ代表BOX-PCR群。
Fig. 5 Canonical correspondence analysis between soil properties and BOX-PCR genomic groups of soybean rhizobia. ρ Groups of BOX-PCR in the figure.
多样性指数 Diversity index | 施肥处理 Treatments | ||||||
---|---|---|---|---|---|---|---|
CK | OM | N1 | N2 | N1P1 | N2P2 | N1+OM | |
丰富度指数 Richness index | 1.485b* | 1.553b | 1.101d | 0.281e | 1.294c | 2.175a | 1.108d |
Simpson指数 Simpson index | 0.767a | 0.815a | 0.562c | 0.431c | 0.682b | 0.707b | 0.706b |
Shannon-Wiener指数 Shannon-Wiener index | 1.416b | 1.249c | 0.993d | 0.622e | 1.444b | 1.572a | 1.404b |
表3 不同施肥处理大豆根瘤菌多样性指数
Table 3 The diversity indexes of rhizobia in different fertilization treatments
多样性指数 Diversity index | 施肥处理 Treatments | ||||||
---|---|---|---|---|---|---|---|
CK | OM | N1 | N2 | N1P1 | N2P2 | N1+OM | |
丰富度指数 Richness index | 1.485b* | 1.553b | 1.101d | 0.281e | 1.294c | 2.175a | 1.108d |
Simpson指数 Simpson index | 0.767a | 0.815a | 0.562c | 0.431c | 0.682b | 0.707b | 0.706b |
Shannon-Wiener指数 Shannon-Wiener index | 1.416b | 1.249c | 0.993d | 0.622e | 1.444b | 1.572a | 1.404b |
多样性指数 Diversity index | 土壤养分 Soil properties | X1 | X2 | X3 | X4 | 相关系数 Correlation coefficient |
---|---|---|---|---|---|---|
丰富度指数 Richness index | X1 | 0.462 | 0.117 | -0.077 | -0.836 | -0.334 |
X2 | 0.118 | 1.166 | 0.002 | -0.604 | 0.682 | |
X3 | 0.074 | 0.009 | -0.309 | 0.253 | 0.027 | |
X4 | -0.226 | -0.640 | -0.071 | 1.100 | 0.163 | |
Shannon-Wiener指数 Shannon-Wiener index | X1 | -0.521 | 0.386 | 0.092 | -0.274 | -0.317 |
X2 | -0.206 | 0.976 | -0.003 | -0.198 | 0.569 | |
X3 | 0.130 | -0.008 | 0.370 | 0.083 | 0.575 | |
X4 | 0.396 | -0.536 | 0.085 | 0.360 | 0.305 | |
Simpson指数 Simpson index | X1 | -0.039 | 0.317 | -0.002 | -0.870 | -0.594 |
X2 | -0.015 | 0.801 | 0.000 | -0.628 | 0.158 | |
X3 | -0.010 | -0.006 | -0.010 | 0.263 | 0.237 | |
X4 | 0.030 | -0.440 | 0.000 | 1.144 | 0.734 |
表4 土壤理化性质对大豆根瘤菌多样性的通径分析
Table 4 Analysis of path coefficients between diversity index of rhizobia and soil properties
多样性指数 Diversity index | 土壤养分 Soil properties | X1 | X2 | X3 | X4 | 相关系数 Correlation coefficient |
---|---|---|---|---|---|---|
丰富度指数 Richness index | X1 | 0.462 | 0.117 | -0.077 | -0.836 | -0.334 |
X2 | 0.118 | 1.166 | 0.002 | -0.604 | 0.682 | |
X3 | 0.074 | 0.009 | -0.309 | 0.253 | 0.027 | |
X4 | -0.226 | -0.640 | -0.071 | 1.100 | 0.163 | |
Shannon-Wiener指数 Shannon-Wiener index | X1 | -0.521 | 0.386 | 0.092 | -0.274 | -0.317 |
X2 | -0.206 | 0.976 | -0.003 | -0.198 | 0.569 | |
X3 | 0.130 | -0.008 | 0.370 | 0.083 | 0.575 | |
X4 | 0.396 | -0.536 | 0.085 | 0.360 | 0.305 | |
Simpson指数 Simpson index | X1 | -0.039 | 0.317 | -0.002 | -0.870 | -0.594 |
X2 | -0.015 | 0.801 | 0.000 | -0.628 | 0.158 | |
X3 | -0.010 | -0.006 | -0.010 | 0.263 | 0.237 | |
X4 | 0.030 | -0.440 | 0.000 | 1.144 | 0.734 |
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