生物多样性 ›› 2015, Vol. 23 ›› Issue (1): 68-78.DOI: 10.17520/biods.2014148

• 研究报告: 微生物多样性 • 上一篇    下一篇

长期施肥对黑土大豆根瘤菌群体结构和多样性的影响

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,*()   

  1. 1 中国农业科学院农业资源与农业区划研究所, 北京 100081
    2 农业部微生物产品质量安全风险评估实验室, 北京 100081
    3 黑龙江省农业科学院土壤肥料与环境资源研究所, 哈尔滨 150006
  • 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:
    现代农业产业技术体系建设专项资助项目(CARS-04);黑龙江省自然科学基金项目(ZD200813-02)

Influence of long-term fertilization on the community structure and diversity of soybean rhizobia in black soil

Dawei Guan1,2, Li Li1,2, Xin Jiang1,2, Mingchao Ma1,2, Fengming Cao1,2, Baoku Zhou3, Jun Li1,2,*()   

  1. 1 Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing 100081
    2 Laboratory of Quality & Safety Risk Assessment for Microbial Products, Ministry of Agriculture, Beijing 100081;
    3 Institute of Soil Fertilizer and Environment Resources, Heilongjiang Academy of Agricultural Sciences, Harbin 150006
  • 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。

关键词: 多样性指数, BOX-PCR, IGS-PCR-RFLP, 典型对应分析, 通径分析

Abstract

The objectives of this study were to explore the effects of long-term fertilization on the community structure and diversity of soybean rhizobia in black soil. BOX-PCR, IGS-PCR-RFLP and 16S rDNA gene sequencing methods were used to analyze the genetic diversity of the 254 soybean rhizobia isolated from 7 treatments at Heilongjiang Long-term Fertilization Experiment Station. The 7 treatments were as follows: no fertilization (CK), organic manure (OM), chemical nitrogen (N1), double chemical nitrogen (N2), chemical nitrogen-phosphorus (N1P1), double chemical nitrogen-phosphorus (N2P2), chemical nitrogen plus organic manure (N1+OM). Soil properties were measured and analyzed to test for relationships with the community and diversity of soybean rhizobia. All of the tested rhizobia belonged to Bradyrhizobium, of which the majority were closely related to B. japonicum and the others closely to B. liaoningense. BOX-PCR fingerprints showed that the tested rhizobia could be divided into 15 groups at 70% similarity level. These groups clustered into 3 communities that correspond to no chemical fertilizer treatments (CK, OM), chemical nitrogen fertilizer treatments (N1, N2, N1+OM), and chemical nitrogen-phosphorus treatments (N1P1, N2P2), respectively. Canonical correspondence analysis showed that pH, available N, and available P of the soil were significantly correlated with the community of rhizobia (P=0.002, 0.004, 0.002, respectively). Soybean rhizobia diversity indices varied significantly among fertilization treatments. Shannon-Wiener and species richness index were highest in N2P2 and Simpson index was highest in the OM treatment. The species richness, Shannon-Wiener and Simpson index in N1 and N2 were significantly lower than those in other treatments. The results of path analysis showed that the pH and available P had the greatest direct positive effects on the 3 indices, and that available N indirectly affected the 3 indices through pH. In conclusion, long-term chemical fertilization results in changes to community structure of soybean rhizobia in black soil, and application of chemical nitrogen fertilizer by itself reduces the diversity of soybean rhizobia, while application of nitrogen- phosphorus has the opposite effect.

Key words: diversity indices, BOX-PCR, IGS-PCR-RFLP, canonical correspondence analysis, path analysis