间作大豆对甘蔗根际土壤细菌及固氮菌多样性的影响

doi:10.3724/SP.J.1258.2014.00090

植物生态学报 ›› 2014, Vol. 38 ›› Issue (9): 959-969.DOI: 10.3724/SP.J.1258.2014.00090

所属专题：生物多样性

间作大豆对甘蔗根际土壤细菌及固氮菌多样性的影响

彭东海^1,^*(),杨建波¹,李健¹,邢永秀^1,²,覃刘东¹,杨丽涛^1,^2,^**(),李杨瑞^1,^2,^**()

¹广西大学农学院/广西大学亚热带农业生物资源保护与利用国家重点实验室, 南宁 530004
²中国农业科学院甘蔗研究中心/广西农业科学院/农业部广西甘蔗生物技术与遗传改良重点实验室/广西甘蔗遗传改良生物技术重点开放实验室/广西农业科学院微生物研究所, 南宁 530007

收稿日期:2014-01-07 接受日期:2014-04-14 出版日期:2014-01-07 发布日期:2014-09-22
通讯作者: 彭东海,杨丽涛,李杨瑞
基金资助:
国家高技术研究发展计划(863计划)(2013AA102604);国家自然科学基金(31171504);国家自然科学基金(31101122);国家自然科学基金(31240056);广西自然科学基金创新团队项目(2011GXNSFF018002);广西八桂学者与特聘专家专项经费(2013);广西科学研究与技术开发计划项目(桂科产1123008-1);广西科学研究与技术开发计划项目(桂科攻1222009);广西农科院团队项目(桂农科2011YT01)

Effects of intercropping with soybean on bacterial and nitrogen-fixing bacterial diversity in the rhizosphere of sugarcane

PENG Dong-Hai^1,^*(),YANG Jian-Bo¹,LI Jian¹,XING Yong-Xiu^1,²,QIN Liu-Dong¹,YANG Li-Tao^1,^2,^**(),LI Yang-Rui^1,^2,^**()

¹College of Agriculture/State Key Laboratory for Subtropical Agri-Bioresources Conservation and Utilization, Guangxi University, Nanning 530004, China
²Guangxi Academy of Agricultural Sciences/Sugarcane Research Center, Chinese Academy of Agricultural Sciences/Guangxi Key Laboratory of Sugarcane Genetic Improvement/Key Laboratory of Sugarcane Biotechnology and Genetic Improvement (Guangxi), Ministry of Agriculture/Microbiology Research Institute, Guangxi Academy of Agricultural Sciences, Nanning 530007, China

Received:2014-01-07 Accepted:2014-04-14 Online:2014-01-07 Published:2014-09-22
Contact: PENG Dong-Hai,YANG Li-Tao,LI Yang-Rui

摘要/Abstract

摘要：

为探讨间作大豆(Glycine max)对甘蔗(Saccharum officinarum)根际土壤细菌及固氮细菌多样性的影响, 收集和开发固氮菌资源, 筛选高效甘蔗联合固氮体系, 选用3个甘蔗栽培品种‘ROC22’、‘GT21’、‘B8’与大豆品种‘Guizao 2’进行间种栽培, 采用巢式PCR特异扩增细菌16S rRNA基因片段和固氮细菌nifH基因片段, 并结合变性梯度凝胶电泳(DGGE)技术, 对间作大豆的甘蔗根际土壤细菌及固氮细菌进行系统演化和多样性分析。聚类分析结果显示, 间作大豆改变了甘蔗根际土壤细菌及固氮细菌原来的群落组成结构, 尤其对固氮菌群落组成的改变更大, 但对群落物种的优势度影响较小。Shannon-Wiener多样性指数和Simpson多样性指数分析结果表明, 甘蔗-大豆间作显著影响甘蔗根际土壤中细菌和固氮菌的多样性, 其中对固氮细菌多样性的影响较大。不同甘蔗品种的根际土壤细菌和固氮菌在间作大豆条件下表现出不同的多样性, ‘ROC22’和‘GT21’间作处理甘蔗根际土壤固氮细菌的Shannon-Wiener多样性指数显著高于单作处理, 而‘ROC22’与大豆间作处理的甘蔗根际土壤固氮菌多样性最为丰富。在大豆生长盛期, 间作处理的甘蔗根际土壤细菌多样性最为丰富, 不同处理间的差异也最大, 随后下降。总体来看, 甘蔗-大豆间作显著地影响根际土壤细菌和固氮菌的群落结构和群落多样性, 有助于对甘蔗合理间作栽培模式的认识和筛选高效甘蔗联合固氮体系。

关键词: 变性梯度凝胶电泳, 间作, 固氮细菌, 大豆, 甘蔗

Abstract:

Aims In order to investigate the effects of intercropping with soybean on the diversities of bacteria and nitrogen- fixing bacteria in the rhizosphere of sugarcane (Saccharum officinarum), to collect and exploit nitrogen-fixing bacterial resources, and to screen for efficient sugarcane-associative nitrogen fixation system, an experiment was conducted by cultivating three sugarcane cultivars ‘ROC22’, ‘GT21’, and ‘B8’ with a soybean (Glycine max) cultivar ‘Guizao 2’.
Methods Rhizospheric soil bacteria and nitrogen-fixing bacteria were measured in the rhizosphere of sugarcane intercropped with soybean by using a nested-PCR combined with denaturing gradient gel electrophoresis (DGGE) technique. The specific bacterial 16S rRNA gene fragments and nitrogen-fixing bacteria nifH gene fragments were amplified for phylogenetic and diversity analyses.
Important findings Cluster analysis of bacterial communities showed that the sugarcane-soybean intercropping changed the community composition of rhizospheric soil bacteria and nitrogen-fixing bacteria, with the effect being greater on nitrogen-fixing bacterial communities than on the soil bacterial communities. The intercropping system had a significant effect on the diversity of the rhizospheric bacteria and nitrogen-fixing bacteria; the impact on the diversity of nitrogen-fixing bacteria was greater than on the soil bacteria, and was only minor on the dominance of the bacterial species. Analyses of Shannon-Wiener index and Simpson index showed that the nitrogen-fixing bacteria differed in diversity among the treatments with different sugarcane varieties, and that the sugarcane cultivar ‘ROC22’-soybean intercropping had the greatest diversity. The diversity of soil bacteria in the rhizosphere of sugarcane varied greatly among different stages of crop growth; the booming stage of soybean had the greatest diversity and variations in the diversity among treatments in soil bacteria in the rhizosphere of sugarcane, and thereafter the two variables decreased. In conclusion, the sugarcane-soybean intercropping system had significant effects on the community structures and diversities of soil bacteria and nitrogen-fixing bacteria in the rhizosphere of sugarcane. This is of help for understanding the beneficial effects of sugarcane-soybean intercropping and for identifying efficient sugarcane-associative nitrogen fixation systems.

Key words: denaturing gradient gel electrophoresis, intercropping, nitrogen-fixing bacteria, soybean, sugarcane

彭东海,杨建波,李健,邢永秀,覃刘东,杨丽涛,李杨瑞. 间作大豆对甘蔗根际土壤细菌及固氮菌多样性的影响. 植物生态学报, 2014, 38(9): 959-969. DOI: 10.3724/SP.J.1258.2014.00090

PENG Dong-Hai,YANG Jian-Bo,LI Jian,XING Yong-Xiu,QIN Liu-Dong,YANG Li-Tao,LI Yang-Rui. Effects of intercropping with soybean on bacterial and nitrogen-fixing bacterial diversity in the rhizosphere of sugarcane. Chinese Journal of Plant Ecology, 2014, 38(9): 959-969. DOI: 10.3724/SP.J.1258.2014.00090

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链接本文: https://www.plant-ecology.com/CN/10.3724/SP.J.1258.2014.00090

https://www.plant-ecology.com/CN/Y2014/V38/I9/959

图/表 9

图1 土壤总DNA提取。1, 空白对照, 时期1; 2, 大豆根际土, 时期1; 3, 空白对照, 时期2; 4, 大豆根际土, 时期2; 5, ‘B8’根际土, 单作, 时期1; 6, ‘B8’根际土, 间作, 时期1; 7, ‘B8’根际土, 单作, 时期2; 8, ‘B8’根际土, 间作, 时期2; 9, ‘B8’根际土, 单作, 时期3; 10, ‘B8’根际土, 间作, 时期3; 11, ‘ROC22’根际土, 单作, 时期1; 12, ‘ROC22’根际土, 间作, 时期1; 13, ‘ROC22’根际土, 单作, 时期2; 14, ‘ROC22’根际土, 间作, 时期2; 15, ‘ROC22’根际土, 单作, 时期3; 16, ‘ROC22’根际土, 间作, 时期3; 17, ‘GT21’根际土, 单作, 时期1; 18, ‘GT21’根际土, 间作, 时期1; 19, ‘GT21’根际土, 单作, 时期2; 20, ‘GT21’根际土, 间作, 时期2; 21, ‘GT21’根际土, 单作, 时期3; 22, ‘GT21’根际土, 间作, 时期3。M, 标准分子量DNA (Mark II, 北京天根)。时期1, 大豆生长盛期(5月22日); 时期2, 大豆成熟期(7月2日); 时期3, 甘蔗伸长期(9月15日)。

Fig. 1 Extraction of total soil DNA. 1, control, stage 1; 2, rhizospheric soil of soybean, stage 1; 3, control, stage 2; 4, rhizospheric soil of soybean, stage 2; 5, rhizospheric soil of ‘B8’, monoculture, stage 1; 6, rhizospheric soil of ‘B8’, intercropping, stage 1; 7, rhizospheric soil of ‘B8’, monoculture, stage 2; 8, rhizospheric soil of ‘B8’, intercropping, stage 2; 9, rhizospheric soil of ‘B8’, monoculture, stage 3; 10, rhizospheric soil of ‘B8’, intercropping, stage 3; 11, rhizospheric soil of ‘ROC22’, monoculture, stage 1; 12, rhizospheric soil of ‘ROC22’, intercropping, stage 1; 13, rhizospheric soil of ‘ROC22’, monoculture, stage 2; 14, rhizospheric soil of ‘ROC22’, intercropping, stage 2; 15, rhizospheric soil of ‘ROC22’, monoculture, stage 3; 16, rhizospheric soil of ‘ROC22’, intercropping, stage 3; 17, rhizospheric soil of ‘GT21’, monoculture, stage 1; 18, rhizospheric soil of ‘GT21’, intercropping, stage 1; 19, rhizospheric soil of ‘GT21’, monoculture, stage 2; 20, rhizospheric soil of ‘GT21’, intercropping, stage 2; 21, rhizospheric soil of ‘GT21’, monoculture, stage 3; 22, rhizospheric soil of ‘GT21’, intercropping, stage 3. M, DNA mark (Mark II, Tiangen). stage 1, the highest at soybean booming stage (22 May); stage 2, soybean maturity (2 July); stage 3, sugarcane elongation stage (15 Sept.).

图2 16S rRNA基因的第二轮PCR扩增产物。图注同图1。

Fig. 2 The products of 16S rRNA gene in second round of PCR amplification. Notes are the same as in Fig. 1.

图3 nifH基因的第2轮PCR扩增产物。图注同图1。+、-分别为正负对照。

Fig. 3 The products of nifH gene in second round of PCR amplification. Notes are the same as in Fig. 1. “+” and “-” are positive and negative controls, respectively.

图4 不同土壤样品中细菌16S rRNA基因的变性梯度凝胶电泳(DGGE)图谱。1-22同图1。

Fig. 4 Denaturing gradient gel electrophoresis (DGGE) bands of bacterial 16S rRNA gene in different soil samples. 1-22 are the same as in Fig. 1.

图5 不同土壤样品中细菌16S rRNA基因变性梯度凝胶电泳(DGGE)图谱的聚类分析。1-22同图1。

Fig. 5 Cluster analysis on denaturing gradient gel electrophoresis (DGGE) bands of bacterial 16S rRNA gene in different soil samples. 1-22 are the same as in Fig. 1.

图6 不同土壤样品中细菌群落的多样性(平均值±标准误差)。不同字母表示不同处理间在p < 0.05水平上差异显著。

Fig. 6 Diversity of bacterial communities in different soil samples (mean ± SE). Different letters represent signi?cant differences among treatmens (p < 0.05).

图7 不同土壤样品中固氮细菌nifH基因的变性梯度凝胶电泳图谱。1-22同图1。

Fig. 7 Denaturing gradient gel electrophoresis bands of nitrogen-fixing bacterial nifH gene in different soil samples. 1-22 are the same as in Fig. 1.

图8 不同土壤样品中固氮菌nifH基因变性梯度凝胶电泳图谱的聚类分析。1-22同图1。

Fig. 8 Cluster analysis on denaturing gradient gel electrophoresis bands of nitrogen-fixing bacterial nifH gene in different soil samples. 1-22 are the same as in Fig. 1.

图9 不同土壤样品中固氮菌群落的多样性(平均值±标准误差)。不同字母表示不同处理间在p < 0.05水平上差异显著。

Fig. 9 Diversity of nitrogen-fixing bacteria in different soil samples (mean ± SE). Different letters represent signi?cant differences among treatments (p < 0.05).

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间作大豆对甘蔗根际土壤细菌及固氮菌多样性的影响

Effects of intercropping with soybean on bacterial and nitrogen-fixing bacterial diversity in the rhizosphere of sugarcane

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