应用磷脂脂肪酸和聚合酶链式反应-变性梯度凝胶电泳分析技术研究湿地植物根际微生物群落多样性

doi:10.3724/SP.J.1258.2013.00078

摘要/Abstract

摘要：

以天津大学校内两个相邻的小型湖泊(青年湖和爱晚湖)为研究区域, 通过采样分析, 利用磷脂脂肪酸(PLFA)和聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)分析技术, 研究了湿地植物种类(芦苇(Phragmites australis)和东方香蒲(Typha orientalis))和生长方式(单生和混生)对根际微生物生物量和群落结构的影响。PLFA分析结果表明, 植物根际微生物生物量大于非根际(爱晚湖芦苇除外); 植物种间的差异较大, 东方香蒲根际沉积物中微生物生物量大于芦苇根际; 种内根际微生物受植物的生长状况影响较大, 采样期间两个湖泊中东方香蒲的生长状况(株高)相似, 根际微生物生物量相差不大, 而爱晚湖芦苇由于与东方香蒲共生, 受到东方香蒲的抑制, 使得根际微生物生物量明显低于单独生长的芦苇; 革兰氏阳性细菌数量小于革兰氏阴性细菌的数量, 且根际的革兰氏阳性细菌与革兰氏阴性细菌的比值小于非根际。沉积物中的细菌群落结构主要与植物种类有关, 同一种植物的根际细菌群落结构差异较小(这些根际细菌聚为一类); 不同植物的根际细菌群落结构差异较大。

关键词: 变性梯度凝胶电泳, 微生物多样性, 磷脂脂肪酸, 植物根际, 湿地

Abstract:

Aims Our objective was to investigate the effect of plant growth pattern (single or mixed) of two emergent plants (i.e., Phragmites australis and Typha orientalis) on the microbial biomass and community structure of the rhizosphere in two adjacent shallow lakes in Tianjin, China.
Methods The microbial biomass and community structure were assayed by using phospholipid fatty acid (PLFA) and polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) techniques. Cluster analysis of the microbial PLFA proﬁles was performed using hierarchical clustering according to the between-groups linkage method using the software package SPSS 13.0 for Windows.
Important findings The PLFA results showed that microbial biomass in the plant rhizosphere was higher than those in the non-rhizosphere, except for the P. australis rhizosphere in Aiwan Lake. The microbial biomass was significantly higher in the T. orientalis rhizosphere sediments than that in the P. australis rhizosphere sediments in both lakes. The microbial biomass of the same plant’s rhizosphere was influenced by pattern of plant growth (single or mixed). As the growth condition (plant height) was similar in the two lakes, there was only minor difference in the microbial biomass of the T. orientalis rhizosphere. When the two plants grew together, the growth of P. australis was significantly inhibited, and significantly lower microbial biomass was assayed than that under the separated growing condition. The gram-positive bacteria (G⁺) were less than that of the gram-negative bacteria (G^-) in all samples. Moreover, the ratio of G⁺ to G^-in the plant rhizosphere was less than in the non-rhizosphere. Microbial community structure was found to be related to the plant species. Little difference in microbial community structure was observed in same plant rhizosphere sediments between the two lakes; however, apparent differences in the bacterial community were detected between the two different plant species.

Key words: denaturing gradient gel electrophoresis (DGGE), microbial diversity, phospholipid fatty acid (PLFA), plant rhizosphere, wetland

王爱丽. 应用磷脂脂肪酸和聚合酶链式反应-变性梯度凝胶电泳分析技术研究湿地植物根际微生物群落多样性. 植物生态学报, 2013, 37(8): 750-757. DOI: 10.3724/SP.J.1258.2013.00078

WANG Ai-Li. Microbial community diversity in the rhizosphere of wetland plants examined by phospholipid fatty acid and polymerase chain reaction denaturing gradient gel electrophoresis. Chinese Journal of Plant Ecology, 2013, 37(8): 750-757. DOI: 10.3724/SP.J.1258.2013.00078

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链接本文: https://www.plant-ecology.com/CN/10.3724/SP.J.1258.2013.00078

https://www.plant-ecology.com/CN/Y2013/V37/I8/750

图/表 6

图1 沉积物样品中磷脂脂肪酸组成。A, 爱晚湖; N, 非根际; PR, 芦苇根际; Q, 青年湖; TR, 东方香蒲根际。

Fig. 1 Composition of phospholipid fatty acid in sediment samples. A, Aiwan Lake; N, non-rhizosphere; PR, Phragmites australis rhizosphere; Q, Qingnian Lake; TR, Typha orientalis rhizosphere.

表1 沉积物中磷脂脂肪酸含量(nmol·g-1干重)

Table 1 Contents of phospholipid fatty acids in sediments (nmol·g-1 dry weight)

	青年湖 Qingnian Lake			爱晚湖 Aiwan Lake
	东方香蒲根际 Typha orientalis rhizosphere	芦苇根际 Phragmites australis rhizosphere	非根际 Non- rhizosphere	东方香蒲根际 Typha orientalis rhizosphere	芦苇根际 Phragmites australis rhizosphere	非根际 Non- rhizosphere
微生物生物量 Microbial biomass	44.17^B(2.19)	12.35^C(1.48)	5.99^D(0.79)	51.17^A(3.19)	3.52^E(0.25)	3.93^E(0.27)
革兰氏阳性细菌 Gram-positive bacteria	8.65^A (0.85)	2.17^B(0.12)	2.10^C(0.08)	26.40^A(2.30)	1.86^C(0.06)	1.78^C(0.05)
革兰氏阴性细菌 Gram-negative bacteria	14.21^B (0.13)	3.81^C(0.05)	-	2.35^A(0.22)	-	-
阳性菌与阴性菌的比值 Ratio of gram-positive bacteria to gram-negative bacteria	0.61^B (0.05)	0.57^B(0.03)	0.83^C(0.02)	8.11^A(0.75)	0.46^E(0.01)	0.70^D(0.01)
细菌生物量 Bacterial biomass	24.91^A(2.15)	6.20^B(0.52)	1.12^E(0.03)	17.39^A(1.45)	1.30^D (0.04)	0.93^F(0.02)
真菌生物量 Fungal biomass	2.99^A (0.25)	0.77^B(0.05)	0.74^A(0.0.2)	0.47^C(0.01)	0.35^D(0.01)	0.75^A(0.03)

图2 不同沉积物样品的变性梯度凝胶电泳分离图。从左到右分别为青年湖东方香蒲根际、青年湖芦苇根际、青年湖非根际、爱晚湖东方香蒲根际、爱晚湖非根际、爱晚湖芦苇根际。图中的数字是条带的编号。

Fig. 2 Denaturing gradient gel electrophoresis separation of different sediment samples. From left to right are Typha orientalis rhizosphere in Qingnian Lake, Phragmites australis rhizosphere in Qingnian Lake, non-rhizosphere in Qingnian Lake, Typha orientalis rhizosphere in Aiwan Lake, non-rhizosphere in Aiwan Lake, Phragmites australis rhizosphere in Aiwan Lake, respectively. The numbers in figure refer to the number of bands.

表2 不同沉积物中微生物群落的多样性指数

Table 2 Diversity index of microbial community in different sediments

	青年湖 Qingnian Lake			爱晚湖 Aiwan Lake
	东方香蒲根际 Typha orientalis rhizosphere	芦苇根际 Phragmites australis rhizosphere	非根际 Non- rhizosphere	东方香蒲根际 Typha orientalis rhizosphere	芦苇根际 Phragmites australis rhizosphere	非根际 Non- rhizosphere
磷脂脂肪酸 Phospholipid fatty acids	2.39^A(0.20)	2.20^A(0.18)	1.72^B(0.15)	2.25^A(0.18)	1.79^B (0.12)	1.35^C(0.09)
变性梯度凝胶电泳 Denaturing gradient gel electrophoresis	2.46^A (0.21)	2.32^A(0.19)	1.68^B(0.15)	2.87^A(0.22)	2.61^A (0.20)	1.66^B(0.13)

图3 沉积物样品中微生物群落磷脂脂肪酸数据聚类分析。图中的数字指聚类分析中的组间平均距离, 单位为cm。

Fig. 3 Cluster analysis of phospholipid fatty acid data of microbial community from sediment samples. The numbers in figure refer to average distance between the groups in cluster analysis and the unit of the number is centimeter.

图4 沉积物样品中微生物群落变性梯度凝胶电泳条带聚类分析。图中的数字指聚类分析中的组间变性梯度凝胶电泳条带的相似程度, 例如0.81代表相似度为81%。

Fig. 4 Cluster analysis of denaturing gradient gel electrophoresis bands of microbial community from sediment samples. The numbers in figure refer to the similarity among denaturing gradient gel electrophoresis bands between the groups in cluster analysis. For example, 0.81 refers to 81% of similarity.

参考文献 26

[1]	Berg G, Roskot N, Steidle A, Eberl L, Zock A, Smalla K (2002). Plant-dependent genotypic and phenotypic diver- sity of antagonistic rhizobacteria isolated from different Verticillium host plants. Applied and Environmental Microbiology, 68, 3328-3338. URL PMID
[2]	Chi J, Yang R, Wang AL (2012). Effect of wetland plant species and growth strategy on the distribution of PAEs and their monoester metabolites in the rhizosphere. Journal of Lake Science, 24, 416-421. (in Chinese with English abstract)
	[ 迟杰, 杨瑞, 王爱丽 (2012). 湿地植物种类和生长方式对根际酞酸酯及其单酯代谢物分布特征的影响. 湖泊科学, 24, 416-421.]
[3]	Dong XL, Reddy GB (2010). Soil bacterial communities in constructed wetlands treated with swine wastewater using PCR-DGGE technique. Bioresource Technology, 101, 1175-1182. DOI URL PMID
[4]	Fan SX, Li PJ, Gong ZQ, Ren WX, He N (2008). Promotion of pyrene degradation in rhizosphere of alfalfa (Medicago sativa L.). Chemosphere, 71, 1593-1598. DOI URL PMID
[5]	Haberl R, Grego S, Langergraber G, Kadlec RH, Cicalini AR, Dias SM, Novais JM, Aubert S, Gerth A, Thomas H, Hebner A (2003). Constructed wetlands for the treatment of organic pollutants. Journal of Soils and Sediments, 3, 109-124.
[6]	Hallberg KB, Johnson BD (2005). Microbiology of a wetland ecosystem constructed to remediate mine drainage from a heavy metal mine. Science of the Total Environment, 338, 53-66. DOI URL PMID
[7]	Heinrich D, Hess D (1985). Chemotactic attraction of Azospirillum lipoferum by wheat roots and characterization of some attractants. Canadian Journal of Microbiology, 31, 26-31.
[8]	Iwamoto T, Tani K, Nakamura K, Suzuki Y, Kitagawa M, Equchi M, Nasu M (2000). Monitoring impact of in suit biostimulation treatment on ground water bacterial community by DGGE. FEMS Microbiology Ecology, 32, 129-141. DOI URL PMID
[9]	Jenkins MB, Lion LW (1993). Mobile bacteria and transport of poly-nuclear aromatic hydrocarbons in porous media. Applied and Environmental Microbiology, 59, 3306-3313. DOI URL PMID
[10]	Li M, Zhou QH, Tao M, Wang Y, Jiang LJ, Wu ZB (2010). Comparative study of microbial community structure in different filter media of constructed wetland. Journal of Environmental Sciences, 22, 127-133. DOI URL
[11]	Li RH, Guan YT, He M, Hu HY, Jiang ZP (2006). Pilot- scale study on riparian mixed plant zones treating polluted river water. Environmental Science, 27, 651-654 (in Chinese with English abstract)
	[ 李睿华, 管运涛, 何苗, 胡洪营, 蒋展鹏 (2006). 河岸混合植物带处理受污染河水中试研究. 环境科学, 27, 651-654.]
[12]	Liu M, Yang Y, Xu S, Liu H, Hou L, Ou D, Liu Q, Cheng S (2006). HCHs and DDTs in salt marsh plants (Scirpus) from the Yangtze estuary and nearby coastal areas, China. Chemosphere, 62, 440-448. DOI URL PMID
[13]	Miglioranza KSB, de Moreno JEA, Moreno VJ (2004). Organochlorine pesticides sequestered in the aquatic macrophyte Schoenoplectus californicus (C.A. Meyer) Soják from a shallow lake in Argentina. Water Research, 38, 1765-1772. DOI URL PMID
[14]	Ruiz-Rueda O, Hallin S, Baneras L (2009). Structure and function of denitrifying and nitrifying bacteria communities in relation to the plant species in a constructed wetland. FEMS Microbiology Ecology, 67, 308-319. DOI URL PMID
[15]	Sirivedhim T, Gray KA (2006). Factors affecting denitrification rates in experimental wetlands: field and laboratory studies. Ecological Engineering, 26, 167-181. DOI URL
[16]	Smalla K, Wieland G, Buchner A, Zock A, Parzy J, Kaiser S, Roskot N, Heuer H, Berg G (2001). Bulk and rhizosphere soil bacterial communities studied by denaturing gradient gel electrophoresis: plant-dependent enrichment and seasonal shifts revealed. Applied and Environmental Microbiology, 67, 4742-4751. URL PMID
[17]	Tawney L, Becker JG, Baldwin AH (2008). A novel dual-compartment, continuous-flow wetland microcosm to assess cis-dichloroethene removal from the rhizosphere. International Journal of Phytoremediation, 10, 455-471. URL PMID
[18]	Toyama T, Sato Y, Inoue D, Sei K, Chang YC, Kikuchi S, Ike M (2009) Biodegradation of bisphenol A and bisphenol F in the rhizosphere sediment of Phragmites australis. Journal of Bioscience and Bioengineering, 108, 147-150. DOI URL PMID
[19]	Wang AL (2011). Dissipation Behaviors of Phthalic Acid Esters in the Rhizosphere of Wetland Plants. PhD dissertation, Tianjin University, Tianjin. (in Chinese with English abstract)
	[ 王爱丽 (2011). 酞酸酯在湿地植物根际环境中的消减行为. 博士学位论文, 天津大学, 天津.]
[20]	Xiang XM, Song CX, Li YS, Sun XY (2004). Microorganism features of Typha Latifolia and Phragmites Australis at rhizosphere. Chinese Journal of Environmental Protection Science, 30, 35-38. (in Chinese with English abstract)
	[ 项学敏, 宋春霞, 李彦生, 孙祥宇 (2004). 湿地植物芦苇和东方香蒲根际微生物特性研究. 环境保护科学, 30, 35-38.]
[21]	Xu XL, Lu XX, Lei XD, Cao LK (2012). Effects of hydrophytes on removal of nitrogen and phosphorus in eutrophic water. Journal of Shanghai Jiaotong University (Agricultural Science), 30, 8-14. (in Chinese with English abstract)
	[ 徐秀玲, 陆欣欣, 雷先德, 曹林奎 (2012). 不同水生植物对富营养化水体中氮磷去除效果的比较. 上海交通大学学报(农业科学版), 30, 8-14.]
[22]	Xue D, Yao HY, Ge DY, Huang CY (2008). Soil microbial community structure in diverse land use systems: a comparative study using Biolog, DGGE, and PLFA analyses. Pedosphere, 18, 653-663. DOI URL
[23]	Yang R (2011) The Studies on Microbial Ecology of the Rhizosphere of Emergent Plants. Master degree dissertation, Tianjin University, Tianjin. (in Chinese with English abstract)
	[ 杨瑞. 挺水植物根际微生物生态研究. 硕士学位论文, 天津大学, 天津.]
[24]	Zelles L (1997). Phospholipid fatty acid profiles in selected members of soil microbial communities. Chemosphere, 35, 275-294. DOI URL PMID
[25]	Zhang QR, Zhou QX, Ren LP, Zhu YG, Sun SL (2006). Ecological effects of crude oil residues on the functional diversity of soil microorganisms in three weed rhizospheres. Journal of Environmental Sciences, 18, 1101-1106. DOI URL
[26]	Zhao XG (2011). Effect of Emergent Plants on the Circulation of Nitrogen and Phosphorus of Eutrophic Lakes. Master degree dissertation, Tianjin University, Tianjin. (in Chinese with English abstract)
	[ 赵旭光. 挺水植物对富营养化湖泊水体中氮磷循环的影响. 硕士学位论文, 天津大学, 天津.]