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[1]林先贵,胡君利,戴珏,等.丛枝菌根真菌群落结构与多样性研究方法概述及实例比较[J].应用与环境生物学报,2017,23(2):343-350.[doi:10.3724/SP.J.1145.2016.04008]
　LIN Xiangui**,HU Junli,DAI Jue,et al.Overview and comparison of research methods for determining the community structure and diversity of arbuscular mycorrhizal fungi[J].Chinese Journal of Applied & Environmental Biology,2017,23(2):343-350.[doi:10.3724/SP.J.1145.2016.04008]
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丛枝菌根真菌群落结构与多样性研究方法概述及实例比较()
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《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
23卷
期数:
2017年第2期
页码:
343-350
栏目:
研究论文
出版日期:
2017-04-25

文章信息/Info

Title:
Overview and comparison of research methods for determining the community structure and diversity of arbuscular mycorrhizal fungi
作者:
林先贵; 胡君利; 戴珏; 王发园; 冯有智
1中国科学院南京土壤研究所，土壤与农业可持续发展国家重点实验室 南京 210008 2上海第二工业大学环境与材料工程学院，上海电子废弃物资源化产学研合作开发中心 上海 201209 3青岛科技大学环境与安全工程学院 青岛 266045
Author(s):
LIN Xiangui1**;  HU Junli1;  DAI Jue1;  2;  WANG Fayuan1;  3 & FENG Youzhi1
1State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China 2Shanghai Cooperative Centre for WEEE Recycling, School of Environmental and Materials Engineering, Shanghai Polytechnic University, Shanghai 201209, China 3College of Environment and Safety Engineering, Qingdao University of Science and Technology, Qingdao 266045, China
关键词:
丛枝菌根真菌; 孢子; 形态鉴定; PCR-DGGE; 454高通量测序
Keywords:
arbuscular mycorrhizal fungi;  spore;  morphological identification;  PCR-DGGE;  454 pyrosequencing
分类号:
Q93-3 : S154.36
DOI:
10.3724/SP.J.1145.2016.04008
摘要:
开展丛枝菌根（Arbuscular mycorrhizal，AM）真菌的群落结构与多样性研究对促进菌物多样性的保护与发掘及生态系统的可持续发展均具有重要意义，而研究方法是实现和推进相关研究的重要基础. 本文首先简要回顾AM真菌的研究方法发展历程，介绍基于孢子形态鉴定的形态学方法和以聚合酶链式反应（Polymerase chain reaction，PCR）、变性梯度凝胶电泳（Denaturing gradient gel electrophoresis，DGGE）、454高通量测序为代表的分子生物学方法的检测原理和分析步骤. 然后，结合本实验室利用孢子计数与鉴定、定量PCR与PCR-DGGE以及454高通量基因测序等3套检测手段解析长期不同施肥影响土壤AM真菌群落结构和多样性的研究案例，比较分析不同方法所得AM真菌群落结构与多样性的研究结果. 综合来看，不同方法既有相互印证和互为补充的效果，又有不尽一致甚至互相矛盾的地方. 最后，着重就AM真菌高通量测序技术的升级及与生物标记物方法、同位素标记技术等的联用提出了研究展望. （图5 参35）
Abstract:
Investigating the community structure and diversity of arbuscular mycorrhizal (AM) fungi is important for accelerating the protection and application of mycodiversity, and for the sustainable development of ecosystems. Nevertheless, establishment of related research methods is the key to realizing and promoting such investigations. In this paper, we first provide a very brief historical survey on research methods of AM fungi, and introduce the principles and steps of both morphological and molecular biological methods, including spore morphological identification, polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE), and 454 pyrosequencing. Then, the results related to the long-term fertilization effects on soil AM fungal community structure and diversity revealed by spore calculation and identification, real-time PCR and PCR-DGGE, and 454 pyrosequencing were integrated for method comparison. Altogether, different methods could not only complement each other or have effects of mutual corroboration, but also have in consistencies and contradictions. Finally, the future development of research methods for AM fungi are proposed, including an upgrade of the pyrosequencing technique, and its combined application with biomarker methods, isotope labeling techniques, and others.

参考文献/References:

1 Smith SE, Read DJ. Mycorrhizal Symbiosis [M]. 3rd ed. London: Academic Press, 2008
2 Wyss P, Bonfante P. Amplification of genomic DNA of arbuscular-mycorrhizal (AM) fungi by PCR using short arbitrary primers [J]. Mycol Res, 1993, 97 (11): 1351-1357
3 Kowalchuk GA, De Souza FA, Van Veen JA. Community analysis of arbuscular mycorrhizal fungi associated with Ammophila arenaria in Dutch coastal sand dunes [J]. Mol Ecol, 2002, 11 (3): 571-581
4 Alkan N, Gadkar V, Coburn J, Yarden O, Kapulnik Y. Quantification of the arbuscular mycorrhizal fungus Glomus intraradices in host tissue using real-time polymerase chain reaction [J]. New Phytol, 2004, 161 (3): 877-885
5 ?pik M, Metsis M, Daniell TJ, Zobel M, Moora M. Large-scale parallel 454 sequencing reveals host ecological group specificity of arbuscular mycorrhizal fungi in a boreonemoral forest [J]. New Phytol, 2009, 184 (2): 424-437
6 郑世学, 董秀丽, 喻子牛, 赵斌. 以新鲜根段进行AM真菌的分子检测及竞争性侵染研究[J]. 菌物学报, 2004, 23 (1): 126-132 [Zheng SX, Dong XL, Yu ZN, Zhao B. Molecular detection of competitive colonization of AM fungi within fresh roots [J]. Mycosystema, 2004, 23 (1): 126-132]
7 龙良鲲, 羊宋贞, 姚青, 朱红惠. AM真菌DNA的提取与PCR-DGGE分析[J]. 菌物学报, 2005, 24 (4): 564-569 [Long LK, Yang SZ, Yao Q, Zhu HH. DNA extraction from arbuscular mycorrhizal fungi and analysis by PCR- denaturing gradient gel electrophoresis [J]. Mycosystema, 2005, 24 (4): 564-569]
8 Zhu HH, Yao Q, Sun XT, Hu YL. Colonization, ALP activity and plant growth promotion of native and exotic arbuscular mycorrhizal fungi at low pH [J]. Soil Biol Biochem, 2007, 39 (4): 942-950
9 Shi P, Abbott LK, Banning NC, Zhao B. Comparison of morphological and molecular genetic quantification of relative abundance of arbuscular mycorrhizal fungi within roots [J]. Mycorrhiza, 2012, 22 (7): 501-513
10 Dumbrell AJ, Ashton PD, Aziz N, Feng G, Nelson M, Dytham C, Fitter AH, Helgason T. Distinct seasonal assemblages of arbuscular mycorrhizal fungi revealed by massively parallel pyrosequencing [J]. New Phytol, 2011, 190 (3): 794-804
11 Dai M, Hamel C. Arnaud M, He Y, Grant C, Lupwayi N, Janzen H, Maihi SS, Yang X, Zhou Z. Arbuscular mycorrhizal fungi assemblages in Chernozem great groups revealed by massively parallel pyrosequencing [J]. Can J Microbiol, 2012, 58 (1): 81-92
12 Lin X, Feng Y, Zhang H, Chen R, Wang J, Zhang J, Chu H. Long-term balanced fertilization decreases arbuscular mycorrhizal fungal diversity in an arable soil in North China revealed by 454 pyrosequencing [J]. Environ Sci Technol, 2012, 46 (11): 5764-5771
13 Merryweather J, Fitter A. The arbuscular mycorrhizal fungi of Hyacinthoides non-scripta I. Diversity of fungal taxa [J]. New Phytol, 1998, 138 (1): 117-129
14 Simon L, Lalonde M, Bruns TD. Specific amplification of 18S fungal ribosomal genes from vesicular-arbuscular endomycorrhizal fungi colonizing roots [J]. Appl Environ Microbiol, 1992, 58 (1): 291-295
15 Helgason T, Daniell TJ, Husband R, Fitter AH, Young JPW. Ploughing up the wood-wide web? [J] Nature, 1998, 394 (6692): 431-431
16 van Tuinen D, Jacquot E, Zhao B, Gollotte A, Gianinazzi-Pearson V. Characterization of root colonization profiles by a microcosm community of arbuscular mycorrhizal fungi using 25S rDNA-targeted nested PCR [J]. Mol Ecol, 1998, 7 (7): 879-887
17 Trouvelot S, van Tuinen D, Hijri M, Gianinazzi-Pearson V. Visualization of ribosomal DNA loci in spore interphasic nuclei of glomalean fungi by fluorescence in situ hybridization [J]. Mycorrhiza, 1999, 8 (4): 203-206
18 Gollotte A, van Tuinen D, Atkinson D. Diversity of arbuscular mycorrhizal fungi colonizing roots of the grass species Agrostis capillaris and Lolium perenne in a field experiment [J]. Mycorrhiza, 2004, 14 (2): 111-117
19 Sato K, Suyama Y, Saito M, Sugawara K. A new primer for discrimination of arbuscular mycorrhizal fungi with polymerase chain reaction-denature gradient gel electrophoresis [J]. Grassland Sci, 2005, 51 (2): 179-181
20 Lee J, Lee S, Young JPW. Improved PCR primers for the detection and identification of arbuscular mycorrhizal fungi [J]. FEMS Microbiol Ecol, 2008, 65 (2): 339-349
21 Krüger M, Stockinger H, Krüger C, Schü?ler A. DNA-based species level detection of Glomeromycota: one PCR primer set for all arbuscular mycorrhizal fungi [J]. New Phytol, 2009, 183 (1): 212-223
22 Gorzelak MA, Holland TC, Xing X, Hart MM. Molecular approaches for AM fungal community ecology: a primer [J]. J Microbiol Meth 2012, 90 (2): 108-114
23 Jiang S, Shi G, Mao L, Pan J, An L, Liu Y, Feng H. Comparison of different PCR primers on detecting arbuscular mycorrhizal communities inside plant roots [J]. Acta Microbiol Sin, 2015, 55 (7): 916-925
24 钦绳武, 顾益初, 朱兆良. 潮土肥力演变与施肥作用的长期定位试验初报[J]. 土壤学报, 1998, 35 (3): 367-375 Qin SW, Gu YC, Zhu ZL, 1998. [A preliminary report on long-term stationary experiment on fertility evolution of fluvo-aquic soil and the effect of fertilization [J]. Acta Pedol Sin, 1998, 35 (3): 367-375]
25 Wang FY, Hu JL, Lin XG, Qin SW, Wang JH. Arbuscular mycorrhizal fungal community structure and diversity in response to long-term fertilization: a field case from China [J]. World J Microbiol Biotechnol, 2011, 27 (1): 67-74
26 Dai J, Hu J, Lin X, Yang A, Wang R, Zhang J, Wong MH. Arbuscular mycorrhizal fungal diversity, external mycelium length, and glomalin-related soil protein content in response to long-term fertilizer management [J]. J Soil Sediment, 2013, 13 (1): 1-11
27 Schü?ler A, Walker C. The Glomeromycota: A Species List with New Families and New Genera [M]. Charleston: Createspace Independent Publishing Platform, 2010. www.amf-phylogeny.com
28 Hu J, Lin X, Wang J, Chu H, Yin R, Zhang J. Population size and specific potential of P-mineralizing and -solubilizing bacteria under long-term P-deficiency fertilization in a sandy loam soil [J]. Pedobiologia, 2009, 53 (1): 49-58
29 金樑, 赵洪, 李博. 我国菌根研究进展及展望[J]. 应用与环境生物学报, 2004, 10 (4): 515-520 [Jin L, Zhao H, Li B. Mycorrhizal studies in China [J]. Chin J Appl Envion Biol, 2004, 10 (4): 515-520]
30 Grilli G, Urcelay C, Galetto L, Davison J, Vasar M, Saks ?, Jairus T, ?pik M. The composition of arbuscular mycorrhizal fungal communities in the roots of a ruderal forb is not related to the forest fragmentation process [J]. Environ Microbiol, 2015, 17 (8): 2709-2720
31 Schmidt PA, Bálint M, Greshake B, Bandow C, R?mbke J, Schmitt I. Illumina metabarcoding of a soil fungal community [J]. Soil Biol Biochem, 2013, 65: 128-132
32 Cui X, Hu J, Wang J, Yang J, Lin X. Reclamation negatively influences arbuscular mycorrhizal fungal community structure and diversity in coastal saline-alkaline land in Eastern China as revealed by Illumina sequencing [J]. Appl Soil Ecol, 2016, 98: 140-149
33 Mosher JJ, Bowman B, Bernberg EL, Shevchenko O, Kan J, Korlach J, Kaplan LA. Improved performance of the PacBio SMRT technology for 16S rDNA sequencing [J]. J Microbiol Meth, 2014, 104: 59-60
34 Grigera MS, Drijber RA, Shores-Morrow RH, Wienhold BJ. Distribution of the arbuscular mycorrhizal biomarker C16: 1cis11 among neutral, glyco and phospholipids extracted from soil during the reproductive growth of corn [J]. Soil Biol Biochem, 2007, 39 (7): 1589-1596
35 Hannula SE, Boschker HTS, de Boer W, Van Veen JA. 13C pulse-labeling assessment of the community structure of active fungi in the rhizosphere of a genetically starch-modified potato (Solanum tuberosum) cultivar and its parental isoline [J]. New Phytol, 2012, 194 (3): 784-799

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更新日期/Last Update: 2017-04-25