用传统分离培养法和高通量测序技术分析印度块菌子囊果内细菌的群落结构

doi:10.17520/biods.2018184

生物多样性 ›› 2018, Vol. 26 ›› Issue (12): 1318-1324. DOI: 10.17520/biods.2018184

• 研究报告: 微生物多样性 • 上一篇下一篇

用传统分离培养法和高通量测序技术分析印度块菌子囊果内细菌的群落结构

邓晓娟¹, 刘建利¹, 闫兴富¹, 刘培贵^2,^*()

1 北方民族大学生物科学与工程学院, 银川 750021
2 中国科学院昆明植物研究所生物多样性与生物地理学重点实验室, 昆明 650201

收稿日期:2018-07-03 接受日期:2018-12-21 出版日期:2018-12-20 发布日期:2019-02-11
通讯作者: 刘培贵
作者简介:# 共同第一作者
基金资助:
北方民族大学重点科研项目(2017KJ27)

Community composition of bacteria associated with ascocarps of Tuber indicum using traditional culture method and Roche 454 high-throughput sequencing

Xiaojuan Deng¹, Jianli Liu¹, Xingfu Yan¹, Peigui Liu^2,^*()

1 College of Biological Science and Engineering, Beifang University of Nationalities, Yinchuan 750021
2 Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201

Received:2018-07-03 Accepted:2018-12-21 Online:2018-12-20 Published:2019-02-11
Contact: Liu Peigui
About author:# 同等贡献作者 Contributed equally to this work

摘要/Abstract

摘要：

块菌是重要的经济真菌, 在其生长发育过程中, 细菌扮演了重要角色。本文利用传统分离培养方法和高通量测序技术分析了印度块菌(Tuber indicum)子囊果内细菌的群落结构。共分离得到细菌532株, 根据物种累积曲线, 选取其中的112株细菌进行了16S rRNA基因序列分析, 共鉴定出4属40种, 其中假单胞菌属(Pseudomonas)菌株占所测菌株数的80%, 不动杆菌属(Acinetobacter)占12.5%, 链霉菌属(Streptomyces)占5%, 贪噬菌属(Variovorax)占2.5%。通过对印度块菌子囊果16S rRNA基因的V1-V3区高通量测序分析, 共获得细菌序列9,862条, 分属于7门43属220种, 其中变形菌门、拟杆菌门和放线菌门的物种占总物种数的99.7%, 是印度块菌子囊果内的优势细菌。黄杆菌属(Flavobacterium)、壤霉菌属(Agromyces)、微杆菌属(Microbacterium)、剑菌属(Ensifer)和寡养食单胞菌属(Stenotrophomonas)的物种数占总物种的86.3%, 是印度块菌子囊果内细菌的优势属。研究结果表明, 采用胰蛋白大豆培养基仅分离得到印度块菌子囊果内少数细菌物种, 而采用高通量测序技术分析发现, 印度块菌子囊果内细菌物种种类丰富, 群落结构复杂。

关键词: 印度块菌, 子囊果, 细菌, 培养, 高通量测序

Abstract

Truffles are important economic fungi, and bacteria play an important role in their growth and development. We investigated the bacterial community composition inside ascocarps of Tuber indicum. Using the traditional culture method, 532 isolates were obtained from ascocarps. Using the rarefaction curve, the 16S rRNA gene was collected and sequenced from 112 purified isolates to identify 4 genera and 40 species. Isolates of the genera Pseudomonas, Acinetobacter, Streptomyces and Variovorax accounted for 80%, 12.5%, 5% and 2.5% of the number of these sequenced isolates, respectively. On the other hand, 9,862 sequences of the bacterial V1-V3 region of 16S rRNA gene, which represented 220 species, were analyzed via Roche 454 high-throughput sequencing. Species of the phyla Proteobacteria, Bacteroidetes, and Actinobacteria were dominant, accounting for 99.7% of all identified species. Genera Flavobacterium, Agromyces, Microbacterium, Ensifer, and Stenotrophomonas were dominant among the bacteria identified with this alternative method, accounting for 86.3% of the number of total species. It was found that relatively few bacterial species were isolated from ascocarps of T. indicum when analyzed via traditional culture method. The bacterial population associated with ascocarps of T. indicum was augmented when analyzed by Roche 454 high-throughput sequencing, which indicates that this latter method provides more comprehensive results.

Key words: Tuber indicum, acocarps, bacteria, culture, high-throughput sequencing

邓晓娟, 刘建利, 闫兴富, 刘培贵 (2018) 用传统分离培养法和高通量测序技术分析印度块菌子囊果内细菌的群落结构. 生物多样性, 26, 1318-1324. DOI: 10.17520/biods.2018184.

Xiaojuan Deng, Jianli Liu, Xingfu Yan, Peigui Liu (2018) Community composition of bacteria associated with ascocarps of Tuber indicum using traditional culture method and Roche 454 high-throughput sequencing. Biodiversity Science, 26, 1318-1324. DOI: 10.17520/biods.2018184.

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链接本文: https://www.biodiversity-science.net/CN/10.17520/biods.2018184

https://www.biodiversity-science.net/CN/Y2018/V26/I12/1318

图/表 4

图1 印度块菌子囊果内可培养细菌

Fig. 1 Culturable bacteria from Tuber indicum ascocarps

图2 基于印度块菌子囊果内可培养细菌16S rRNA基因序列构建的最大简约树。分支上的数字为≥ 50%的靴带值。

Fig. 2 One of most parsimonious trees based on the analysis of 16S rRNA gene sequences of culturable bacteria from Tuber indicum ascocarps. Numbers above branches indicate bootstrap support above 50%.

图3 印度块菌子囊果内细菌的序列数量和物种数量之间的Rarefaction曲线分析

Fig. 3 Rarefaction curve between species and sequences of bacteria from Tuber indicum ascocarps

图4 印度块菌子囊果内细菌物种在不同门的分布(a)和在不同属的分布(b)

Fig. 4 Distribution of bacterial species in phylum (a) and at genus level (b)

参考文献 27

1	Barbieri E, Bertini L, Rossi I, Ceccaroli P, Saltarelli R, Guidi C, Zambonelli A, Stocchi V (2005) New evidence for bacterial diversity in the ascoma of the ectomycorrhizal fungus Tuber borchii Vittad. FEMS Microbiology Letters, 247, 23-35.
2	Barbieri E, Guidi C, Bertaux J, Frey-Klett P, Garbaye J, Ceccaroli P, Saltarelli R, Zambonelli A, Stocchi V (2007) Occurrence and diversity of bacterial communities in Tuber magnatum during truffle maturation. Environmental Microbiology, 9, 2234-2246.
3	Citterio B, Cardoni P, Potenza L, Amicucci A, Stocchi V, Gola G, Nuti M (1995) >Isolation of bacteria from sporocarps of Tuber magnatum Pico, Tuber borchii Vitt. and Tuber maculatum Vitt. In: Biotechnology of Ectomycorrhizae (eds Stocchi V, Bonfante P, Nuti M), pp. 241-248. Plenum Press, New York.
4	Citterio B, Malatesta M, Battistelli S, Marcheggiani F, Baffone W, Saltarelli R, Stocchi V, Gazzanelli G (2001) Possible involvement of Pseudomonas fluorescens and Bacillaceae in structural modifications of Tuber borchii fruit bodies. Canadian Journal of Microbiology, 47, 264-268.
5	Deveau A, Antony-Babu S, Le Tacon F, Robin C, Frey-Klett P (2016) Temporal changes of bacterial communities in the Tuber melanosporum ectomycorrhizosphere during ascocarp development. Mycorrhiza, 26, 389-399.
6	Dib-Bellahouel S, Fortas Z (2014) Activity of the desert truffle Terfezia boudieri Chatin, against associated soil microflora. African Journal of Microbiology Research, 8, 3008-3016.
7	Dorofeev AG, Grigor’eva NV, Kozlov MN, Kevbrina MV, Aseeva VG, Nikolav YA (2014) Approaches to cultivation of “nonculturable” bacteria: Cyclic cultures. Microbiology, 83, 450-461.
8	Fu Y, Li X, Li Q, Wu H, Xiong C, Geng Q, Sun H, Sun Q (2016) Soil microbial communities of three major Chinese truffles in Southwest China. Canadian Journal of Microbiology, 62, 970-979.
9	Gryndler M, Soukupová L, Hršelová H, Gryndlerová H, Borovičika J, Streiblová E, Jansa J (2013) A quest for indigenous truffle helper prokaryotes. Environment Microbiology Reports, 5, 346-352.
10	Gurtler V, Stanisich VA (1996) New approaches to typing and identification of bacteria using the 16S-23S rDNA spacer region. Microbiology, 142, 3-16.
11	Hall TA (1999) BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41, 95-98.
12	Liu PG, Chen J, Deng XJ, Wang XH, Qiao P, Zhang JP, Wan SP, Geng LY, Zhao FL, Zhao WQ, Wang XJ (2016) Truffles from China. Science Press, Beijing. (in Chinese)
	[刘培贵, 陈娟, 邓晓娟, 王向华, 乔鹏, 张介平, 万山平, 耿丽英, 赵峰岚, 赵文青, 王晓进 (2016) 中国的块菌. 科学出版社, 北京.]
13	Mavromatis K, Land M L, Brettin T S, Quest D J, Copeland A, Clum A, Goodwin L, Woyke T, Lapidus A, Klenk HP, Cottingham RW, Kyrpides NC (2012) The fast changing landscape of sequencing technologies and their impact on microbial genome assemblies and annotation. PLoS ONE, 7, e48837.
14	Mello A, Miozzi L, Vizzini A, Napoli C, Kowalchuk G, Bonfante P (2010) Bacterial and fungal communities associated with Tuber magnatum—productive niches. Plant Biosystems, 144, 323-332.
15	Navarro-Ródenas A, Berná L M, Lozano-Carrillo C, Andrino A, Morte A (2016) Beneficial native bacteria improve survival and mycorrhization of desert truffle mycorrhizal plants in nursery conditions. Mycorrhiza, 26, 769-779.
16	Picceri GG, Leonardi P, Iotti M, Gallo M, Baldi F, Zambonelli A, Amicucci A, Vallorani L, Piccoli G, Ciccimarra G, Arshakyan M, Burattini S, Falcieri E, Chiarantini L (2018) Bacteria-produced ferric exopolysaccharide nanoparticles as iron delivery system for truffles (Tuber borchii). Applied Microbiology and Biotechnology, 102, 1429-1441.
17	Qiao P, Tian W, Liu PG, Yu GQ, Chen J, Deng XJ, Wan SP, Wang R, Wang Y, Guo HG (2018) Phylogeography and population genetic analyses reveal the speciation of the Tuber indicum complex. Fungal Genetics and Biology, 113, 14-23.
18	Sbrana C, Bagnoli G, Bedini S, Filippi C, Giovanetti M, Nuti MP (2000) Adhesion to hyphal matrix and antifungal activity of Pseudomonas strains isolated from Tuber borchii ascocarps. Canadian Journal of Microbiology, 46, 259-268.
19	Sbrana C, Agnolucci M, Bedini S, Lepera A, Toffanin A, Giovannetti M, Nuti MP (2002) Diversity of culturable bacterial populations associated to Tuber borchii ectomycorrhizas and their activity on T. borchii mycelial growth. FEMs Microbiology Letters, 211, 195-201.
20	Siqueira JF Jr, Fouad AF, Rôças IN (2012) Pyrosequencing as a tool for better understanding of human microbiomes. Journal of Oral Microbiology, 4, 10743.
21	Streiblová E, Gryndlerová H, Gryndler M (2012) Truffle brûlé: An efficient fungal life strategy. FEMS Microbiology Ecology, 80, 1-8.
22	Soudzilovskaia NA, Douma JC, Akhmetzhanova AA, Van Bodegom PM, Cornwell WK, Moens EJ, Treseder KK, Tibbett M, Wang YP, Cornelissen JHC (2015) Global patterns of plant root colonization intensity by mycorrhizal fungi explained by climate and soil chemistry. Global Ecology and Biogeography, 24, 371-382.
23	Splivallo R, Deveau A, Valdez N, Kirchhoff N, Frey-Klett P, Karlovsky P (2015) Bacteria associated with truffle fruiting bodies contribute to truffle aroma. Environmental Microbiology, 17, 2647-2660.
24	Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular Biology and Evolution, 24, 1596-1599.
25	Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The Clustal_X Windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research, 25, 4876-4882.
26	Wan SP, Liu PG (2014) Diversity of culturable bacteria associated with ascocarps of a Chinese white truffle. Plant Diversity and Resources, 36, 29-36.
27	Yan WR, Zhao TC, Xiao TB, Xiao M, Zhao ZX, Chen MC (2013) Applications of biocontrol bacterial in plant disease control. Genomics and Applied Biology, 32, 533-539.

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用传统分离培养法和高通量测序技术分析印度块菌子囊果内细菌的群落结构

Community composition of bacteria associated with ascocarps of Tuber indicum using traditional culture method and Roche 454 high-throughput sequencing

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