Biodiversity Science ›› 2018, Vol. 26 ›› Issue (12): 1318-1324.doi: 10.17520/biods.2018184

• Original Papers • Previous Article     Next Article

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 Liu2, *()   

  1. 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:2019-02-11
  • Liu Peigui
  • About author:# 同等贡献作者 Contributed equally to this work

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

Fig. 1

Culturable bacteria from Tuber indicum ascocarps"

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%."

Fig. 3

Rarefaction curve between species and sequences of bacteria from Tuber indicum ascocarps"

Fig. 4

Distribution of bacterial species in phylum (a) and at genus level (b)"

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.
[1] Gui-Feng GAO. (2020) Techniques and methods of microbiomics and their applications . Chin J Plant Ecol, 44(生态技术与方法专辑): 0-0.
[2] Ben-Feng HAN Xin Zhou Xue Zhang. (2020) Verification of virus identity and host association using genomics technologies . Biodiv Sci, 28(5): 0-0.
[3] Hong LUO. (2020) Establishment of in vitro regeneration system of Helenium aromaticum . Chin Bull Bot, 55(3): 0-0.
[4] Sha Deng,Yanni Wu,Kunlin Wu,Lin Fang,Lin Li,Songjun Zeng. (2020) Breeding characteristics and artificial propagation of 14 species of Wild Plant with Extremely Small Populations (WPESP) in China . Biodiv Sci, 28(3): 385-400.
[5] Li Ping,Dong Yahui,Li Chenglong,He Yulong,Li Mingjun. (2020) Optimization of Cell Suspension Culture Conditions of Achyranthes bidentata . Chin Bull Bot, 55(1): 90-95.
[6] Tang Jiali, Qiu Jie, Huang Xuehui. (2020) The Development of Genomics Technologies Drives New Progress in Horticultural Plant Research . Chin Bull Bot, 55(1): 1-4.
[7] Qi Lu,Qiang Hu,Xiaogang Shi,Senlong Jin,Sheng Li,Meng Yao. (2019) Metabarcoding diet analysis of snow leopards (Panthera uncia) in Wolong National Nature Reserve, Sichuan Province . Biodiv Sci, 27(9): 960-969.
[8] Jun Liu, Ning Wang, Daizong Cui, Lei Lu, Min Zhao. (2019) Community structure and diversity of soil bacteria in different habitats of Da Liangzihe National Forest Park in the Lesser Khinggan Mountains . Biodiv Sci, 27(8): 911-918.
[9] Jun Liu, Ning Wang, Daizong Cui, Lei Lu, Min Zhao. (2019) Diversity of bacterial resources in the Greater and Lesser Khinggan Mountains . Biodiv Sci, 27(8): 903-910.
[10] Cao Ning, Xue Dayuan. (2019) On biodiversity conservation by Zhuang traditional culture: A case study in Jingxi City of Guangxi Zhuang Autonomous Region . Biodiv Sci, 27(7): 728-734.
[11] YAN Ya-Nan, YE Xiao-Qi, WU Ming, YAN Ming, ZHANG Xin-Li. (2019) Diversity and potassium-solubilizing activity of rhizosphere potassium-solubilizing bacteria of invasive Solidago canadensis . Chin J Plant Ecol, 43(6): 543-556.
[12] Zhang Wenting,He Yanhong,Shu Ning,Xing Jingjing,Liu Baojun,Bao Manzhu,Liu Guofeng. (2019) Plant Regeneration and Rapid Propagation System of Lilium bakerianum var. aureum . Chin Bull Bot, 54(6): 773-778.
[13] Zhang Xue, Li Xing’an, Su Qinzhi, Cao Qina, Li Chenyi, Niu Qingsheng, Zheng Hao. (2019) A curated 16S rRNA reference database for the classification of honeybee and bumblebee gut microbiota . Biodiv Sci, 27(5): 557-566.
[14] Liu Xiaomei, Sun Lili, Fu Xiangdong, Liao Hong. (2019) An Effective Method for the Rooting of Tea Cuttings . Chin Bull Bot, 54(4): 531-538.
[15] Chen Zhixiang, Yao Xueying, Stephen R. Downie, Wang Qizhi. (2019) Assembling and analysis of Sanicula orthacantha chloroplast genome . Biodiv Sci, 27(4): 366-372.
Full text