Abstract
Plants are colonized various microorganisms including endophytes. These microbes can play an important role in agricultural production as they promote plant growth and/or enhance the resistance of their host plant against diseases and environmental stress conditions. Although culture-independent molecular approaches such as DNA barcoding have greatly enhanced our understanding of bacterial and fungal endophyte communities, there are some methodical problems when investigating endophyte diversity. One main issue are sequence contaminations such as plastid-derived rRNA gene sequences which are co-amplified due to their high homology to bacterial 16S rRNA genes. The same is true for plant and fungal ITS sequences. The application of highly specific-primers suppressing co-amplification of these sequence contaminations is a good solution for this issue. Here, we describe a detailed protocol for assessing bacterial and fungal endophyte diversity in plants using these primers in combination with next-generation sequencing.
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References
Hardoim PR, van Overbeek LS, Berg G, Pirttila AM, Compant S, Campisano A et al (2015) The hidden world within plants, ecological and evolutionary considerations for defining functioning of microbial endophytes. Microbiol Mol Biol Rev 79:293–320
Lodewyckx C, Vangronsveld J, Porteous F, Moore ERB, Taghavi S, Mezgeay M et al (2002) Endophytic bacteria and their potential applications. Crit Rev Plant Sci 21:583–606
Bulgarelli D, Garrido-Oter R, Münch PC, Weiman A, Dröge J, Pan Y et al (2015) Structure and function of the bacterial root microbiota in wild and domesticated barley. Cell Host Microbe 17:392–403
Gottel NR, Castro HF, Kerley M, Yang Z, Pelletier DA, Podar M et al (2011) Distinct microbial communities within the endosphere and rhizosphere of Populus deltoides roots across contrasting soil types. Appl Environ Microbiol 77:5934–5944
Wemheuer F, Wemheuer B, Kretzschmar D, Pfeiffer B, Herzog S, Daniel R et al (2016) Impact of grassland management regimes on bacterial endophyte diversity differs with grass species. Lett Appl Microbiol 62:323. doi:10.1111/lam.12551
Robinson RJ, Fraaije BA, Clark IM, Jackson RW, Hirsch PR, Mauchline TH (2016) Endophytic bacterial community composition in wheat (Triticum aestivum) is determined by plant tissue type, developmental stage and soil nutrient availability. Plant Soil 405:381
Toju H, Tanabe AS, Yamamoto S, Sato H (2012) High-coverage ITS primers for the DNA-based identification of ascomycetes and basidiomycetes in environmental samples. PLoS One 7:e40863
White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protoc 18:315–322
Chelius MK, Triplett EW (2001) The diversity of Archaea and Bacteria in association with the roots of Zea mays L. Microb Ecol 41:252–263
Lane DJ (1991) 16s/23s rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds) Nucleic acid techniques in bacterial systematics. John Wiley & Sons, New York, NY, pp 115–175
Nübel U, Engelen B, Felske A, Snaidr J, Wieshuber A, Amann RI et al (1996) Sequence heterogeneities of genes encoding 16S rRNAs in Paenibacillus polymyxa detected by temperature gradient gel electrophoresis. J Bacteriol 178:5636–5643
Edgar RC (2010) Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26:2460–2461
Abarenkov K, Henrik Nilsson R, Larsson K-H, Alexander IJ, Eberhardt U, Erland S et al (2010) The UNITE database for molecular identification of fungi – recent updates and future perspectives. New Phytol 186:281–285
Cole JR, Wang Q, Cardenas E, Fish J, Chai B, Farris RJ et al (2009) The Ribosomal Database Project, improved alignments and new tools for rRNA analysis. Nucleic Acids Res 37:D141–D145
Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P et al (2013) The SILVA ribosomal RNA gene database project, improved data processing and web-based tools. Nucleic Acids Res 41:D590–D596
R Core Team (2014) R, a language and environment for statistical computing. Vienna, R Foundation for Statistical Computing. Available at: http://www.R-project.org/
Araujo WL, Marcon J, Maccheroni W Jr, Van Elsas JD, Van Vuurde JW, Azevedo JL (2002) Diversity of endophytic bacterial populations and their interaction with Xylella fastidiosa in citrus plants. Appl Environ Microbiol 68:4906–4914
Edgar RC (2013) UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nat Methods 10:996–998
Edgar RC, Haas BJ, Clemente JC, Quince C, Knight R (2011) UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 27:2194–2200
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Wemheuer, B., Wemheuer, F. (2017). Assessing Bacterial and Fungal Diversity in the Plant Endosphere. In: Streit, W., Daniel, R. (eds) Metagenomics. Methods in Molecular Biology, vol 1539. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6691-2_6
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DOI: https://doi.org/10.1007/978-1-4939-6691-2_6
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