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Isothermal DNA amplification facilitates the identification of a broad spectrum of bacteria, fungi and protozoa in Eleutherococcus sp. plant tissue cultures

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Abstract

In vitro cultures of Eleutherococcus sieboldianus originating from surface sterilized leaf explants were found to be associated with several microorganisms. The associations included bacteria, fungi and protozoa within the rhizosphere and inside root hairs. To determine if this phenomenon is unique to this species, plant tissue cultures of E. gracilistylus and E. senticosus were included in our studies for comparison. A methodology consisting of isothermal amplification, cloning and sequencing was established for analysing 16S ribosomal DNA of cultivated and non-cultivated bacteria from different tissue types. The same methodology was used to obtain internal transcribed spacer regions and 18S regions of fungal and protozoan rDNA. Comparative analyses of sequencing data resulted in the identification of various genera within the Firmicutes and γ-proteobacteria kingdoms and a broad spectrum of fungal genera related to several uncultured fungi. In addition, amoebal and chrysophyte species were detected. Most of the species were identified in different plant organs and in in vitro culture cell types indicating the microorganisms are systemically distributed. The presence of identical microorganisms in different plant species argues for an evolutionary long-lasting and stable association between the plant genus and the microinhabitants.

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Acknowledgments

We like to thank Felix Kirchner and Maritha Lippmann, financially supported by the EU project JCA4-CT-2001-10057, for excellent technical assistance, and Andreas Brune (MPI for Terrestrial Microbiology, Marburg) for fruitful discussion and for the calculation of sequence alignments. We owe many thanks to Dave Westenberg, Missouri University of Science and Technology Rolla, USA, for correcting this manuscript. This work was financially supported by the SFB 395 project to P. Müller.

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  1. Fachbereich Biologie, Zellbiologie, Philipps Universität Marburg, Karl-von-Frisch-Str. 8, 35032, Marburg, Germany

    Peter Müller

  2. Fachbereich Biologie, Spezielle Botanik, Philipps Universität Marburg, Karl-von-Frisch-Str. 8, 35032, Marburg, Germany

    Matthias Döring

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Müller, P., Döring, M. Isothermal DNA amplification facilitates the identification of a broad spectrum of bacteria, fungi and protozoa in Eleutherococcus sp. plant tissue cultures. Plant Cell Tiss Organ Cult 98, 35–45 (2009). https://doi.org/10.1007/s11240-009-9536-8

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