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Applied and Environmental Microbiology, March 2004, p. 1787-1794, Vol. 70, No. 3
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.3.1787-1794.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Analysis of the Endophytic Actinobacterial Population in the Roots of Wheat (Triticum aestivum L.) by Terminal Restriction Fragment Length Polymorphism and Sequencing of 16S rRNA Clones

Vanessa M. Conn and Christopher M. M. Franco^*

Department of Medical Biotechnology, Flinders University, Bedford Park, South Australia 5042, Australia

Received 17 September 2003/ Accepted 3 December 2003

The endophytic actinobacterial population in the roots of wheat grown in three different soils obtained from the southeast part of South Australia was investigated by terminal restriction fragment length polymorphism (T-RFLP) analysis of the amplified 16S rRNA genes. A new, validated approach was applied to the T-RFLP analysis in order to estimate, to the genus level, the actinobacterial population that was identified. Actinobacterium-biased primers were used together with three restriction enzymes to obtain terminal restriction fragments (TRFs). The TRFs were matched to bacterial genera by the T-RFLP Analysis Program, and the data were analyzed to validate and semiquantify the genera present within the plant roots. The highest diversity and level of endophytic colonization were found in the roots of wheat grown in a dark loam from Swedes Flat, and the lowest were found in water-repellent sand from Western Flat. This molecular approach detected a greater diversity of actinobacteria than did previous culture-dependent methods, with the predominant genera being Mycobacterium (21.02%) in Swedes Flat, Streptomyces (14.35%) in Red Loam, and Kitasatospora (15.02%) in Western Flat. This study indicates that the soil that supported a higher number of indigenous organisms resulted in wheat roots with higher actinobacterial diversity and levels of colonization within the plant tissue. Sequencing of 16S rRNA clones, obtained using the same actinobacterium-biased PCR primers that were used in the T-RFLP analysis, confirmed the presence of the actinobacterial diversity and identified a number of Mycobacterium and Streptomyces species.

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* Corresponding author. Mailing address: Department of Medical Biotechnology, Flinders University, Bedford Park, South Australia 5042, Australia. Phone: 61 8 8204 5764. Fax: 61 8 8204 4101. E-mail: Chris.Franco{at}flinders.edu.au.

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Applied and Environmental Microbiology, March 2004, p. 1787-1794, Vol. 70, No. 3
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.3.1787-1794.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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