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Applied and Environmental Microbiology, April 2002, p. 1595-1603, Vol. 68, No. 4
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.4.1595-1603.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Molecular Method To Assess the Diversity of Burkholderia Species in Environmental Samples

Joana Falcão Salles,1 Francisco Adriano De Souza,2,3 and Jan Dirk van Elsas1*

Plant Research International, 6700 AA Wageningen,1 NIOO-CTO, 6666 ZG Heteren, The Netherlands,2 Embrapa Agrobiologia, Seropédica, Rio de Janeiro, Brazil3

Received 21 June 2001/ Accepted 27 November 2001

In spite of the importance of many members of the genus Burkholderia in the soil microbial community, no direct method to assess the diversity of this genus has been developed so far. The aim of this work was the development of soil DNA-based PCR-denaturing gradient gel electrophoresis (DGGE), a powerful tool for studying the diversity of microbial communities, for detection and analysis of the Burkholderia diversity in soil samples. Primers specific for the genus Burkholderia were developed based on the 16S rRNA gene sequence and were evaluated in PCRs performed with genomic DNAs from Burkholderia and non-Burkholderia species as the templates. The primer system used exhibited good specificity and sensitivity for the majority of established species of the genus Burkholderia. DGGE analyses of the PCR products obtained showed that there were sufficient differences in migration behavior to distinguish the majority of the 14 Burkholderia species tested. Sequence analysis of amplicons generated with soil DNA exclusively revealed sequences affiliated with sequences of Burkholderia species, demonstrating that the PCR-DGGE method is suitable for studying the diversity of this genus in natural settings. A PCR-DGGE analysis of the Burkholderia communities in two grassland plots revealed differences in diversity mainly between bulk and rhizosphere soil samples; the communities in the latter samples produced more complex patterns.

* Corresponding author. Mailing address: Plant Research International, P.O. Box 16, 6700 AA Wageningen, The Netherlands. Phone: 31 317 476210. Fax: 31 317 410113. E-mail: J.D.vanElsas{at}plant.wag-ur.nl.

Applied and Environmental Microbiology, April 2002, p. 1595-1603, Vol. 68, No. 4
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.4.1595-1603.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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