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Applied and Environmental Microbiology, May 2006, p. 3103-3110, Vol. 72, No. 5
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.5.3103-3110.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Diazotrophic Burkholderia Species Associated with Field-Grown Maize and Sugarcane

L. Perin,^1,2, L. Martínez-Aguilar,^3, R. Castro-González,³ P. Estrada-de los Santos,^3, T. Cabellos-Avelar,³ H. V. Guedes,² V. M. Reis,² and J. Caballero-Mellado^3*

Universidade Federal Rural do Rio de Janeiro, km 45, BR 465, C.P. 74505, Seropédica, Rio de Janeiro, Brazil,¹ Embrapa Agrobiology, km 47, BR 465, C.P. 74505, Seropédica, Rio de Janeiro, Brazil,² Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Ap. Postal 565-A, Cuernavaca, Morelos, Mexico³

Received 21 July 2005/ Accepted 15 February 2006

Until recently, diazotrophy was known in only one of the 30 formally described species of Burkholderia. Novel N₂-fixing plant-associated Burkholderia species such as B. unamae, B. tropica, and B. xenovorans have been described, but their environmental distribution is scarcely known. In the present study, the occurrence of N₂-fixing Burkholderia species associated with different varieties of sugarcane and maize growing in regions of Mexico and Brazil was analyzed. Only 111 out of more than 900 isolates recovered had N₂-fixing ability as demonstrated by the acetylene reduction assay. All 111 isolates also yielded a PCR product with primers targeting the nifH gene, which encodes a key enzyme in the process of nitrogen fixation. These 111 isolates were confirmed as belonging to the genus Burkholderia by using a new 16S rRNA-specific primer pair for diazotrophic species (except B. vietnamiensis) and closely related nondiazotrophic Burkholderia. In Mexico, many isolates of B. unamae (predominantly associated with sugarcane) and B. tropica (more often associated with maize) were recovered. However, in Brazil B. tropica was not identified among the isolates analyzed, and only a few B. unamae isolates were recovered from one sugarcane variety. Most Brazilian diazotrophic Burkholderia isolates (associated with both sugarcane and maize plants) belonged to a novel species, as revealed by amplified 16S rRNA gene restriction profiles, 16S rRNA gene sequencing, and protein electrophoresis. In addition, transmissibility factors such as the cblA and esmR genes, identified among clinical and environmental isolates of opportunistic pathogens of B. cenocepacia and other species of the B. cepacia complex, were not detected in any of the plant-associated diazotrophic Burkholderia isolates analyzed.

L. Perin and L. Martínez-Aguilar contributed equally to this study.

Present address: Center of Microbial and Plant Genetics, Katholieke Univesiteit Leuven, Kasteelpark Arenberg 20, B-3001 Heverlee, Belgium.

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