Plant and Soil 257: 35–47, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands. 35 Diazotrophic bacteria associated with banana (Musa spp.). Luc´ ıa Mart´ ınez 1 , Jes´ us Caballero-Mellado 1 , Jos´ e Orozco 2 & Esperanza Mart´ ınez-Romero 1,3 1 Centro de Investigaci´ on sobre Fijaci´ on de Nitr´ ogeno, UNAM. Ap. P. 565-A., Cuernavaca, Mor. M´ exico. 2 Instituto Nacional de Investigaciones Forestales, Agr´ ıcolas y Pecuarias (INIFAP) , Km 35 Carretera Colima-Manzanillo, Ap. P. 88, C.P. 28100, Tecom´ an, Col., M´ exico. 3 Corresponding author ∗ Received 4 July 2002. Accepted in revised form 1 April 2003 Key words: banana, diazotrophs, endophytes, Klebsiella, nitrogen fixation, Rhizobium Abstract Nitrogen-fixing bacteria were isolated from surface sterilized banana (Musa spp.) plants and constituted a minor proportion of banana endophytic bacteria. Some isolates were characterized by alloenzyme profiles, biochemical tests, 16S rRNA and rpoB partial gene sequences, plasmid profiles and plant colonization. A large group of enterobacterial isolates that could not be clearly affiliated, most of them ascribed to group I (with characteristics of Enterobacter cloacae) were the diazotrophs most frequently found in banana. Different Klebsiella spp. and Rhizobium sp. were identified as well. Klebsiella spp. were isolated from inside the roots and stems of plants grown in the two geographical regions sampled and from tissue culture-derived plantlets. Rhizobium sp. isolates were obtained only from Colima where bananas are grown extensively. Group I isolates and Rhizobium sp. could be re-isolated from surface-sterilized banana derived from tissue culture at five months after inoculation and significant increases in stem and leave fresh weight were obtained with some of the isolates. Introduction The isolation and characterization of diazotrophic en- dophytic bacteria from surface sterilized plants is a growing field of research interest and a large phylo- genetic diversity of bacteria associated with agronom- ically important crops such as rice, sugar cane and maize has been found. A rationale for the study of en- dophytic diazotrophs is to extend biological nitrogen fixation to important non-legume crops, thereby dis- placing the use of chemical N fertilizers. The inside of plants constitute adequate habitats for bacterial colon- ization and novel bacterial groups may be encountered therein. Nevertheless, although beneficial effects of associated diazotrophic bacteria on their hosts are known, only in a few cases is the growth promotion due to nitrogen fixation (James, 2000). Plant growth may be stimulated by bacterially-produced phytohor- mones (Okon and Labandera-González, 1994; Tien et al., 1979; Yanni et al., 2001), vitamin-related products (Phillips et al., 1999) or by pathogen supres- ∗ FAX No: 777-317-5581. E-mail:emartine@cifn.unam.mx sion activities (Bashan and de Bashan, 2002; Haque and Ghaffar, 1993). A current problem in the analysis of the effects of the inoculated endophytes on plants is the growth pro- motion mediated by the resident bacteria that masks the effects of the applied inocula. To eliminate endo- phytic bacteria, plants derived from tissue culture with or without antibiotic treatments, are sometimes used (Holland and Polacco, 1994; Leifert et al., 1991). Bananas are propagated vegetatively and tissue culture is a common source of plants for agricultural fields in many places (Robles-González and Orozco- Romero, 1996). Agricultural practices in commercial crops of banana include chemical nitrogen fertilization to increase yield using over 200 kg N ha −1 in many countries including Brazil (Weber et al., 1999). Ba- nana is the most widely consumed fruit in the world and in some countries it is the main source of nutrients. Commercial bananas have a narrow genetic diversity (Powledge, 1996). When this work started there was no information on banana associated nitrogen-fixing bacteria, since then some banana bacterial diazotrophs