Genotypic Analysis Of Novel Rhizobial Isolates From Diverse Legume Species Of Agronomic And Ecological Interest Native To Spain India Nepal And Myanmar

P. Vinuesa1'3, A. Willems2, E. Martínez-Romero3, M. León-Barrios5, M. Thynn1'4,

'FB Biologie, Dept of Cell Biology and Applied Botany, Philipps-Universitát Marburg, D-35032, Germany; 2Dept of Microbiology, Ghent University, Belgium; 3Centro de Investigación sobre Fijación de Nitrógeno, UN AM, Cuernavaca, Morelos, Mexico; 4Dept of Microbiology and Cellular Biology, Univ. of La Laguna, Tenerife, Spain; 6Dept of Botany, Univ. of Mandalay, Myanmar; 7Dept of Botany, Maharshi Dayanand Saraswati University, Ajmer, India; 4Dept of Botany, Tribhuvan University, Kiritipur, Kathmandu, Nepal

In the frame of a new INCO-DEV Cooperation Project, we are using molecular DNA fingerprinting techniques and computer-assisted pattern analysis to characterize the diversity of rhizobial strains isolated from the nodules of plants of notable agronomic and ecological interest native to the Canary Islands, India, Myanmar (former Burma) and Nepal. Our database currently contains over 150 isolates from these countries and diverse host genera such as Abrus, Acacia, Adenocarpus, Calopogonium, Chamaecytisus, Crotalaria, Erythrina, Glycine, Lupinus, Prosopis, Sesbania, Spartocytisus, Teline and Vigna. All isolates were first subjected to rep-PCR genomic fingerprinting, a technique suited to distinguish strains of a particular species. Selected strains were then subjected to PCR-RFLP analysis of the rrs, rrl and rDNA IGS regions (Vinuesa et al. 1998, 1999). The resulting patterns were compared with those of most of the currently described rhizobial species, allowing a high-resolution phylogenetic placement of the new isolates to be performed. We are particularly advanced in the elucidation of the genetic and taxonomic affinities of the strains isolated from endemic woody legumes (Papilionoideae:Genisteae) of the Canary Islands. As previously reported (Jarabo-Lorenzo et al. 2000; Vinuesa et al. 1998, 1999), most of these strains form a clade closely related to, but distinct from B. japonicum and B. liaoningense strains. Based on our more recent DNA-DNA hybridization data, MLEE analysis of seven enzyme loci, ITS and partial nodC sequencing, the strains within this clade constitute a distinct genomic species, which is present also in Moroccan soils. These strains do not nodulate Glycine max, nor do B. japonicum strains nodulate the Canarian shrub legumes (Genisteae). The poor correlation found between the Bradyrhizobium strain groupings obtained based on the analysis phenotypic and genotypic traits suggests that a revision of the minimal standards proposed by Graham et al. (1991) for rhizobial species descriptions should be made, giving stronger support to the genotypic data, particularly due to the wealth of sequence data that has accumulated in recent years for many rhizobial genes and strains. Several Acacia and Prosopis isolates from the arid Rajastan region were found to be highly related to S. saheli, others to R. etli and R. hainanense. The Nepalese Glycine max isolates were all closely related, having ITS sequences very similar (> 98%) to those of B. japonicum USDA62 and USDA122. The most divergent isolates were those from tropical legumes growing in SE Myanmar, which represent new species of uncertain taxonomic status. References

Graham et al. (1991) Int. J. Syst. Bacteriol. 41, 582-587 Jarabo-Lorenzo et al. (2000) Syst. Appl. Microbiol. 23, 418-425 Vinuesa et al. (1998) Appl. Environ. Microbiol. 64, 2096-2104

Vinuesa et al. (1999) In Martinez-Romero E, Hernández, G (eds) Highlights on Nitrogen Fixation

Research, pp. 275-279, Plenum Publishing Corporation, NY

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