International Journal of Systematic Bacteriology (1 999), 49, 51-65 Printed in Great Britain Diversity of rhizobia associated with Amorpha fruticosa isolated from Chinese soils and description of Mesorhizobium amorphae sp. nov. E. T. Wang,lt3 P. van Berkum,2 X. H. SU~,~ D. Beyene,2 W. X. Chen3 and E. Martinez-Romerol Author for correspondence : E. T. Wang. Tel : + 52 73 13 1697. Fax: + 52 73 17558 1. e-mail: ewang@cifn.unam.mx 1 Centro de lnvestigacidn sobre Fijaci6n de Nitrdgeno, UNAM, Apdo Postal 565-A, Cuernavaca, Morelos, Mexico * Alfalfa and Soybean Research Laboratory, Ag ricu I tura I Research Service, US Department of Agriculture, Beltsvi I le, MD 20705, USA Microbiology, College of Biology, China Agricultural University, Beijing 100094, People’s Republic of China 3 Department of Fifty-five Chinese isolates from nodules of Amorpha fruticosa were characterized and compared with the type strains of the species and genera of bacteria which form nitrogen-f ixing symbioses with leguminous host plants. A polyphasic approach, which included RFLP of PCR-amplified 165 rRNA genes, multilocus enzyme electrophoresis (MLEE), DNA-DNA hybridization, 165 rRNA gene sequencing, electrophoretic plasmid profiles, cross-nodulation and a phenotypic study, was used in the comparative analysis. The isolates originated from several different sites in China and they varied in their phenotypic and genetic characteristics. The majority of the isolates had moderate to slow growth rates, produced acid on YMA and harboured a 930 kb symbiotic plasmid (pSym). Five different RFLP patterns were identified among the 16s rRNA genes of all the isolates. Isolates grouped by PCR-RFLP of the 165 rRNA genes were also separated into groups by variation in MLEE profiles and by DNA-DNA hybridization. A representative isolate from each of these DNA homology groups had a separate position in a phylogenetic tree as determined from sequencing analysis of the 165 rRNA genes. A new species, Mesorhizobium amorphae, is proposed for the majority of the isolates, which belonged to a moderately slow- to slow-growing, acid-producing group based upon their distinct phylogenetic position, their unique electrophoretic type, their low DNA homology with reference strains representing the species within the genus Mesorhizobium and their distinct phenotypic features. Strain ACCC 19665 was chosen as the type strain for M. amorphae sp. nov. Keywords : Mesorhizobium amorphae, Amorpha fruticosa, polyphasic taxonomy, diversity, rhizobia INTRODUCTION Low-input sustainable agriculture depends upon the management of leguminous crops because these plants form nitrogen-fixing symbioses with five genera of bacteria belonging to the family Rhizobiaceae (Chen et al., 1988; de Lajudie et al., 1994; Dreyfus et al., 1988; Abbreviations: ET, electrophoretic type; IA, index of association; MLEE, multilocus enzyme electrophoresis. The GenBank accession numbers for the 165 rRNA gene sequences of isolates ACCC 1 9665T, ACCC 19667, H1 5003, HL56 and SH283012, and of M. tianshanense A-1 BST are AF041442-AF041447, respectively. Jarvis et al., 1997; Jordan, 1984). Because of their value in agriculture, many legume species are intro- duced and are cultivated outside their native range. False indigo, Amorpha fruticosa, is a leguminous shrub native to the South-Eastern and Mid-Western United States (Allen & Allen, 198 1) and has been cultivated in Asia for more than half a century. Amorpha fruticosa is useful as a windbreak and as soil cover for erosion control, but also provides food for wildlife. The pods yield amorphin, which is toxic to aphids, cinch bugs and cucumber beetles, and a repellent against cattle flies. In China, the leaves are used as green manure and the seeds are a source of oil used in the manufacture of glycerol. Additional products from this plant may 00806 0 1999 IUMS 51