Characterization of bacteria isolated from wild legumes in the north-western regions of China

1 Department of Microbiology, College of Biological Science, China Agricultural University, Beijing 100094, PR China 2 Centro de Investigación sobre Fijación de Nitrógeno, UNAM Cuernavaca PA 565-A, Morelos, Mexico 3 Department of Agrochemistry, NorthWestern Agricultural University, Yangling 712100, Shaanxi Province, PR China Author for correspondence: Wen Xin Chen. Tel: +86 10 62891854. Fax: +86 10 62891055. e-mail: wenxin_chen{at}263.net ABSTRACT Nodule isolates from 11 species of wild legumes in north-western China were characterized by numerical taxonomy, PCR-based 16S rRNA gene RFLP and sequence analyses, DNA-DNA hybridization, restriction patterns of nodDAB and nifH genes, and symbiotic properties. Based on the results of numerical taxonomy, most of the 35 new isolates were grouped into five clusters (clusters 7, 9, 12, 14 and 15). Clusters 7 and 12 were identified as Mesorhizobium amorphae and Agrobacterium tumefaciens , respectively, based on their high DNA homologies with the reference strains for these species, their 16S rRNA gene analysis and their phenotypic features. Results of 16S rDNA PCR-RFLP analysis showed that cluster 9 belonged to Rhizobium . Clusters 14 and 15 were identified as Mesorhizobium based on their moderately slow-growing, acid-producing characters and the high similarity of their 16S rDNA PCR-RFLP patterns to those of Mesorhizobium species. These two clusters were genomic species distinct from all described species based on analysis of DNA relatedness within this genus. The isolates in cluster 12 ( Agrobacterium tumefaciens ) failed to nodulate their original host and other selected hosts and they did not hybridize to nif or nod gene probes. The possibility of opportunistic nodulation of these isolates is discussed. Identical restriction patterns were obtained in the nif or nod gene hybridization studies from the three isolates within cluster 15, which were isolated from the same host species. The isolates from different host plants in each of clusters 9 and 14 produced different nodDAB RFLP patterns, but similar nifH RFLP patterns appeared (one band for each isolate). Different patterns were observed among different clusters from both the nod and nif gene hybridization studies. Cross-nodulation was recorded among the isolates and the host plants in the same cluster and promiscuous properties were found among some of the hosts tested. Key Words: wild leguminous plants • diversity of rhizobia • polyphasic taxonomy