System. Appl. Microbiol. 22, 378-386 (1999) SVSTEI\A4fIC AND © Urban & Fischer Verlag ___ _______________________________ Phylogeny and Diversity of Bradyrhizobium Strains Isolated from the Root Nodules of Peanut (Arachis hypogaeaJ in Sichuan, China XIAOPING ZHANG 1 ,2, GISELLE NICKI, SEPPO KAIJALAINENI, ZEWDU TEREFEWORK1, LARS PAULIN 3 , SCOTT W. TIGHE\ PETER H. GRAHAMS, and KRISTINA LINDSTROM! lDepartment of Applied Chemistry and Microbiology, Biocenter 1, University of Helsinki, Finland 2Department of Applied Microbiology, Sichuan Agricultural University, Sichuan, China 3Institute of Biotechnology, Biocenter 1, University of Helsinki, Finland 4Analytical Services Inc. Willston, U.S.A. SDepartment of Soil, Climate and Water, 1991 Upper Buford Circle, University of Minnesota, St. Paul, U.S.A. Received February 5,1999 Summary Twenty-two rhizobial strains isolated from the root nodules of two Chinese peanut cultivars (Arachis hypogaea L. Tianfu no. 3 and a local cultivar) growing at four different sites in the Sichuan province, Southwest China, were characterized by growth rate, rep-PCR, PCR-RFLP of 16S rDNA, partial se- quencing of ribosomal genes, and fatty acid - methyl ester analysis (FAME), and compared with strains representing Bradyrhizobium ;apanicum, B. elkanii and other unclassified Bradyrhizobium sp. All peanut isolates from Sichuan were bradyrhizobia. Dendrograms constructed using the rep-PCR finger- prints grouped the strains mainly according to their geographic and cultivar origin. Based on PCR-RFLP and partial sequence analysis of 16S rDNA it appears that peanut bradyrhizobial strains from Sichuan are similar to peanut strains from Africa and Israel, and closely related to B. ;aponicum. In contrast, analysis of FAME data using two-dimensional principal component analysis indicated that Bradyrhizo bium sp. (Arachis) were similar to, but slightly different from other bradyrhizobia. The presence and level of fatty acid 16:1 w5c was the distinguishing feature. The results of PCR-RFLP of the 16S rRNA gene, the partial sequence analysis of 16S rDNA, and FAME were in good agreement. Key words: Bradyrhizobium - peanut - China - 16S rRNA - rep-PCR - FAME - taxonomy Introduction Peanut (Arachis hypogaea L.) is an important cash crop for farmers in China. The plant is not indigenous, but has been cultivated there for about 500 years, and it is usually nodulated by indigenous root-nodule bacteria. Improved symbiotic effectiveness can be achieved by in- oculation with selected strains from the indigenous pop- ulations (HUANG, 1987, 1988). The rhizobial strains nodulating peanut are generally slow-growing, but effec- tive fast growing strains have been reported in Sichuan (Hu, et al. 1991). The peanut growing area in Sichuan spans several dif- ferent soil types, is centered in the warm temperate and subtropical region, and involves several different geno- types. Because China is the center of origin for many 0723-2020/99/22/03-378 $ 12.00/0 wild legume hosts, indigenous rhizobial populations may vary enormously. Variations in the symbiotic properties of these organisms were evaluated by ZHANG et al. (1996), but little is known about the genetic variation in peanut bradyrhizobial populations indigenous to China. There is also a need to determine the exact taxonomic position of the peanut (brady)rhizobia. Molecular methods for analysing the diversity and phylogeny of Rhizobium and Bradyrhizobium include plasmid profiling (BROCKMAN, 1989), DNA-DNA hy- bridization, DNA-rRNA hybridization (GRAHAM et al., 1991), restriction fragment length polymorphism (RFLP) (KAlJALAINEN and LINDSTROM, 1989, LAGUERRE et al. 1993, 1994), sequence analysis of 16S ribosomal genes