Genetic diversity amongBradyrhizobiumisolates that effectively nodulate peanut (Arachis hypogaea)

Abstract

Symbiotic gene diversity and other measures of genetic diversity were examined in Bradyrhizobium isolates that form an effective symbiosis with peanut (Arachis hypogaea). Initially, restriction fragment length polymorphism (RFLP) analysis using a nitrogenase (nif) gene probe was performed on 33 isolates along with one Bradyrhizobium elkanii and two Bradyrhizobium japonicum strains. Considerable diversity was observed among the RFLP patterns of many of the isolates, especially those from South America. Some isolates, however, were found to have similar nif and subsequent nod (nodulation) gene RFLP patterns, indicating symbiotic gene relatedness. With some noted exceptions, symbiotic gene relatedness correlated with relatedness based on total DNA homology and ribotyping analyses. Symbiotic gene relatedness also correlated with symbiotic effectiveness. The RFLP and DNA homology analyses indicate that bradyrhizobia effective with peanut are genetically diverse and consist of at least three different species. This diversity, however, was not particularly evident with partial 16S rRNA gene sequencing. Sequences obtained from the isolates were very similar to each other as well as to sequences previously reported for other Bradyrhizobium strains.

RFLP analysis of the rRNA operon of a Brazilian collection of bradyrhizobial strains from 33 legume species

“…Also, in an analysis of a large collection of indigenous rhizobia in China, no B. elkanii strains were detected (Dr Wen-Xin Chen, personal communication). In conclusion, despite the low variability reported for the 16S rRNA region of Bradyrhizobium in some studies (So et al, 1994;Urtz & Elkan, 1996;Moreira et al, 1998;Molouba et al, 1999;van Berkum & Fuhrmann, 2000;Chen et al, 2000;Willems et al, 2001), and despite the coefficients used in some of the analyses being different, the strains from our study were joined at a very low level of similarity, 28 %, showing greater variability than any previous report. For example, an analysis of African indigenous soybean bradyrhizobia by RFLP-PCR with five restriction enzymes grouped the strains with a similarity of 70 % (Abaidoo et al, 2000), while in strains from native leguminous species of Senegal, the similarity with five restriction enzymes was approximately 74 %, decreasing to 55 % when a strain from Aeschynomene was included (Doignon-Bourcier et al, 1999).…”

“…Indeed, bradyrhizobia seem to represent the majority of isolates from leguminous trees in Brazilian tropical forests (Moreira, 1991(Moreira, , 2000. In addition, a high level of diversity among strains has been reported in a few studies performed in South America (Moreira et al, 1993;Urtz & Elkan, 1996;Chen et al, 2000;Fernandes et al, 2003;Hara, 2003;Menna, 2005 (FEPAGRO, 1999) were used in this study. Information about the original source of the strains, the origin of the strains and their numbers in other collections is available in Supplementary Table S1 available in IJSEM Online.…”

“…Partial or complete 16S rRNA gene sequences, which have also been used extensively for studying the phylogeny of rhizobia (e.g. Young et al, 1991;Oyaizu et al, 1992;Yanagi & Yamasato, 1993;van Rossum et al, 1995;Urtz & Elkan, 1996;Moreira et al, 1998;Vinuesa et al, 1998;Wang et al, 1999;Chen et al, 2000; JaraboLorenzo et al, 2000), have contributed to the recent descriptions of four new genera and several rhizobial species. However, there are reports showing that, despite a high level of diversity in morphological, physiological and genetic properties, diversity is low in the 16S rRNA gene sequences of strains of Bradyrhizobium investigated so far (So et al, 1994, Urtz & Elkan, 1996Molouba et al, 1999;Vinuesa et al, 1998;Chen et al, 2000;van Berkum & Fuhrmann, 2000;Willems et al, 2001;Qian et al, 2003).…”

“…Young et al, 1991;Oyaizu et al, 1992;Yanagi & Yamasato, 1993;van Rossum et al, 1995;Urtz & Elkan, 1996;Moreira et al, 1998;Vinuesa et al, 1998;Wang et al, 1999;Chen et al, 2000; JaraboLorenzo et al, 2000), have contributed to the recent descriptions of four new genera and several rhizobial species. However, there are reports showing that, despite a high level of diversity in morphological, physiological and genetic properties, diversity is low in the 16S rRNA gene sequences of strains of Bradyrhizobium investigated so far (So et al, 1994, Urtz & Elkan, 1996Molouba et al, 1999;Vinuesa et al, 1998;Chen et al, 2000;van Berkum & Fuhrmann, 2000;Willems et al, 2001;Qian et al, 2003).The 23S rRNA is a long fragment of about 2?3 kb; it therefore contains more information than the 16S rRNA and has proven to be useful in the speciation of several genera of bacteria (Ludwig & Schleifer, 1994), including rhizobia (Tesfaye et al, 1997;Terefework et al, 1998;Tesfaye & Holl, 1998;Qian et al, 2003). Furthermore, as the rate of sequence change seems to be faster in the 23S rRNA than in the 16S rRNA gene (Olsen & Woese, 1993), the former may be more valuable for delineating close relationships (Wang & Martínez-Romero, 2000).…”

“…However, the diversity observed among bradyrhizobial strains is usually not reflected in 16S rRNA gene sequence analysis; therefore, few Bradyrhizobium species have been described so far. As bradyrhizobia seem to constitute a major part of the rhizobial isolates in the tropics (Norris, 1965;Moreira, 2000) and as rhizobia might be more diverse in the tropics (Oyaizu et al, 1992;Moreira et al, 1993;Urtz & Elkan, 1996;Vinuesa et al, 1998Vinuesa et al, , 2005a, a large collection of Bradyrhizobium strains would add valuable information about the diversity of ribosomal genes within the genus.The genetic characterization in this study was performed with 119 strains from the Brazilian official culture collection SEMIA of rhizobial strains. The strains had been isolated from 33 legume species (Table 1), most from the tropics, representing nine tribes and including all three subfamilies (Supplementary Table S2).…”