Novel Alphaproteobacterial Root Nodule Symbiont Associated with Lupinus texensis

Abstract

Phylogenetic analysis of rRNA gene, recA, nodA, nifD, and nifH sequences suggested that nitrogen-fixing symbionts from two populations of Lupinus texensis acquired the capacity for nodule symbiosis separately from other rhizobia in the alphaproteobacteria. Their closest 16S rRNA relatives were the nonsymbiotic taxa Chelatococcus, Bosea, and Balneomonas.Legume nodule symbionts are found in several lineages in the alphaproteobacteria and at least two lineages in the betaproteobacteria (5, 17). Four genera of alphaproteobacteria (Rhizobium, Mesorhizobium, Sinorhizobium, and Bradyrhizobium) constitute the predominant symbionts for most legume species in habitats throughout the world. However, additional nodule-forming lineages of alphaproteobacteria that may have a more restricted geographic distribution or limited host range have been discovered in recent years (8,14,16,22,28,31,32). This suggests that further sampling of other host taxa and locations is likely to uncover additional lineages of nodule bacteria.The legume genus Lupinus includes approximately 275 species of herbs and shrubs (11). Studies of most Lupinus species within their native range (4,15,21,24), as well as outside their native distribution (6,26,30), indicate that strains of Bradyrhizobium are the predominant nodule symbionts. We sampled nodule bacteria in the native range of Lupinus texensis in Texas (13) and found that none of the isolates resembled Bradyrhizobium symbionts associated with other Lupinus species. Twenty-eight root nodules were collected from multiple plants at two sites 20 km apart (Williamson County and Travis County), and one bacterial isolate per nodule was obtained as previously described (25). DNA purification and PCR experiments used standard protocols (5). Amplification of the 5Ј intervening sequence region of the 23S rRNA gene (27) indicated that all 28 isolates had a unique size variant (473 bp) that differed from length variants found in North American Bradyrhizobium strains (510 or 537 bp) (18,20,21). A 431-bp 5Ј portion of the 23S rRNA gene was sequenced in five Texas isolates (GenBank accession numbers EF191402 to EF191406), and each sequence was the same. Blast searches indicated that this sequence was distantly related to all currently known lineages of legume nodule symbionts. The closest match among rhizobia was Bradyrhizobium japonicum USDA 110 (80% similarity). Other genera of alphaproteobacterial and betaproteobacterial symbionts had similarities ranging from 62 to 77%.To screen the remaining L. texensis nodule isolates, PCR assays were performed with a primer pair (LUTf1 and 23LUTr2; Table 1) matching unique portions of the L. texensis symbiont 5Ј 23S rRNA sequence. All isolates yielded the same 167-bp amplification product. Other genera of nodule bacteria (Bradyrhizobium, Mesorhizobium, Sinorhizobium, and Rhizobium) failed to yield an amplification product with these primers.A nearly full-length portion of the 16S rRNA gene (1,408 bp) was sequenced for two isolates (Lut5, Lut6). Both isolates had the same...

Characterization of Rhizobia for the Improvement of Soybean Cultivation at Cold Conditions in Central Europe

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