The occurrence of alternative Nod factor (NF)-independent symbiosis between legumes and rhizobia was first demonstrated in some Aeschynomene species that are nodulated by photosynthetic bradyrhizobia lacking the canonical nodABC genes. In this study, we revealed that a large diversity of non-photosynthetic bradyrhizobia, including B. elkanii, was also able to induce nodules on the NFindependent Aeschynomene species, A. indica. Using cytological analysis of the nodules and the nitrogenase enzyme activity as markers, a gradient in the symbiotic interaction between bradyrhizobial strains and A. indica could be distinguished. This ranged from strains that induced nodules that were only infected intercellularly to rhizobial strains that formed nodules in which the host cells were invaded intracellularly and that displayed a weak nitrogenase activity. In all nonphotosynthetic bradyrhizobia, the type III secretion system (T3SS) appears required to trigger nodule organogenesis. In contrast, genome sequence analysis revealed that apart from a few exceptions, like the Bradyrhizobium ORS285 strain, photosynthetic bradyrhizobia strains lack a T3SS. Furthermore, analysis of the symbiotic properties of an ORS285 T3SS mutant revealed that the T3SS could have a positive or negative role for the interaction with NF-dependent Aeschynomene species, but that it is dispensable for the interaction with all NF-independent Aeschynomene species tested. Taken together, these data indicate that two NF-independent symbiotic processes are possible between legumes and rhizobia: one dependent on a T3SS and one using a so far unknown mechanism.

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ABSTRACTThe diversity of bacteria nodulatingAeschynomene americanaL. in Thailand was determined from phenotypic characteristics and multilocus sequence analysis of the 16S rRNA gene and 3 housekeeping genes (dnaK,recA, andglnB). The isolated strains were nonphotosynthetic bacteria and were assigned to the genusBradyrhizobium, in whichB. yuanmingensewas the dominant species. Some of the other species, includingB. japonicum,B. liaoningense, andB. canariense, were minor species. These isolated strains were divided into 2 groups—nod-containing and divergentnod-containing strains—based on Southern blot hybridization and PCR amplification ofnodABCgenes. The divergentnodgenes could not be PCR amplified and failed to hybridizenodgene probes designed fromB. japonicumUSDA110, but hybridized to probes from other bradyrhizobial strains under low-stringency conditions. The grouping based on sequence similarity ofnodgenes was well correlated with the grouping based on that ofnifHgene, in which thenod-containing and divergentnod-containing strains were obviously distinguished. The divergentnod-containing strains and photosynthetic bradyrhizobia shared closenifHsequence similarity and an ability to fix nitrogen in the free-living state. Surprisingly, the strains isolated fromA. americanacould nodulateAeschynomeneplants that belong to different cross-inoculation (CI) groups, includingA. afrasperaandA. indica. This is the first discovery of bradyrhizobia (nonphotosynthetic andnod-containing strain) originating from CI group 1 nodulating roots ofA. indica(CI group 3). An infection process used to establish symbiosis onAeschynomenedifferent from the classical one is proposed.

Plant associations by bradyrhizobia have been detected not only in leguminous plants, but also in non-leguminous species including rice. Bradyrhizobium sp. SUTN9-2 was isolated from Aeschynomene americana L., which is a leguminous weed found in the rice fields of Thailand. This strain promoted the highest total rice (Oryza sativa L. cultivar Pathum Thani 1) dry weight among the endophytic bradyrhizobial strains tested, and was, thus, employed for the further characterization of rice-Bradyrhizobium interactions. Some known bacterial genes involved in bacteria-plant interactions were selected. The expression of the type III secretion component (rhcJ), type IV secretion component (virD4), and pectinesterase (peces) genes of the bacterium were up-regulated when the rice root exudate was added to the culture. When SUTN9-2 was inoculated into rice seedlings, the peces, rhcJ, virD4, and exopolysaccharide production (fliP) genes were strongly expressed in the bacterium 6–24 h after the inoculation. The gene for glutathione-S-transferase (gst) was slightly expressed 12 h after the inoculation. In order to determine whether type III secretion system (T3SS) is involved in bradyrhizobial infections in rice plants, wild-type SUTN9-2 and T3SS mutant strains were inoculated into the original host plant (A. americana) and a rice plant (cultivar Pathum Thani 1). The ability of T3SS mutants to invade rice tissues was weaker than that of the wild-type strain; however, their phenotypes in A. americana were not changed by T3SS mutations. These results suggest that T3SS is one of the important determinants modulating rice infection; however, type IV secretion system and peces may also be responsible for the early steps of rice infection.