Both incompatible and compatible rhizobia inhabit the intercellular spaces of leguminous root nodules.

In addition to rhizobia, many types of co-existent bacteria are found in leguminous root nodules, but their habitats are unclear. To investigate this phenomenon, we labeled Bradyrhizobium diazoefficiens USDA122 and Bradyrhizobium sp. SSBR45 with Discosoma sp. red fluorescent protein (DsRed) or enhanced green fluorescent protein (eGFP). USDA122 enhances soybean growth by forming effective root nodules, but SSBR45 does not form any nodules. Using low-magnification laser scanning confocal microscopy, we found that infected cells in the central zone of soybean nodules appeared to be occupied by USDA122. Notably, high-magnification microscopy after co-inoculation of non-fluorescent USDA122 and fluorescence-labeled SSBR45 also revealed that SSBR45 inhabits the intercellular spaces of healthy nodules. More unexpectedly, co-inoculation of eGFP-labeled USDA122 and DsRed-labeled SSBR45 (and vice versa) revealed the presence of USDA122 bacteria in both the symbiosomes of infected cells and in the apoplasts of healthy nodules. We then next inspected nodules formed after a mixed inoculation of differently-labeled USDA122, without SSBR45, and confirmed the inhabitation of the both populations of USDA122 in the intercellular spaces. In contrast, infected cells were occupied by single-labeled USDA122. We also observed Mesorhizobium loti in the intercellular spaces of active wild-type nodules of Lotus japonicus using transmission electron microscopy. Compatible intercellular rhizobia have been described during nodule formation of several legume species and in some mutants, but our evidence suggests that this type of colonization may occur much more commonly in leguminous root nodules.

Characterization of photosynthetic Bradyrhizobium sp. strain SSBR45 isolated from the root nodules of Aeschynomene indica.

We isolated a novel strain of Bradyrhizobium sp., SSBR45, from the nodulated roots of Aeschynomene indica and labeled it with Discosoma sp. red fluorescent protein (dsRED) or enhanced green fluorescent protein (eGFP) and determined its draft genomic sequence. The labeled SSBR45 stimulated the growth of A. indica markedly on a nitrogen-free medium, as observed by visualizing the fluorescent root nodules. The nodulated roots also exhibited high acetylene reduction activities. The SSBR45 genome included genes involved in nitrogen fixation, photosynthesis, and type IV secretion system; however, it did not consist of canonical nodABC genes and type III secretion system genes. SSBR45, a novel species of the genus Bradyrhizobium, consisted of an average nucleotide identity and average amino acid identity of 87% and 90%, respectively, with the closest strain B. oligotrophicum S58.