Efficient but Elusive Rhizobia Fix Nitrogen in the Wild Legumes Bituminaria bituminosa and Coronilla Viminalis

In ecological restoration of degraded natural habitats, revegetation with wild native plants is a priority. Legumes play a key role in this process through nitrogen (N)-fixing symbiosis with rhizobia, obtaining N for their growth and improving soil fertility, which benefits other nonleguminous plants in the environment. This study explores the rhizobia of two wild legumes, Coronilla viminalis and Bituminaria bituminosa, found in a degraded habitat in Lanzarote (Canary Islands). We found these legumes nodulated by highly efficient N-fixing mesorhizobia harboring the symbiovars canariensis and hedysari in Mesorhizobium species distinct from those originally reported to carry these symbiovars. However, isolating these rhizobia was challenging. Despite the good plant development and the pink color of root nodules indicating effective N-fixation, these rhizobia could not be cultured in most cases. This suggests the presence of unculturable or "fastidious" rhizobia in the nodules, with requirements poorly mimicked in conventional rhizobial media. Additionally, the presence of fast-growing non-rhizobial endophytes in the nodules complicates the isolation of slower-growing rhizobia, which requires special care during the isolation protocol to avoid endophytes and extend incubation times. The difficulty of cultivating the rhizobia of these two wild legumes suggests that their diversity may be greater than described here.