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In the symbiosis between rhizobia and legumes, host plants can form symbiotic root nodules with multiple rhizobial strains, potentially showing different symbiotic performances in nitrogen fixation. Here, we investigated the presence of mixed nodules, containing rhizobia with different degrees of mutualisms, and evaluate their relative fitness in the  Sinorhizobium meliloti – Medicago sativa  model symbiosis. We used three  S. meliloti  strains, the mutualist strains Rm1021 and BL225C and the non-mutualist AK83. We performed competition experiments involving both  in vitro  and  in vivo  symbiotic assays with  M. sativa  host plants. We show the occurrence of a high number (from 27 to 100%) of mixed nodules with no negative effect on both nitrogen fixation and plant growth. The estimation of the relative fitness as non-mutualist/mutualist ratios in single nodules shows that in some nodules the non-mutualist strain efficiently colonized root nodules along with the mutualist ones. In conclusion, we can support the hypothesis that in  S. meliloti – M. sativa  symbiosis mixed nodules are formed and allow non-mutualist or less-mutualist bacterial partners to be less or not sanctioned by the host plant, hence allowing a potential form of cheating behavior to be present in the nitrogen fixing symbiosis.

Mixed Nodule Infection in Sinorhizobium meliloti–Medicago sativa Symbiosis Suggest the Presence of Cheating Behavior