Manganese (Mn) toxicity is a very common soil stress around the world, which is responsible for low soil fertility. This manuscript evaluates the effect of the endophytic bacterium Pseudomonas sp. Q1 on different rhizobial-legume symbioses in the absence and presence of Mn toxicity. Three legume species, Cicer arietinum (chickpea), Trifolium subterraneum (subterranean clover), and Medicago polymorpha (burr medic) were used. To evaluate the role of 1-aminocyclopropane-1-carboxylate (ACC) deaminase produced by strain Q1 in these interactions, an ACC deaminase knockout mutant of this strain was constructed and used in those trials. The Q1 strain only promoted the symbiotic performance of Rhizobium leguminosarum bv. trifolii ATCC 14480T and Ensifer meliloti ATCC 9930T, leading to an increase of the growth of their hosts in both conditions. Notably, the acdS gene disruption of strain Q1 abolished the beneficial effect of this bacterium as well as causing this mutant strain to act deleteriously in those specific symbioses. This study suggests that the addition of non-rhizobia with functional ACC deaminase may be a strategy to improve the pasture legume–rhizobial symbioses, particularly when the use of rhizobial strains alone does not yield the expected results due to their difficulty in competing with native strains or in adapting to inhibitory soil conditions.

Rhizobia are soil bacteria able to form symbioses with legumes and fix atmospheric nitrogen, converting it into a form that can be assimilated by the plant. The biological nitrogen fixation is a possible strategy to reduce the environmental pollution caused by the use of chemical N-fertilizers in agricultural fields. Successful colonization of the host root by free-living rhizobia requires that these bacteria are able to deal with adverse conditions in the soil, in addition to stresses that may occur in their endosymbiotic life inside the root nodules. Stress response genes, such as otsAB , groEL , clpB , rpoH play an important role in tolerance of free-living rhizobia to different environmental conditions and some of these genes have been shown to be involved in the symbiosis. This review will focus on stress response genes that have been reported to improve the symbiotic performance of rhizobia with their host plants. For example, chickpea plants inoculated with a Mesorhizobium strain modified with extra copies of the groEL gene showed a symbiotic effectiveness approximately 1.5 fold higher than plants inoculated with the wild-type strain. Despite these promising results, more studies are required to obtain highly efficient and tolerant rhizobia strains, suitable for different edaphoclimatic conditions, to be used as field inoculants.

Evidences for an involvement of the bacterial type IV secretion system (T4SS) in the symbiotic relationship between rhizobia and legumes have been pointed out by several recent studies. However, information regarding this secretion system in Mesorhizobium is still very scarce. The aim of the present study was to investigate the phylogeny and expression of the traG gene, which encodes a substrate receptor of the T4SS. In addition, the occurrence and genomic context of this and other T4SS genes, namely, genes from tra/trb and virB/virD4 complexes, were also analyzed in order to unveil the structural and functional organization of T4SS in mesorhizobia. The location of the T4SS genes in the symbiotic region of the analyzed rhizobial genomes, along with the traG phylogeny, suggests that T4SS genes could be horizontally transferred together with the symbiosis genes. Regarding the T4SS structural organization in Mesorhizobium, the virB/virD4 genes were absent in all chickpea (Cicer arietinum L.) microsymbionts and in the Lotus symbiont Mesorhizobium japonicum MAFF303099T. Interestingly, the presence of genes belonging to another secretion system (T3SS) was restricted to these strains lacking the virB/virD4 genes. The traG gene expression was detected in M. mediterraneum Ca36T and M. ciceri LMS-1 strains when exposed to chickpea root exudates and also in the early nodules formed by M. mediterraneum Ca36T, but not in older nodules. This study contributes to a better understanding of the importance of T4SS in mutualistic symbiotic bacteria.

Aims: The aim of this study was to investigate whether the overexpression of NifA and NodD regulators contribute to the symbiotic improvement of chickpea mesorhizobia. Methods: The native strains V-15b, ST-2 and PMI-6 were transformed with extra copies of nifA or nodD genes and several plants trial were performed. Results: Plant growth assays showed that nifA overexpression was able to improve the symbiotic effectiveness of V-15b, while nodD overexpression lead to the improvement of ST-2 and PMI-6. Hydroponic assays showed that plants inoculated with V15bnifA+ and PMI6nodD+ started developing nodules earlier than those inoculated with the corresponding control strains. In addition, the number of nodules was always higher in plants inoculated with the strains overexpressing the symbiotic genes. Analysis of histological sections of nodules formed by V15bnifA+ showed a more developed fixation zone when compared to control. On the other hand, nodules induced by PMI6nodD+ did not show a senescent zone, which was observed in nodules from plants inoculated with the control strain. Plants inoculated with PMI6nodD+ and ST2nodD+ showed a higher number of infection threads than the corresponding control inoculations. Conclusion: These results indicate that overexpressing nifA and nodD may be an important tool to achieve the improvement of the symbiotic performance of mesorhizobia.