These results, which are in agreement with observations of competition for root colonization, where mutants lacking the thin flagellum were equally competitive as the parental strains, while mutants lacking the thick flagellum or both were less competitive (Althabegoiti

et al., 2010), suggest a complex role of flagellins in competitiveness. On the one hand, the effects of motility on competitiveness depended on the water status of the rooting substrate, and on the other, mutants devoid of the thin flagellum indicated that flagellin activities unrelated to motility Aloxistatin purchase might have exerted an influence. Flagellins are pathogen-related molecular patterns able to elicit plant defense responses (Nicaise et al., 2009). However, the active portion is a 22-amino acid peptide near the N-terminus called flg22, which is not conserved in rhizobial flagellins (Gómez-Gómez & Boller, 2002) including FliCI-II or FliC1-4 (J. Pérez-Giménez, unpublished data). Another possible role related to competitiveness might be in bacterial adhesion to roots; however, studies in Rhizobium leguminosarum indicated that flagellin is not an adhesin (Smit et al.,

1989). Furthermore, flagellin expression in the vermiculite is unknown. Thus, more studies are required to evaluate the nature of flagellin activities in B. japonicum. In soils at field capacity, rhizobial motility may be scarce (Madsen & Alexander, 1982; Liu et al., 1989; McDermott & Graham, 1989; López-García et al., 2002; Horiuchi et al., 2005), because chemoattractant MAPK inhibitor diffusion is slower due to the lower water potential, paths are impaired due to the tortuosity and size of the soil pores, and bacterial movement is retarded due to attachment/detachment to and from soil particles Idoxuridine (Watt et al., 2006; Tufenkji, 2007). Our results with the nonmotile double mutants are in agreement with these observations, indicating that the effect of swimming on competition for nodulation would

be restricted to situations of water saturation of the soil pores (which, in field crops, occur after irrigation or rainfall). However, much work still remains to be carried out to understand the different performances of each flagellum in laboratory and field experiments. Among the main factors that may play a role in the field situation are the physiological state of the rhizobia at the time of inoculation, the expression of each flagellum in the environment, their activities apart from motility, and the influence of soil factors such as micro- and macrobiota, organic matter, porosity, structure, and climate, all of which are absent in the lab system. Nevertheless, our results underscore the importance of inoculant application methods in field crops to benefit from rhizobial motility in the competition for nodulation (López-García et al., 2002, 2009; Althabegoiti et al., 2008).