Two Broad Host Range Rhizobial Strains Isolated From Relict Legumes Have Various Complementary Effects on Symbiotic Parameters of Co-inoculated Plants

Abstract

Two bacterial strains Ach-343 and Opo-235 were isolated, respectively from nodules of Miocene-Pliocene relict legumes <i>Astragalus chorinensis</i> Bunge and <i>Oxytropis popoviana</i> Peschkova originated from Buryatia (Baikal Lake region, Russia). For identification of these strains the sequencing of 16S rRNA (<i>rrs</i>) gene was used. Strain Opo-235 belonged to the species <i>Mesorhizobium japonicum</i>, while the strain Ach-343 was identified as <i>M. kowhaii</i> (100 and 99.9% <i>rrs</i> similarity with the type strains MAFF 303099^T and ICMP 19512^T, respectively). Symbiotic genes of these strains as well as some genes that promote plant growth (<i>acdS</i>, gibberellin- and auxin-synthesis related genes) were searched throughout the whole genome sequences. The sets of plant growth-promoting genes found were almost identical in both strains, whereas the sets of symbiotic genes were different and complemented each other with several <i>nod</i>, <i>nif</i>, and <i>fix</i> genes. Effects of mono- and co-inoculation of <i>Astragalus sericeocanus</i>, <i>Oxytropis caespitosa</i>, <i>Glycyrrhiza uralensis</i>, <i>Medicago sativa</i>, and <i>Trifolium pratense</i> plants with the strains <i>M. kowhaii</i> Ach-343 and <i>M. japonicum</i> Opo-235 expressing fluorescent proteins mCherry (red) and EGFP (green) were studied in the gnotobiotic plant nodulation assay. It was shown that both strains had a wide range of host specificity, including species of different legume genera from two tribes (Galegeae and Trifolieae). The effects of co-microsymbionts on plants depended on the plant species and varied from decrease, no effect, to increase in the number of nodules, nitrogen-fixing activity and plant biomass. One of the reasons for this phenomenon may be the discovered complementarity in co-microsymbionts of symbiotic genes responsible for the specific modification of Nod-factors and nitrogenase activity. Localization and co-localization of the strains in nodules was confirmed by the confocal microscopy. Analysis of histological and ultrastructural organization of <i>A. chorinensis</i> and <i>O. popoviana</i> root nodules was performed. It can be concluded that the strains <i>M. kowhaii</i> Ach-343 and <i>M. japonicum</i> Opo-235 demonstrate lack of high symbiotic specificity that is characteristic for primitive legume-rhizobia systems. Further study of the root nodule bacteria having complementary sets of symbiotic genes will contribute to clarify the evolutionary paths of legume-rhizobia relationships and the mechanisms of effective integration between partners.