Microbial Inoculants with Autochthonous Bacteria for Biodiverse Legume Pastures in Portuguese Agro-Forestry Ecosystems

One key point to achieve the success of microorganisms like rhizobia as biofertilizers (inoculants) is the characterization and selection of autochthonous strains. The aim of this work was to study the nitrogen-fixing efficiency of autochthonous Rhizobium strains, using as hosts several agronomic important annual Trifolium species (Trifolium subterraneum, Trifolium incarnatum, Trifolium suaveolens, and Trifolium vesiculosum), and compare them with a peat commercial inoculant . For this purpose, three preselected strains (89Ts2a, 123Ts2a, and 149Ts2) isolated from several Trifolium spp. grown in the south of Portugal were characterized beyond their symbiotic performance, also for their “in vitro” PPB activities (solubilization of mineral phosphate and production of siderophores). In addition, molecular identification was performed using the sequence of 16S rRNA and recA genes. Results of shoot dry weight indicated that autochthonous Rhizobium strains were highly efficient in nitrogen fixation. For T. subterraneum and T. incarnatum, strain 123TS2a had the best performance, and for T. suaveolens and T. vesiculosum, the best performance was accomplished by strain 89TS2a. These two strains were combined in a mixed inoculum that was shown to promote higher yields than the commercial peat inoculant in the majority of Trifolium species tested. Results of “in vitro” activities indicated that two strains (89Ts2 and 123Ts2a) could solubilize phosphate, and the three strains could produce siderophores. All these strains had strong phylogenetic relationship with Rhizobium leguminosarum and are closely related to biovar trifolii and viciae.