Colonization and beneficial effects on annual ryegrass by mixed inoculation with plant growth promoting bacteria

Multi-strain inoculants have increased potential to accomplish a diversity of plant needs, mainly attributed to its multi-functionality. This work evaluated the ability of a mixture of three bacteria to colonize and induce a beneficial response on the pasture crop annual ryegrass. Pseudomonas G1Dc10 and Paenibacillus G3Ac9 were previously isolated from annual ryegrass and were selected for their ability to perform multiple functions related to plant growth promotion. Sphingomonas azotifigens DSMZ 18530^T was included due to nitrogen fixing ability. The effects of the bacterial mixture were assessed in gnotobiotic plant inoculation assays and compared with single and dual inoculation treatments. Triple inoculation with 3 × 10⁸ bacteria significantly increased plant dry weight and leaf pigments, indicating improved photosynthetic performance. Plant lipid biosynthesis was enhanced by 65%, mainly due to the rise of linolenic acid, an omega-3 fatty acid with high dietary value. Electrolyte leakage, an indicator of plant membrane stability under stress, was decreased pointing to a beneficial effect by inoculation. Plants physiological condition was more favoured by triple inoculation than by single, although benefits on biomass were only evident relative to non-inoculated plants. The colonization behaviour and coexistence in plant tissues were assessed using FISH and GFP-labelling, combined with confocal microscopy and a cultivation-based approach for quantification. The three strains occupied the same sites, localizing preferentially along root hairs and in stem epidermis. Endophytic colonization was observed as bacteria entered root and stem inner tissues. This study reveals the potential of this mixture of strains for biofertilization, contributing to improve crop productivity and nutritional value.