Annual ryegrass is a fast-growing cool-season grass broadly present in the Portuguese "montado", a typically Mediterranean agro-forestry-pastoral ecosystem. A culture-dependent approach was used to investigate natural associations of this crop with potentially beneficial bacteria, aiming to identify strains suitable for biofertilization purposes. Annual ryegrass seedlings were used to trap bacteria from three different soils in laboratory conditions. Using a nitrogen-free microaerophilic medium, 147 isolates were recovered from the rhizosphere, rhizoplane, and surface-sterilized plant tissues, which were assigned to 12 genera in classes Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Bacilli and Actinobacteria. All isolates were able to grow in the absence of nitrogen and several of them were able to perform in vitro activities related to plant growth promotion. Isolates of the genera Sphingomonas and Achromobacter were found to be the most effective stimulators of annual ryegrass growth under nitrogen limitation (47-92% biomass increases). Major enhancements were obtained with isolates G3Dc4 (Achromobacter sp.) and G2Ac10 (Sphingomonas sp.). The latest isolate was also able to increment plant growth in nitrogen-supplemented medium, as well as the phosphate solubilizer and siderophore producer, G1Dc10 (Pseudomonas sp.), and the cellulose/pectin hydrolyser, G3Ac9 (Paenibacillus sp.). This study represents the first survey of annual ryegrass-associated bacteria in the "montado" ecosystem and unveiled a set of strains with potential for use as inoculants.

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 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 3×10 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.

The novel strain belongs to the plant-associated Burkholderia cluster and is a promising candidate for exploitation as plant inoculant in field conditions.