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.
Keywords: Biofertilizers; Nitrogen fixation; Plant growth promoting activities; Plant inoculation; Plant-associated bacteria.
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