title: Colonization and beneficial effects on annual ryegrass by mixed inoculation with plant growth promoting bacteria.
abstract: 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 18530Twas 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 3×108bacteria 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.
authors.affiliation: Instituto Nacional de Investigação Agrária e Veterinária, I. P., Av. da República, 2780-159 Oeiras, Portugal; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal.
authors.forename: Nádia L
authors.initials: NL
authors.lastname: Castanheira
authors.affiliation: iBET-Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal.
authors.forename: Ana Catarina
authors.initials: AC
authors.lastname: Dourado
authors.affiliation: Instituto Nacional de Investigação Agrária e Veterinária, I. P., Av. da República, 2780-159 Oeiras, Portugal.
authors.forename: Isabel
authors.initials: I
authors.lastname: Pais
authors.affiliation: Instituto Nacional de Investigação Agrária e Veterinária, I. P., Av. da República, 2780-159 Oeiras, Portugal.
authors.forename: José
authors.initials: J
authors.lastname: Semedo
authors.affiliation: Instituto Nacional de Investigação Agrária e Veterinária, I. P., Av. da República, 2780-159 Oeiras, Portugal.
authors.forename: Paula
authors.initials: P
authors.lastname: Scotti-Campos
authors.affiliation: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal.
authors.forename: Nuno
authors.initials: N
authors.lastname: Borges
authors.affiliation: UCBIO, REQUIMTE, Chemistry Department, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal.
authors.forename: Gilda
authors.initials: G
authors.lastname: Carvalho
authors.affiliation: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal; iBET-Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal.
authors.forename: Maria Teresa
authors.initials: MT
authors.lastname: Barreto Crespo
authors.affiliation: Instituto Nacional de Investigação Agrária e Veterinária, I. P., Av. da República, 2780-159 Oeiras, Portugal. Electronic address: paula.fareleira@iniav.pt.
authors.forename: Paula
authors.initials: P
authors.lastname: Fareleira
mesh_terms: D018533:Biomass; D017404:In Situ Hybridization, Fluorescence; D050356:Lipid Metabolism; D008129:Lolium / Q000737:chemistry / Q000254:growth & development* / Q000382:microbiology*; D018613:Microscopy, Confocal; D056507:Paenibacillus / Q000254:growth & development*; D010788:Photosynthesis; D063245:Plant Development; D018517:Plant Roots / Q000382:microbiology; D011549:Pseudomonas / Q000254:growth & development*; D020578:Sphingomonas / Q000254:growth & development*; D013559:Symbiosis
keywords: Annual ryegrass colonization; Fluorescence in situ hybridization; Paenibacillus; Plant growth promotion; Pseudomonas; Sphingomonas