Production of the Phytohormone Indole-3-Acetic Acid by Estuarine Species of the Genus Vibrio

Strains of Vibrio spp. isolated from roots of the estuarine grasses Spartina alterniflora and Juncus roemerianus produce the phytohormone indole-3-acetic acid (IAA). The colorimetric Salkowski assay was used for initial screening of IAA production. Gas chromatography-mass spectroscopy (GC-MS) was then employed to confirm and quantify IAA production. The accuracy of IAA quantification by the Salkowski assay was examined by comparison to GC-MS assay values. Indole-3-acetamide, an intermediate in IAA biosynthesis by the indole-3-acetamide pathway, was also identified by GC-MS. Multilocus sequence typing of concatenated 16S rRNA, recA, and rpoA genes was used for phylogenetic analysis of environmental isolates within the genus Vibrio. Eight Vibrio type strains and five additional species-level clades containing a total of 16 environmental isolates and representing five presumptive new species were identified as IAA-producing Vibrio species. Six additional environmental isolates similar to four of the Vibrio type strains were also IAA producers. To our knowledge, this is the first report of IAA production by species of the genus Vibrio or by bacteria isolated from an estuarine environment.Estuaries along the east coast of temperate North America are ecologically valuable, productive systems dominated by only a few species of plants. Spartina alterniflora (smooth cord grass; hereinafter referred to as Spartina) is a keystone species responsible for very high rates of primary production in Atlantic coast marshes and is a major contributor to the global cycling of several elements (10, 14, 15, 35, 38, 39, 45). Juncus roemerianus (black needle rush; hereinafter referred to as Juncus) is a common subdominant species (28) residing in areas of higher elevation, lower salinity, and less frequent tidal inundation. The roots of these macrophytes are associated with a diverse assemblage of microorganisms, including N₂-fixing and sulfate-reducing bacteria, which greatly contribute to their productivity (30, 31).The phytohormone indole-3-acetic acid (IAA) is the most commonly occurring naturally produced auxin and the most thoroughly studied plant growth regulator. IAA directs several aspects of plant growth and development (37), including the induction and regulation of a variety of processes: e.g., cell division, root extension, vascularization, apical dominance, and tropisms (6, 32). The effects of IAA on plant root tissue are concentration dependent and can be species specific. Responses to increasing IAA concentrations advance from the stimulation of primary root tissue to the development of lateral and adventitious roots and finally to the complete cessation of root growth (1, 6, 16, 29, 32, 37, 44).Many microorganisms interact with and affect their environment through the production and transudation of signal compounds (17). The findings of numerous studies (see, e.g., references 8, 23, 25, and 37) demonstrate that a variety of plant-associated terrestrial bacteria produce and exude IAA. Auxin synthesis by cyanobacteria has also been reported previously (40). IAA is thought to reduce the integrity of plant cell walls by upregulating the production of cellulases and hemicelluloses, resulting in the leakage of some simple sugars and other nutrients that would benefit root-associated microorganisms (17). Likewise, root growth would be an advantage to resident bacteria due to the increased availability of root exudates and root surface for growth. Microorganisms that produce IAA can influence the host plant and function as pathogens, symbionts, or growth regulators, depending on how their IAA production influences the concentration of the plant''s endogenous IAA pool and on the sensitivity of the plant to auxin. Organisms such as Erwinia chrysanthemi, Pseudomonas savastanoi, and Agrobacterium tumefaciens are phytopathogens of many host plants (11, 21, 23, 46). Other organisms, including Azospirillum brasilense and Pseudomonas putida GR12-2, have proven beneficial to plants, and many IAA producers have been shown to stimulate increases in root mass and/or length (20, 37, 44).The aim of the present study was to assess IAA synthesis by Vibrio strains isolated from the roots of highly productive salt marsh grasses. The Salkowski assay was used to perform an initial screening for the presence of IAA, gas chromatography-mass spectroscopy (GC-MS) verified and quantified IAA production, and multilocus sequence typing (MLST) analysis classified all isolates within the genus Vibrio.