Genome survey and characterization of endophytic bacteria exhibiting a beneficial effect on growth and development of poplar trees

Abstract

The association of endophytic bacteria with their plant hosts has a beneficial effect for many different plant species. Our goal is to identify endophytic bacteria that improve the biomass production and the carbon sequestration potential of poplar trees (Populus spp.) when grown in marginal soil and to gain an insight in the mechanisms underlying plant growth promotion. Members of the Gammaproteobacteria dominated a collection of 78 bacterial endophytes isolated from poplar and willow trees. As representatives for the dominant genera of endophytic gammaproteobacteria, we selected Enterobacter sp. strain 638, Stenotrophomonas maltophilia R551-3, Pseudomonas putida W619, and Serratia proteamaculans 568 for genome sequencing and analysis of their plant growth-promoting effects, including root development. Derivatives of these endophytes, labeled with gfp, were also used to study the colonization of their poplar hosts. In greenhouse studies, poplar cuttings (Populus deltoides x Populus nigra DN-34) inoculated with Enterobacter sp. strain 638 repeatedly showed the highest increase in biomass production compared to cuttings of noninoculated control plants. Sequence data combined with the analysis of their metabolic properties resulted in the identification of many putative mechanisms, including carbon source utilization, that help these endophytes to thrive within a plant environment and to potentially affect the growth and development of their plant hosts. Understanding the interactions between endophytic bacteria and their host plants should ultimately result in the design of strategies for improved poplar biomass production on marginal soils as a feedstock for biofuels.

FIG. 1.

Taxonomic breakdown of 16S rRNA gene sequences of the cultivable endophytic community composition as isolated from hybrid poplar H11-11 and willow trees. Taxonomic classifications were determined according to Wang et al. (44). The central pie shows percentages by phyla; each outer annulus progressively breaks these down to finer taxonomic levels, with families, genera, and species in the outermost annuli. Numbers indicate the relative abundance, expressed as a percentage, of the different taxonomic groups.

FIG. 2.

Growth indexes for poplar cuttings inoculated with different endophytic bacteria. Growth indexes were determined 10 weeks after the inoculating and planting of the cuttings in sandy soil. Per condition, seven plants were used. Plants were grown in the greenhouse. Noninoculated plants were used as references. Bars indicate standard errors. Growth indexes were calculated as (Mt − M0)/M0 after 10 weeks of growth in the presence or absence of endophytic inoculum. M0, plant's weight (g) at week 0; Mt, plant's weight (g) after 10 weeks. The statistical significance of the increased biomass production of inoculated plants, compared to that of noninoculated control plants, was confirmed at the 5% level (**) using the Dunnett test.

FIG. 3.

Effects of S. proteamaculans 568 on the rooting and shoot formation of poplar DN-34. Plants were incubated hydroponically in half-strength Hoagland's solution in the absence (control) or presence (568) of strain 568. Root and shoot development are presented after 1 week (A) and 10 weeks (B).

FIG. 4.

Endophytic colonization of the poplar DN-34 roots by gfp-labeled derivatives of S. proteamaculans 568, Enterobacter sp. strain 638, and P. putida W619. (A) Colonization of the surface of a poplar root by a gfp-labeled derivative of P. putida W619. The picture was taken by fluorescence microscopy. Arrows indicate the positions of microcolonies on the root surface. (B) Interior view of a translateral section of a poplar root colonized by a gfp-labeled derivative of P. putida W619. The picture was taken with the help of the apotome feature of the fluorescence microscope. Arrows indicate the zones of dense interior colonization. (C and D) Interior views of a section of a poplar root colonized by a gfp-labeled derivative of S. proteamaculans 568 (C) and Enterobacter sp. strain 638 (D). The root tissue was stained with 0.05% methyl violet to decrease autofluorescence.

FIG. 5.

Metabolic pathways involved in the production of plant growth hormones (IAA, diacetyl, acetoin, and 2,3-butanediol) found on the genomes of selected endophytic bacteria. The metabolic pathways were constructed using PRIAM predictions mapped on the KEGG database (http://www.genome.ad.jp). For each organism, differently colored arrows are used to indicate the presence of the putative pathways: S. maltophilia R551-3 (red), P. putida W619 (green), B. vietnamiensis G4 (orange), Enterobacter sp. strain 638 (dark blue), and S. proteamaculans 568 (light blue). Black arrows indicate known pathway steps that could not be identified. Dashed arrows correspond to the presence of putative enzymes (PRIAM E value below 10⁻³⁰). In the tryptophan-dependent IAA synthesis, the enzymes involved are tryptophan transaminase Lao1 (1), indolepyruvate decarboxylase IpdC (2), indole-3-acetaldehyde dehydrogenase DhaS (3), tryptophan decarboxylase Dcd1 (4), amine oxidase (5), tryptophan 2-monooxygenase IaaM (6), deaminase IaaH (7), nitrile hydratase (8), nitrilase YhcX (9), and indole-3-acetaldehyde reductase AdhC (10). Besides the production of IAA, the main pathway for tryptophan metabolism is via tryptophan 2,3-dioxygenase KynA (11). In butanoate metabolism, the production of acetoin from pyruvate is catalyzed by the acetolactate synthase AlsS (12) and the acetolactate decarboxylase AlsD (13). The genome of Enterobacter sp. strain 638 encodes the acetoin dehydrogenase ButA (14) that is able to catalyze the conversion of acetoin into diacetyl. It is unknown if this compound has plant growth stimulatory effects. The 2,3-butanediol dehydrogenase ButB (15) involved in the conversion of acetoin into 2,3-butanediol was not found encoded on the genomes of the endophytic bacteria but is present on the poplar genome.