Dynamic nitrogen fixation in an aerobic endophyte of Populus.

Biological nitrogen fixation by microbial diazotrophs can contribute significantly to nitrogen availability in non-nodulating plant species. In this study of molecular mechanisms and gene expression relating to biological nitrogen fixation, the aerobic nitrogen-fixing endophyte Burkholderia vietnamiensis, strain WPB, isolated from Populus trichocarpa served as a model for endophyte-poplar interactions. Nitrogen-fixing activity was observed to be dynamic on nitrogen-free medium with a subset of colonies growing to form robust, raised globular like structures. Secondary ion mass spectrometry (NanoSIMS) confirmed that N-fixation was uneven within the population. A fluorescent transcriptional reporter (GFP) revealed that the nitrogenase subunit nifH is not uniformly expressed across genetically identical colonies of WPB and that only ~11% of the population was actively expressing the nifH gene. Higher nifH gene expression was observed in clustered cells through monitoring individual bacterial cells using single-molecule fluorescence in situ hybridization. Through 15N2 enrichment, we identified key nitrogenous metabolites and proteins synthesized by WPB and employed targeted metabolomics in active and inactive populations. We cocultivated WPB Pnif-GFP with poplar within a RhizoChip, a synthetic soil habitat, which enabled direct imaging of microbial nifH expression within root epidermal cells. We observed that nifH expression is localized to the root elongation zone where the strain forms a unique physical interaction with the root cells. This work employed comprehensive experimentation to identify novel mechanisms regulating both biological nitrogen fixation and beneficial plant-endophyte interactions.

Potential Biocontrol Activities of Populus Endophytes against Several Plant Pathogens Using Different Inhibitory Mechanisms.

The plant microbiome can be used to bolster plant defense against abiotic and biotic stresses. Some strains of endophytes, the microorganisms within plants, can directly inhibit the growth of plant fungal pathogens. A previously isolated endophyte from wild Populus (poplar), WPB of the species Burkholderia vietnamiensis, had robust in vitro antifungal activity against pathogen strains that are highly virulent and of concern to Pacific Northwest agriculture: Rhizoctonia solani AG-8, Fusarium culmorum 70110023, and Gaemannomyces graminis var. tritici (Ggt) ARS-A1, as well as activity against the oomycete, Pythium ultimum 217. A direct screening method was developed for isolation of additional anti-fungal endophytes from wild poplar extracts. By challenging pathogens directly with dilute extracts, eleven isolates were found to be inhibitory to at least two plant pathogen strains and were therefore chosen for further characterization. Genomic analysis was conducted to determine if these endophyte strains harbored genes known to be involved in antimicrobial activities. The newly isolated Bacillus strains had gene clusters for production of bacillomycin, fengicyn, and bacillibactin, while the gene cluster for the synthesis of sessilin, viscosin and tolaasin were found in the Pseudomonas strains. The biosynthesis gene cluster for occidiofungin (ocf) was present in the Burkholderia vietnamiensis WPB genome, and an ocf deletion mutant lost inhibitory activity against 3 of the 4 pathogens. The new isolates lacked the gene cluster for occidiofungin implying they employ different modes of action. Other symbiotic traits including nitrogen fixation, phosphate solubilization, and the production of auxins and siderophores were investigated. Although it will be necessary to conduct in vivo tests of the candidates with pathogen-infected agricultural crops, the wild poplar tree microbiome may be a rich source of beneficial endophyte strains with potential for biocontrol applications against a variety of pathogens and utilizing varying modes of action.

Endophyte-Promoted Phosphorus Solubilization in Populus.

Phosphorus is one of the essential nutrients for plant growth, but it may be relatively unavailable to plants because of its chemistry. In soil, the majority of phosphorus is present in the form of a phosphate, usually as metal complexes making it bound to minerals or organic matter. Therefore, inorganic phosphate solubilization is an important process of plant growth promotion by plant associated bacteria and fungi. Non-nodulating plant species have been shown to thrive in low-nutrient environments, in some instances by relying on plant associated microorganisms called endophytes. These microorganisms live within the plant and help supply nutrients for the plant. Despite their potential enormous environmental importance, there are a limited number of studies looking at the direct molecular impact of phosphate solubilizing endophytic bacteria on the host plant. In this work, we studied the impact of two endophyte strains of wild poplar (Populus trichocarpa) that solubilize phosphate. Using a combination of x-ray imaging, spectroscopy methods, and proteomics, we report direct evidence of endophyte-promoted phosphorus uptake in poplar. We found that the solubilized phosphate may react and become insoluble once inside plant tissue, suggesting that endophytes may aid in the re-release of phosphate. Using synchrotron x-ray fluorescence spectromicroscopy, we visualized the nutrient phosphorus inside poplar roots inoculated by the selected endophytes and found the phosphorus in both forms of organic and inorganic phosphates inside the root. Tomography-based root imaging revealed a markedly different root biomass and root architecture for poplar samples inoculated with the phosphate solubilizing bacteria strains. Proteomics characterization on poplar roots coupled with protein network analysis revealed novel proteins and metabolic pathways with possible involvement in endophyte enriched phosphorus uptake. These findings suggest an important role of endophytes for phosphorus acquisition and provide a deeper understanding of the critical symbiotic associations between poplar and the endophytic bacteria.

The Effect of Microbial Endophyte Consortia on Pseudotsuga menziesii and Thuja plicata Survival, Growth, and Physiology Across Edaphic Gradients.

Increased frequency of droughts and degraded edaphic conditions decreases the success of many reforestation efforts in the Pacific Northwest. Microbial endophyte consortia have been demonstrated to contribute to plant growth promotion and protection from abiotic and biotic stresses - specifically drought conditions - across a number of food crops but for limited tree species. Our research aimed to investigate the potential to improve establishment of economically and ecologically important conifers through a series of in situ field trials and ex situ simulations. Microbial endophyte consortia from Salicaceae, previously shown to confer drought tolerance, and conifer endophyte strains with potentially symbiotic traits were selected for trials with Douglas-fir (Pseudotsuga menziesii) and western redcedar (Thuja plicata). Reductive experimentation was used to subject seedlings to a spectrum of simulated drought levels or presence/absence of fertilizer, testing hypotheses that endophyte consortia impart improved drought resistance and growth promotion, respectively. Inoculation from Salicaceae consortia significantly (p ≤ 0.05) improved survival among seedlings of both species subject to increasing drought stress, with T. plicata seedlings surviving at twofold higher rates in extreme drought conditions. Both species demonstrated improved growth 540 days after inoculation of seed with conifer derived consortia. In the carefully controlled greenhouse experiments with both species, seedling Fv/Fm and SPAD values remained significantly (p ≤ 0.05) more stable in inoculated treatment groups as stress increased. Our findings confirm that multi-strain consortia may be applied as seed or field amendment to conifers, and the approach is efficient in garnering a positive growth response and can mitigate abiotic stressors.

Variable Nitrogen Fixation in Wild Populus.

The microbiome of plants is diverse, and like that of animals, is important for overall health and nutrient acquisition. In legumes and actinorhizal plants, a portion of essential nitrogen (N) is obtained through symbiosis with nodule-inhabiting, N2-fixing microorganisms. However, a variety of non-nodulating plant species can also thrive in natural, low-N settings. Some of these species may rely on endophytes, microorganisms that live within plants, to fix N2 gas into usable forms. Here we report the first direct evidence of N2 fixation in the early successional wild tree, Populus trichocarpa, a non-leguminous tree, from its native riparian habitat. In order to measure N2 fixation, surface-sterilized cuttings of wild poplar were assayed using both 15N2 incorporation and the commonly used acetylene reduction assay. The 15N label was incorporated at high levels in a subset of cuttings, suggesting a high level of N-fixation. Similarly, acetylene was reduced to ethylene in some samples. The microbiota of the cuttings was highly variable, both in numbers of cultured bacteria and in genetic diversity. Our results indicated that associative N2-fixation occurred within wild poplar and that a non-uniformity in the distribution of endophytic bacteria may explain the variability in N-fixation activity. These results point to the need for molecular studies to decipher the required microbial consortia and conditions for effective endophytic N2-fixation in trees.