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.

Effect of Arsenic on EPS Synthesis, Biofilm Formation, and Plant Growth-Promoting Abilities of the Endophytes Pseudomonas PD9R and Rahnella laticis PD12R.

Phytoremediation, a cost-effective, eco-friendly alternative to conventional remediation, could expand efforts to remediate arsenic-contaminated soils. As with other pollutants, the plant microbiome may improve phytoremediation outcomes for arsenic-contaminated sites. We used in vitro and in silico methods to compare the arsenic resistance mechanisms, synthesis of extracellular polymeric substances (EPS), biofilm formation, and plant growth-promoting abilities of the endophytes Pseudomonas sp. PD9R and Rahnella laticis PD12R. PD12R, which tolerates arsenate (As(V)) and arsenite (As(III)) to concentrations fivefold greater than PD9R, synthesizes high volumes of EPS in response to arsenic, and sequesters arsenic in the capsular EPS and cells. While arsenic exposure induced EPS synthesis in both strains, only PD12R continued to form biofilms at high As(III) and As(V) concentrations. The effects of endophyte inoculation on Arabidopsis growth varied by strain and As(V) concentration, and PD9R had positive effect on plants exposed to low levels of arsenic. Comparative genomic analyses exploring the EPS synthesis and arsenic resistance mechanisms against other Pseudomonas and Rahnella strains suggest that both strains possess atypical arsenic resistance mechanisms from other plant-associated strains, while the configuration of the EPS synthesis systems appeared to be more broadly distributed among plant- and non-plant-associated strains.

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.

Endophytes Increased Fruit Quality with Higher Soluble Sugar Production in Honeycrisp Apple (Malus pumila).

Endophytes are fungi, bacteria, or yeast symbionts that live in the intercellular spaces or vascular tissues of host plants. Investigations indicate that endophytes isolated from the Salicaceae family (Populus and Salix) hosts provide several benefits that promote plant growth, including but not limited to di-nitrogen fixation, plant hormone production, nutrient acquisition, stress tolerance, and defense against phytopathogens. In exchange, the microorganisms receive domicile and photosynthates. Considering the known characteristics of nitrogen fixation and plant hormone production, we hypothesized that apple trees grown under nitrogen-limited conditions would show improved biometrics with endophyte inoculation. Our research objectives were to investigate the endophyte effects on plant physiology and fruiting. We examined these effects through ecophysiology metrics involving rates of photosynthesis, stomatal conductance and density, transpiration, biomass accretion, chlorophyll content and fluorescence, and fruit soluble sugar content and biomass. Our results showed evidence of the endophytes' colonization in apple trees, decreased stomatal density, delayed leaf senescence, and increased lateral root biomass with endophytes. A highlight of the findings was a significant increase in both fruit soluble sugar content and biomass. Future research into the mechanistic underpinnings of this phenomenon stands to offer novel insights on how microbiota may alter carbohydrate metabolism under nitrogen-deficient conditions.

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.

Corrigendum: An In vitro Study of Bio-Control and Plant Growth Promotion Potential of Salicaceae Endophytes.

[This corrects the article DOI: 10.3389/fmicb.2017.00386.].

Salicaceae Endophytes Modulate Stomatal Behavior and Increase Water Use Efficiency in Rice.

Bacterial and yeast endophytes isolated from the Salicaceae family have been shown to promote growth and alleviate stress in plants from different taxa. To determine the physiological pathways through which endophytes affect plant water relations, we investigated leaf water potential, whole-plant water use, and stomatal responses of rice plants to Salicaceae endophyte inoculation under CO2 enrichment and water deficit. Daytime stomatal conductance and stomatal density were lower in inoculated plants compared to controls. Leaf ABA concentrations increased with endophyte inoculation. As a result, transpirational water use decreased significantly with endophyte inoculation while biomass did not change or slightly increased. This response led to a significant increase in cumulative water use efficiency at harvest. Different endophyte strains produced the same results in host plant water relations and stomatal responses. These stomatal responses were also observed under elevated CO2 conditions, and the increase in water use efficiency was more pronounced under water deficit conditions. The effect on water use efficiency was positively correlated with daily light integrals across different experiments. Our results provide insights on the physiological mechanisms of plant-endophyte interactions involving plant water relations and stomatal functions.

Do Endophytes Promote Growth of Host Plants Under Stress? A Meta-Analysis on Plant Stress Mitigation by Endophytes.

Endophytes are microbial symbionts living inside plants and have been extensively researched in recent decades for their functions associated with plant responses to environmental stress. We conducted a meta-analysis of endophyte effects on host plants' growth and fitness in response to three abiotic stress factors: drought, nitrogen deficiency, and excessive salinity. Ninety-four endophyte strains and 42 host plant species from the literature were evaluated in the analysis. Endophytes increased biomass accumulation of host plants under all three stress conditions. The stress mitigation effects by endophytes were similar among different plant taxa or functional groups with few exceptions; eudicots and C4 species gained more biomass than monocots and C3 species with endophytes, respectively, under drought conditions. Our analysis supports the effectiveness of endophytes in mitigating drought, nitrogen deficiency, and salinity stress in a wide range of host species with little evidence of plant-endophyte specificity.

Genomic features of bacterial adaptation to plants.

Plants intimately associate with diverse bacteria. Plant-associated bacteria have ostensibly evolved genes that enable them to adapt to plant environments. However, the identities of such genes are mostly unknown, and their functions are poorly characterized. We sequenced 484 genomes of bacterial isolates from roots of Brassicaceae, poplar, and maize. We then compared 3,837 bacterial genomes to identify thousands of plant-associated gene clusters. Genomes of plant-associated bacteria encode more carbohydrate metabolism functions and fewer mobile elements than related non-plant-associated genomes do. We experimentally validated candidates from two sets of plant-associated genes: one involved in plant colonization, and the other serving in microbe-microbe competition between plant-associated bacteria. We also identified 64 plant-associated protein domains that potentially mimic plant domains; some are shared with plant-associated fungi and oomycetes. This work expands the genome-based understanding of plant-microbe interactions and provides potential leads for efficient and sustainable agriculture through microbiome engineering.

Enhanced Degradation of TCE on a Superfund Site Using Endophyte-Assisted Poplar Tree Phytoremediation.

Trichloroethylene (TCE) is a widespread environmental pollutant common in groundwater plumes associated with industrial manufacturing areas. We had previously isolated and characterized a natural bacterial endophyte, Enterobacter sp. strain PDN3, of poplar trees, that rapidly metabolizes TCE, releasing chloride ion. We now report findings from a successful three-year field trial of endophyte-assisted phytoremediation on the Middlefield-Ellis-Whisman Superfund Study Area TCE plume in the Silicon Valley of California. The inoculated poplar trees exhibited increased growth and reduced TCE phytotoxic effects with a 32% increase in trunk diameter compared to mock-inoculated control poplar trees. The inoculated trees excreted 50% more chloride ion into the rhizosphere, indicative of increased TCE metabolism in planta. Data from tree core analysis of the tree tissues provided further supporting evidence of the enhanced rate of degradation of the chlorinated solvents in the inoculated trees. Test well groundwater analyses demonstrated a marked decrease in concentration of TCE and its derivatives from the tree-associated groundwater plume. The concentration of TCE decreased from 300 µg/L upstream of the planted area to less than 5 µg/L downstream of the planted area. TCE derivatives were similarly removed with cis-1,2-dichloroethene decreasing from 160 µg/L to less than 5 µg/L and trans-1,2-dichloroethene decreasing from 3.1 µg/L to less than 0.5 µg/L downstream of the planted trees. 1,1-dichloroethene and vinyl chloride both decreased from 6.8 and 0.77 µg/L, respectively, to below the reporting limit of 0.5 µg/L providing strong evidence of the ability of the endophytic inoculated trees to effectively remove TCE from affected groundwater. The combination of native pollutant-degrading endophytic bacteria and fast-growing poplar tree systems offers a readily deployable, cost-effective approach for the degradation of TCE, and may help mitigate potential transfer up the food chain, volatilization to the atmosphere, as well as direct phytotoxic impacts to plants used in this type of phytoremediation.

A Field Trial of TCE Phytoremediation by Genetically Modified Poplars Expressing Cytochrome P450 2E1.

A controlled field study was performed to evaluate the effectiveness of transgenic poplars for phytoremediation. Three hydraulically contained test beds were planted with 12 transgenic poplars, 12 wild type (WT) poplars, or left unplanted, and dosed with equivalent concentrations of trichloroethylene (TCE). Removal of TCE was enhanced in the transgenic tree bed, but not to the extent of the enhanced removal observed in laboratory studies. Total chlorinated ethene removal was 87% in the CYP2E1 bed, 85% in the WT bed, and 34% in the unplanted bed in 2012. Evapotranspiration of TCE from transgenic leaves was reduced by 80% and diffusion of TCE from transgenic stems was reduced by 90% compared to WT. Cis-dichloroethene and vinyl chloride levels were reduced in the transgenic tree bed. Chloride ion accumulated in the planted beds corresponding to the TCE loss, suggesting that contaminant dehalogenation was the primary loss fate.

An In vitro Study of Bio-Control and Plant Growth Promotion Potential of Salicaceae Endophytes.

Microbial communities in the endosphere of Salicaceae plants, poplar (Populus trichocarpa) and willow (Salix sitchensis), have been demonstrated to be important for plant growth promotion, protection from biotic and abiotic stresses, and degradation of toxic compounds. Our study aimed to investigate bio-control activities of Salicaceae endophytes against various soil borne plant pathogens including Rhizoctonia solani AG-8, Fusarium culmorum, Gaeumannomyces graminis var. tritici, and Pythium ultimum. Additionally, different plant growth promoting traits such as biological nitrogen fixation (BNF), indole-3-acetic acid (IAA) biosynthesis, phosphate solubilization, and siderophore production were assessed in all bio-control positive strains. Burkholderia, Rahnella, Pseudomonas, and Curtobacterium were major endophyte genera that showed bio-control activities in the in-vitro assays. The bio-control activities of Burkholderia strains were stronger across all tested plant pathogens as compared to other stains. Genomes of sequenced Burkholderia strains WP40 and WP42 were surveyed to identify the putative genes involved in the bio-control activities. The ocf and hcnABC gene clusters responsible for biosynthesis of the anti-fungal metabolites, occidiofungin and hydrogen cyanide, are present in the genomes of WP40 and WP42. Nearly all endophyte strains showing the bio-control activities produced IAA, solubilized tricalcium phosphate, and synthesized siderophores in the culture medium. Moreover, some strains reduced acetylene into ethylene in the acetylene reduction assay, a common assay used for BNF. Salicaceae endophytes could be useful for bio-control of various plant pathogens, and plant growth promotion possibly through the mechanisms of BNF, IAA production, and nutrient acquisition.

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.

Latent homology and convergent regulatory evolution underlies the repeated emergence of yeasts.

Convergent evolution is common throughout the tree of life, but the molecular mechanisms causing similar phenotypes to appear repeatedly are obscure. Yeasts have arisen in multiple fungal clades, but the genetic causes and consequences of their evolutionary origins are unknown. Here we show that the potential to develop yeast forms arose early in fungal evolution and became dominant independently in multiple clades, most likely via parallel diversification of Zn-cluster transcription factors, a fungal-specific family involved in regulating yeast-filamentous switches. Our results imply that convergent evolution can happen by the repeated deployment of a conserved genetic toolkit for the same function in distinct clades via regulatory evolution. We suggest that this mechanism might be a common source of evolutionary convergence even at large time scales.

Biological nitrogen fixation and biomass accumulation within poplar clones as a result of inoculations with diazotrophic endophyte consortia.

Sustainable production of biomass for bioenergy relies on low-input crop production. Inoculation of bioenergy crops with plant growth-promoting endophytes has the potential to reduce fertilizer inputs through the enhancement of biological nitrogen fixation (BNF). Endophytes isolated from native poplar growing in nutrient-poor conditions were selected for a series of glasshouse and field trials designed to test the overall hypothesis that naturally occurring diazotrophic endophytes impart growth promotion of the host plants. Endophyte inoculations contributed to increased biomass over uninoculated control plants. This growth promotion was more pronounced with multi-strain consortia than with single-strain inocula. Biological nitrogen fixation was estimated through (15)N isotope dilution to be 65% nitrogen derived from air (Ndfa). Phenotypic plasticity in biomass allocation and branch production observed as a result of endophyte inoculations may be useful in bioenergy crop breeding and engineering programs.

Diversity of rhizobia associated with leguminous trees growing in South Korea.

This study was carried out to examine the diversity of 34 isolates collected from 11 species of leguminous trees growing in South Korea. Phylogenetic relationships between these 34 isolates and reference strains of the Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium and Ensifer/Sinorhizobium were analysed by using 16S rRNA gene sequences. Twenty-one isolates were related to Mesorhizobium, four isolates to Rhizobium, and nine isolates to Bradyrhizobium. But none of isolates were related to Sinorhizobium/Ensifer and Azorhizobium. Robinia pseudoacacia and Amorpha fruticosa were nodulated by various genotypes of rhizobia out of them, most of the isolates belonged to the genus Mesorhizobium. The isolates from Lespedeza bicolar belonged to diverse genera of Mesorhizobium, Rhizobium, and Bradyrhizobium. The isolates from Maackia amurensis and Lespedeza maximowiezii var. tomentella were phylogenetically related to the genera of Bradyrhizobium. PCR-based RAPD method and phylogenetic analysis of the 16S rRNA results revealed a high phylogenetic diversity of rhizobial strains nodulating leguminous trees in South Korea. Also, the relationships between host and bacterial phylogenies showed that only Robinia pseudoacacia, and Wisteria floribunda have significantly unique branch length than expected by chance based on phylogenetic tree.

Novel endophytic yeast Rhodotorula mucilaginosa strain PTD3 II: production of xylitol and ethanol in the presence of inhibitors.

A systematic study was conducted characterizing the effect of furfural, 5-hydroxymethylfurfural (5-HMF), and acetic acid concentration on the production of xylitol and ethanol by a novel endophytic yeast, Rhodotorula mucilaginosa strain PTD3. The influence of different inhibitor concentrations on the growth and fermentation abilities of PTD3 cultivated in synthetic nutrient media containing 30 g/l xylose or glucose were measured during liquid batch cultures. Concentrations of up to 5 g/l of furfural stimulated production of xylitol to 77 % of theoretical yield (10 % higher compared to the control) by PTD3. Xylitol yields produced by this yeast were not affected in the presence of 5-HMF at concentrations of up to 3 g/l. At higher concentrations of furfural and 5-HMF, xylitol and ethanol yields were negatively affected. The higher the concentration of acetic acid present in a media, the higher the ethanol yield approaching 99 % of theoretical yield (15 % higher compared to the control) was produced by the yeast. At all concentrations of acetic acid tested, xylitol yield was lowered. PTD3 was capable of metabolizing concentrations of 5, 15, and 5 g/l of furfural, 5-HMF, and acetic acid, respectively. This yeast would be a potent candidate for the bioconversion of lignocellulosic sugars to biochemicals given that in the presence of low concentrations of inhibitors, its xylitol and ethanol yields are stimulated, and it is capable of metabolizing pretreatment degradation products.

Phytoremediation of chlorpyrifos by Populus and Salix.

Chlorpyrifos is one of the commonly used organophosphorus insecticides that are implicated in serious environmental and human health problems. To evaluate plant potential for uptake of chlorpyrifos, several plant species of poplar (Populus sp.) and willow (Salix sp.) were investigated. Chlorpyrifos was taken up from nutrient solution by all seven plant species. Significant amounts of chlorpyrifos accumulated in plant tissues, and roots accumulated higher concentrations of chlorpyrifos than did shoots. Chlorpyrifos did not persist in the plant tissues, suggesting further metabolism of chlorpyrifos in plant tissue. To our knowledge, this work represents the first report for phytoremediation of chlorpyrifos using poplar and willow plants.