Assembly and potential transmission of the Lens culinaris seed microbiome.

Soil is an important source of bacteria and fungi for the plant, but seeds can also provide microbial inocula through heritable or stochastic assembly. Seed-associated microbial communities can potentially interact with the host plant through multiple generations. Here, we assessed the impact of two different soil types on the seed microbiome assembly of seven lentil (Lens culinaris) genotypes under environmentally controlled conditions and examined the vertical transmission of bacterial communities from seed to seed across two generations. Bulk soil microbiomes and seed microbiomes were characterized using high-throughput amplicon sequencing of the bacterial 16S rRNA gene. Our results revealed that bacterial communities in the two soils differed significantly and that bacterial communities associated with seeds were significantly impacted by genotype (15%) in one of the soils. Co-occurrence of amplicon sequence variants between generations suggests that members of the genera Cutibacterium, Methylobacterium, Sphingomonas, Streptococcus and Tepidimonas are transmitted and preserved in lentil genotypes irrespective of the soil in which they were grown. Increasing our knowledge of how microbial communities carried by seeds are assembled, transmitted and preserved offers a promising way for future breeding programs to consider microbial communities when selecting for more resilient and productive cultivars.

Impact of diesel and biodiesel contamination on soil microbial community activity and structure.

Soil contamination as a result of oil spills is a serious issue due to the global demand for diesel fuel. As an alternative to diesel, biodiesel has been introduced based on its high degradability rates and potential for reducing of greenhouse gases emissions. This study assessed the impacts diesel and biodiesel contamination on soil microbial community activity and structure. Our results suggest higher microbial activity in biodiesel contaminated soils and analysis of PLFA profiles confirmed shifts in microbial community structure in response to contamination. High-throughput 16S rRNA amplicon sequencing also revealed a lower bacterial richness and diversity in contaminated soils when compared to control samples, supporting evidence of the detrimental effects of hydrocarbons on soil microbiota. Control samples comprised mostly of Actinobacteria, whereas Proteobacteria were predominantly observed in diesel and biodiesel contaminated soils. At genus level, diesel and biodiesel amendments highly selected for Rhodococcus and Pseudomonas spp., respectively. Moreover, predicted functional profiles based on hydrocarbon-degrading enzymes revealed significant differences between contaminated soils mostly due to the chemical composition of diesel and biodiesel fuel. Here, we also identified that Burkholderiaceae, Novosphingobium, Anaeromyxobacter, Pseudomonas and Rhodococcus were the main bacterial taxa contributing to these enzymes. Together, this study supports the evidence of diesel/biodiesel adverse effects in soil microbial community structure and highlights microbial taxa that could be further investigated for their biodegradation potential.

Complete Genome Sequence of a Pseudomonas simiae Strain with Biocontrol Potential against Aphanomyces Root Rot.

Aphanomyces euteiches is a soilborne plant pathogen. It causes severe root rot in leguminous crop species. We report the complete genome sequence of a biocontrol strain, Pseudomonas simiae K-Hf-L9. The strain inhibited Aphanomyces euteiches mycelia and zoospores and suppressed root rot in field peas grown under controlled growth chamber conditions.

Crop, genotype, and field environmental conditions shape bacterial and fungal seed epiphytic microbiomes.

Seeds are reproductive structures able to carry and transfer microorganisms that play an important role in plant fitness. Genetic and external factors are reported to be partly responsible for the plant microbiome assemblage, but their contribution in seeds is poorly understood. In this study, wheat, canola, and lentil seeds were analyzed to characterize diversity, structure, and persistence of seed-associated microbial communities. Five lines and 2 generations of each crop were subjected to high-throughput amplicon sequencing of the 16S rRNA and internal transcribed spacer (ITS) regions. Bacterial and fungal communities differed most by crop type (30% and 47% of the variance), while generation explained an additional 10% and 15% of the variance. The offspring (i.e., generation harvested in 2016 at the same location) exhibited a higher number of common amplicon sequence variants (ASVs) and less variability in microbial composition. Additionally, in every sample analyzed, a "core microbiome" was detected consisting of 5 bacterial and 12 fungal ASVs. Our results suggest that crop, genotype, and field environmental conditions contributed to the seed-associated microbial assemblage. These findings not only expand our understanding of the factors influencing the seed microbiome but may also help us to manipulate and exploit the microbiota naturally carried by seeds.

A Meta-Analysis to Determine the State of Biological Control of Aphanomyces Root Rot.

The increasing incidence and prevalence of the pathogen Aphanomyces euteiches in various pulse-growing regions worldwide necessitates the development of effective management strategies, including biological control agents. Numerous labs have undertaken research examining biological control methods to evaluate aphanomyces root rot suppression in multistep processes that include isolation of inhibitory organisms, lab assays, growth chamber assays, and field trials. Given the emergence of various biocontrol agents and the need to mitigate aphanomyces yield losses, we have undertaken a meta-analysis approach to analyze the effectiveness of biocontrol agents in relation to application method, biocontrol agent richness, biocontrol agent type, the type of study, and reporting system-oriented moderator variables. An effect size, calculated as a natural log response ratio, resulted in a summary weighted mean of -0.411, suggesting the overall effectiveness of biocontrol agents (p < .001). Aphanomyces root rot suppression using biological treatments showed significant heterogeneity for all moderator variables, confirming that the studies do not share a common effect size and the use of a random effect model was appropriate. Across studies, meta-analyses revealed that soil amendments, biocontrol agent application as a seed coating and suspension, bacterial and fungal biocontrol agents, mixed applications, growth chamber and field studies, and qualitative and quantitative reporting systems were all associated with significantly positive outcomes for aphanomyces root rot suppression. Our findings suggest that there is potential promise for biological control of aphanomyces root rot, and more field trials need to be conducted to demonstrate the efficacy level observed under growth chamber conditions. Moreover, we identified a lack of detailed understanding of the mechanism(s) of biological control of aphanomyces root rot as a research priority.

Hydrocarbon-degrading genes in root endophytic communities on oil sands reclamation covers.

In response to environmental regulations, the Canadian oil sands industry aims to reclaim all disturbed areas to equivalent land capability prior to mining operations. However, tailing sands used in reclamation contain residual hydrocarbons and plants growing in these areas may rely on hydrocarbon-degrading endophytic bacteria to survive. This study assessed the hydrocarbon-degrading potential (genes: CYP153, alkB and nah) of culturable and unculturable endophytic bacteria associated with annual barley (Hordeum vulgare) and sweet clover (Melilotus albus) plants in an oil sands reclamation area. Our results suggest higher CYP153 gene copy numbers in sweet clover when compared to barley. Yet, no significant differences were detected in 16S rRNA, alkB and nah genes. In addition, total hydrocarbons, pH, total soil carbon, organic carbon and total nitrogen play an important role in determining hydrocarbon-degrading potential in these communities. The assessment of culturable hydrocarbon-degrading bacteria revealed 42 isolates (total of 316) that were positive for at least one hydrocarbon-degrading gene. Most of these isolates were positive for alkB, and closely match the database for Pantoea, Pseudomonas and Enterobacter spp. Thus, to improve oil sands reclamation strategies, plant inoculation with select hydrocarbon-degrading endophytes could be used to increase plant tolerance and hydrocarbon degradation in these areas.

Bacterial microbiome associated with the rhizosphere and root interior of crops in Saskatchewan, Canada.

Rhizosphere and root associated bacteria are key components of plant microbiomes and influence crop production. In sustainable agriculture, it is important to investigate bacteria diversity in various plant species and how edaphic factors influence the bacterial microbiome. In this study, we used high-throughput sequencing to assess bacterial communities associated with the rhizosphere and root interior of canola, wheat, field pea, and lentil grown at four locations in Saskatchewan, Canada. Rhizosphere bacteria communities exhibited distinct profiles among crops and sampling locations. However, each crop was associated with distinct root endophytic bacterial communities, suggesting that crop species may influence the selection of root bacterial microbiome. Proteobacteria, Actinobacteria, and Bacteroidetes were the dominant phyla in the root interior, whereas Gemmatimonadetes, Firmicutes, and Acidobacteria were prevalent in the rhizosphere soil. Pseudomonas and Stenotrophomonas were predominant in the rhizosphere and root interior, whereas Acinetobacter, Arthrobacter, Rhizobium, Streptomyces, Variovorax, and Xanthomonas were dominant in the root interior of all crops. The relative abundance of specific bacterial groups in the rhizosphere correlated with soil pH and silt and organic matter contents; however, there was no correlation between root endophytes and analyzed soil properties. These results suggest that the root microbiome may be modulated by plant factors rather than soil characteristics.

Potential use of endophytic root bacteria and host plants to degrade hydrocarbons.

Microbe-assisted phytoremediation depends on competent root-associated microorganisms that enhance remediation efficiency of organic compounds. Endophytic bacteria are a key element of the root microbiome and may assist plant degradation of contaminants. The aim of this study was to investigate the application of four hydrocarbon-degrading endophytic strains previously isolated from an oil sands reclamation area. Strains EA1-17 (Stenotrophomonas sp.), EA2-30 (Flavobacterium sp.), EA4-40 (Pantoea sp.), and EA6-5 (Pseudomonas sp.) were inoculated in white sweet clover growing on soils amended with diesel at 5,000, 10,000, and 20,000 mg·kg-1. Our results indicate that plant growth inhibition caused by diesel fuel toxicity was overcome in inoculated plants, which showed significantly higher plant biomass. Analysis of soil F2 and F3 hydrocarbon fractions also revealed that these soils were remediated by inoculated plants when diesel was applied at 10,000 mg·kg-1 and 20,000 mg·kg-1. In addition, quantification of hydrocarbon-degrading genes suggests that all bacterial strains successfully colonized sweet clover plants. Overall, the endophytic strain EA6-5 (Pseudomonas sp.), which harbored hydrocarbon-degrading genes, was the most effective candidate in phytoremediation experiments and could be a strategy to increase plant tolerance and hydrocarbon degradation in contaminated (e.g., diesel fuel) soils.

Microbial communities associated with barley growing in an oil sands reclamation area in Alberta, Canada.

Microbial communities that colonize the plant rhizosphere and the root interior can ameliorate plant stress and promote growth. These plant-microbe associations are being investigated to assist in reclamation soils in northern Alberta. This study assessed the diversity of bacterial species associated with barley plants growing at different cover managements and slope positions in an oil sands reclamation area. Phospholipid fatty acid analysis of the microbial communities indicated that both cover type and slope, in addition to soil total and organic carbon, NH4+, and organic matter, were significant determinants of microbial community composition. However, analysis of denaturing gel gradient electrophoresis (DGGE) banding patterns revealed that while most bulk and rhizosphere soils differentiated by cover management, no clustering was observed in endophytes. In addition, techniques to assess culture-dependent endophytic bacteria revealed a dominance of the class Gammaproteobacteria, in which Enterobacteriaceae (44%), Xanthomonaceae (30%), and Pseudomonaceae (26%) were the most abundant families in this class. Several endophytic isolates also matched those from DGGE profiles. The results of this study suggest that plants growing on oil sands reclamation covers host a wide range of bacterial endophytes, which should be assessed as to their potential to assist plant establishment and growth at such sites.

Bacterial Root Microbiome of Plants Growing in Oil Sands Reclamation Covers.

Oil sands mining in northern Alberta impacts a large footprint, but the industry is committed to reclaim all disturbed land to an ecologically healthy state in response to environmental regulations. However, these newly reconstructed landscapes may be limited by several factors that include low soil nutrient levels and reduced microbial activity. Rhizosphere microorganisms colonize plant roots providing hosts with nutrients, stimulating growth, suppressing disease and increasing tolerance to abiotic stress. High-throughput sequencing techniques can be used to provide a detailed characterization of microbial community structure. This study used 16S rRNA amplicon sequencing to characterize the bacterial root microbiome associated with annual barley (Hordeum vulgare) and sweet clover (Melilotus albus) growing in an oil sands reclamation area. Our results indicate that Proteobacteria dominated the endosphere, whereas other phyla such as Acidobacteria and Gemmatimonadetes were restricted to the rhizosphere, suggesting that plants have the ability to select for certain soil bacterial consortia. The bacterial community in the endosphere compartments were less rich and diverse compared to the rhizosphere. Furthermore, it was apparent that sweet clover plants were more selective, as the community exhibited a lower richness and diversity compared to barley. Members of the family Rhizobiaceae, such as Sinorhizobium and Rhizobium were mainly associated with clover, whereas Acholeplasma (wall-less bacteria transmitted by insects) was unique to barley. Genera from the Enterobacteriaceae family, such as Yersinia and Lentzea were also mostly detected in barley, while other genera such Pseudomonas and Pantoea were able to successfully colonize both plants. Endophytic bacterial profiles varied within the same plant species at different sampling locations; however, these differences were driven by factors other than slope positions or cover management. Our results suggest that bacterial endophytic communities of plants growing in land reclamation systems are a subset of the rhizosphere community and selection is driven by plant factors.

Endophytic root bacteria associated with the natural vegetation growing at the hydrocarbon-contaminated Bitumount Provincial Historic site.

The Bitumount Provincial Historic site is the location of 2 of the world's first oil-extracting and -refining operations. Despite hydrocarbon levels ranging from 330 to 24 700 mg·(kg soil)-1, plants have been able to recolonize the site through means of natural revegetation. This study was designed to achieve a better understanding of the plant-root-associated bacterial partnerships occurring within naturally revegetated hydrocarbon-contaminated soils. Root endophytic bacterial communities were characterized from representative plant species throughout the site by both high-throughput sequencing and culturing techniques. Population abundance of rhizosphere and root endosphere bacteria was significantly influenced (p < 0.05) by plant species and sampling location. In general, members of the Actinomycetales, Rhizobiales, Pseudomonadales, Burkholderiales, and Sphingomonadales orders were the most commonly identified orders. Community structure of root-associated bacteria was influenced by both plant species and sampling location. Quantitative real-time polymerase chain reaction was used to determine the potential functional diversity of the root endophytic bacteria. The gene copy numbers of 16S rRNA and 2 hydrocarbon-degrading genes (CYP153 and alkB) were significantly affected (p < 0.05) by the interaction of plant species and sampling location. Our findings suggest that some of the bacterial communities detected are known to exhibit plant growth promotion characteristics.

Naturally occurring phenanthrene degrading bacteria associated with seeds of various plant species.

Seeds of 11 of 19 plant species tested yielded naturally occurring phenanthrene degrading bacteria when placed on phenanthrene impression plates. Seed associated phenanthrene degrading bacteria were mostly detected on caragana, Canada thistle, creeping red fescue, western wheatgrass, and tall wheat grass. Based on 16S rRNA analysis the most common bacteria isolated from these seeds were strains belonging to the genera Enterobacteria, Erwinia, Burkholderia, Pantoea, Pseudomonas, and Sphingomonas. These plants may provide an excellent source of pre-adapted bacterial-plant associations highly suitable for use in remediation of contaminated soil environments.

Gene expression patterns in wheat coleorhiza under cold- and biological stratification.

This study assessed germination of wheat seeds under cold and biological stratification and determined the expression level of gibberellins (GA) and abscisic acid (ABA) genes in coleorhiza. Both cold and biological stratification significantly (P<0.05) enhanced the rate and efficacy of germination. The spatial distance between the fungal endophyte and the seed can be a determining factor of biological stratification as seeds in direct contact with fungal endophyte showed the highest rate and efficacy of germination. Consistently high expression of GA3ox2 gene was found in wheat coleorhiza throughout the tested period of germination. The expression of ABA biosynthesis gene, TaNCED, was substantially higher in cold stratification seeds, reflecting the role of abscisic acid in stress-adaptation. Overall, this study provides molecular evidence of the importance of coleorhiza in germinating wheat seeds, in addition to reporting that the spatial distance between symbiotic partners may be a critical factor driving mycovitality.

Impact of arbuscular mycorrhizal fungal inoculants on subsequent arbuscular mycorrhizal fungi colonization in pot-cultured field pea (Pisum sativum L.).

The use of commercial inoculants containing non-resident arbuscular mycorrhizal fungi (AMF) is an emerging technology in field crop production in Canada. The objective of this study was to assess the impact of AMF inoculants containing either a single species (Glomus irregulare) or mixed species (G. irregulare, Glomus mosseae, and Glomus clarum) on AMF root colonization and consequent plant growth parameters of field pea grown using pot cultures. Field pea was grown in both sterilized and non-sterile (i.e., natural) field-collected soil containing resident AMF and received three inoculation treatments: uninoculated control, G. irregulare only, and a mixture of AMF species of G. irregulare, G. mosseae, and G. clarum. After 42 days, the AMF community assembled in field pea roots was assessed by cloning and sequencing analysis on the LSU-ITS-SSU rDNA gene, together with a microscopic assessment of colonization, biomass production, nutrient uptake, and N(2) fixation. The identity of AMF inoculants had a significant effect on field pea performance. The mixed species AMF inoculant performed better than the single species G. irregulare alone by promoting mycorrhizal colonization, field pea biomass, N and P uptake, and N(2) fixation and did not result in a significant compositional change of the AMF community that subsequently assembled in field pea roots. In contrast, the single species G. irregulare inoculant did not significantly enhance field pea biomass, N and P uptake, and N(2) fixation, although a significant compositional change of the subsequent AMF community was observed. No significant interactions affecting these measurements were detected between the resident AMF and the introduced AMF inoculants. The observation that the mixed species AMF inoculant promoted plant growth parameters without necessarily affecting the subsequent AMF community may have important implications regarding the use of non-resident AMF inoculants in agricultural production.

Shifts in root-associated microbial communities of Typha latifolia growing in naphthenic acids and relationship to plant health.

Naphthenic acids (NAs) are a complex mixture of organic acid compounds released during the extraction of crude oil from oil sands operations. The accumulation of toxic NAs in tailings pond water (TPW) is of significant environmental concern, and phytoremediation using constructed wetlands is one remediation option being assessed. Since root-associated microorganisms are an important factor during phytoremediation of organic compounds, this study investigated the impact of NAs on the microbial communities associated with the macrophyte Typha latifolia (cattail). Denaturing gradient gel electrophoresis revealed that the impact of NAs on microbial communities was niche dependent, with endophytic communities being the most stable and bulk water communities being the least stable. The type of NA used was significant to microbial response, with commercial NAs causing greater adverse changes than TPW NAs. In general, plant beneficial bacteria such as diazotrophs were favoured in cattails grown in TPW NAs, while potentially deleterious bacteria such as denitrifying Dechlorospirillum species increased in commercial NA treatments. These findings suggest that NAs may affect plant health by impacting root-associated microbial communities. A better understanding of these impacts may allow researchers to optimize those microbial communities that support plant health, and thus further optimize wetland treatment systems.

Ultrahigh-resolution mass spectrometry of simulated runoff from treated oil sands mature fine tailings.

There is interest in using mature fine tailings (MFT) in reclamation strategies of oil sands mining operations. However, simulated runoff from different dried MFT treatments is known to have elevated levels of salts, toxic ions, and naphthenic acids, and alkaline pH and it is phytotoxic to the emergent macrophyte, common reed (Phragmites australis). Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) of the acidic species in the runoff confirmed that the distribution of oil sands naphthenic acids and associated oil sand acids was dependent on the MFT treatment. Furthermore, FT-ICR MS studies of the acidic species in hydroponic systems revealed that there was no plant-mediated change in the electrospray ionization mass spectra of the runoff. O(o)-containing species were prevalent (>90%), O(o)S(s) were predominant (<10% relative abundance), and O(o)N(n) were least abundant in all runoff water samples. O(o)S(s) species were predominant in all the samples investigated. The heteroatomic classes present in runoff water at greater than 1% relative abundance include: O(2)N(1), O(3)N(1), O(2), O(2)S(1) O(3), O(3)S(1), O(4), O(4)S(1), O(5), O(5)S(1), O(6), O(6)S(1), O(7), O(7)S(1), O(8) and O(8)S(1). Assuming the same response factor for all O(o) species, the O(4) class, presumably dicarboxylic acids, was generally more prevalent than the O(2) class in all samples. The O(2) class is indicative of classical naphthenic acids. However, dicarboxylic acids will form negative ions more readily than the monocarboxylic acids as there are two acidic hydrogens available for formation of these species.

Phytotoxicity and naphthenic acid dissipation from oil sands fine tailings treatments planted with the emergent macrophyte Phragmites australis.

During reclamation the water associated with the runoff or groundwater flushing from dry stackable tailings technologies may become available to the reclaimed environment within an oil sands lease. Here we evaluate the performance of the emergent macrophyte, common reed (Phragmites australis), grown in chemically amended mature fine tailings (MFT) and simulated runoff/seepage water from different MFT drying treatments. The present study also investigated the phytotoxicity of the concentration of oil sands naphthenic acids (NAs) in different MFT drying chemical treatments, in both planted and unplanted systems. We demonstrate that although growth was reduced, the emergent macrophyte common reed was capable of growing in diluted unamended MFT runoff, as well as in diluted runoff from MFT amended with either 0.25% lime and gypsum or 0.5% gypsum. Common reed can thus assist in the dewatering process of oil sands MFT. However, simulated runoff or seepage waters from chemically amended and dried MFT were phytotoxic, due to combined levels of salts, naphthenic acids and pH. Phytoremediation of runoff water/ground water seepage from dry-land applied MFT will thus require pre-treatment in order to make conditions more favorable for plant growth.

Differences in phytotoxicity and dissipation between ionized and nonionized oil sands naphthenic acids in wetland plants.

Naphthenic acids (NAs) are composed of alkyl-substituted acyclic and cycloaliphatic carboxylic acids and, because they are acutely toxic to fish, are of toxicological concern. During the caustic hot-water extraction of oil from the bitumen in oil sands deposits, NAs become concentrated in the resulting tailings pond water. The present study investigated if dissipation of NAs occurs in the presence of hydroponically grown emergent macrophytes (Typha latifolia, Phragmites australis, and Scirpus acutus) to determine the potential for phytoremediation of these compounds. Plants were grown with oil sands NAs (pKa approximately 5-6) in medium at pH 7.8 (predominantly ionized NAs) and pH 5.0 (predominantly nonionized NAs) to determine if, by altering their chemical form, NAs may be more accessible to plants and, thus, undergo increased dissipation. Whereas the oil sands NA mixture in its nonionized form was more toxic to wetland plants than its ionized form, neither form appeared to be sequestered by wetland plants. The present study demonstrated that plants may selectively enhance the dissipation of individual nonionized NA compounds, which contributes to toxicity reduction but does not translate into detectable total NA dissipation within experimental error and natural variation. Plants were able to reduce the toxicity of a NA system over 30 d, increasing the median lethal concentration (LC50; % of hydroponic solution) of the medium for Daphnia magna by 23.3% +/- 8.1% (mean +/- standard error; nonionized NAs) and 37.0% +/- 2.7% (ionized NAs) as determined by acute toxicity bioassays. This reduction in toxicity was 7.3% +/- 2.6% (nonionized NAs) and 45.0% +/- 6.8% (ionized NAs) greater than that in unplanted systems.