Spores of arbuscular mycorrhizal fungi host surprisingly diverse communities of endobacteria.

Arbuscular mycorrhizal fungi (AMF) are ubiquitous plant root symbionts, which can house two endobacteria: Ca. Moeniiplasma glomeromycotorum (CaMg) and Ca. Glomeribacter gigasporarum (CaGg). However, little is known about their distribution and population structure in natural AMF populations and whether AMF can harbour other endobacteria. We isolated AMF from two environments and conducted detailed analyses of endobacterial communities associated with surface-sterilised AMF spores. Consistent with the previous reports, we found that CaMg were extremely abundant (80%) and CaGg were extremely rare (2%) in both environments. Unexpectedly, we discovered an additional and previously unknown level of bacterial diversity within AMF spores, which extended beyond the known endosymbionts, with bacteria belonging to 10 other phyla detected across our spore data set. Detailed analysis revealed that: CaGg were not limited in distribution to the Gigasporaceae family of AMF, as previously thought; CaMg population structure was driven by AMF host genotype; and a significant inverse correlation existed between the diversity of CaMg and diversity of all other endobacteria. Based on these data, we generate novel testable hypotheses regarding the function of CaMg in AMF biology by proposing that they might act as conditional mutualists of AMF.

Evidence of a selective and bi-directional relationship between arbuscular mycorrhizal fungal and bacterial communities co-inhabiting plant roots.

Arbuscular mycorrhizal fungi (AMF) provide plants with vital mineral nutrients and co-exist inside the roots alongside a complex community of bacterial endophytes. These co-existing AMF and bacterial root communities have been studied individually and are known to be influenced in structure by different environmental parameters. However, the extent to which they are affected by environmental parameters and by each other is completely unknown. The current study addressed this knowledge gap by characterising AMF and bacterial communities inside plant roots from a natural and an agricultural ecosystem. Using multivariate modelling, the relative contribution of environmental parameters in structuring the two communities was quantified at different spatial scales. Using this model, it was possible to then remove the contribution of environmental parameters and show that the co-existing AMF and bacterial communities were significantly correlated with each other, explaining up to 36% of each other's variance. Notably, this was not due to the presence of know AMF endobacteria, as removal of endobacterial reads maintained the significance of correlation. These findings provide the first empirical evidence of a selective and bi-directional relationship between AMF and bacteria co-inhibiting plant roots and indicate that a significant fraction of this covariation is due to biological and ecological interactions between them.

Antibiotic resistance in grass and soil.

Antibiotic resistance is currently one of the greatest threats to human health. The global overuse of antibiotics in human medicine and in agriculture has resulted in the proliferation and dissemination of a multitude of antibiotic resistance genes (ARGs). Despite a large proportion of antibiotics being used in agriculture, little is understood about how this may contribute to the overall antibiotic resistance crisis. The use of manure in agriculture is a traditional and widespread practice and is essential for returning nutrients to the soil; however, the impact of continuous manure application on the environmental microbiome and resistome is unknown. The use of antibiotics in animal husbandry in therapeutic and sub-therapeutic doses creates a selective pressure for ARGs in the gut microbiome of the animal, which is then excreted in the faeces. Therefore, the application of manure to agricultural land is a potential route for the transmission of antibiotic-resistant bacteria from livestock to crops, animals and humans. It is of vital importance to understand the mechanisms behind ARG enrichment and its maintenance both on the plant and within the soil microbiome to mitigate the spread of this resistance to animals and humans. Understanding this link between human health, animal health, plant health and the environment is crucial to inform implementation of new regulations and practice regarding antibiotic use in agriculture and manure application, aimed at ensuring the antibiotic resistance crisis is not aggravated.

Holistic Evaluation of Field-Scale Denitrifying Bioreactors as a Basis to Improve Environmental Sustainability.

Denitrifying bioreactors convert nitrate-nitrogen (NO-N) to di-nitrogen and protect water quality. Herein, the performance of a pilot-scale bioreactor (10 m long, 5 m wide, 2 m deep) containing seven alternating cells filled with either sandy loam soil or lodgepole pine woodchip and with a novel "zig-zag" flow pattern was investigated. The influent water had an average NO-N concentration of 25 mg L. The performance of the bioreactor was evaluated in two scenarios. In Scenario 1, only NO-N removal was evaluated; in Scenario 2, NO-N removal, ammonium-N (NH-N), and dissolved reactive phosphorus (DRP) generation was considered. These data were used to generate a sustainability index (SI), which evaluated the overall performance taking these parameters into account. In Scenario 1, the bioreactor was a net reducer of contaminants, but it transformed into a net producer of contaminants in Scenario 2. Inquisition of the data using these scenarios meant that an optimum bioreactor design could be identified. This would involve reduction to two cells: a single sandy loam soil cell followed by a woodchip cell, which would remove NO-N and reduce greenhouse gas (GHG) emissions and DRP losses. An additional post-bed chamber containing media to eliminate NH-N and surface capping to reduce GHG emissions further is advised. Scenario modeling, such as that proposed in this paper, should ideally include GHG in the SI, but because different countries have different emission targets, future work should concentrate on the development of geographically appropriate weightings to facilitate the incorporation of GHG into a SI.

Differential impact of swine, bovine and poultry manure on the microbiome and resistome of agricultural grassland.

Land spreading of animal manure is an essential process in agriculture. Despite the importance of grassland in global food security the potential of the grass phyllosphere as a reservoir of antimicrobial resistance (AMR) is unknown. Additionally, the comparative risk associated with different manure sources is unclear. Due to the One Health nature of AMR there is an urgent need to fully understand the risk associated with AMR at the agriculture - environmental nexus. We performed a grassland field study to assess and compare the relative and temporal impact of bovine, swine and poultry manure application on the grass phyllosphere and soil microbiome and resistome over a period of four months, using 16S rRNA amplicon sequencing and high-throughput quantitative PCR (HT-qPCR). The soil and grass phyllosphere contained a diverse range of antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs). Manure treatment was found to introduce ARGs belonging to clinically important antimicrobial classes, such as aminoglycoside and sulphonamide into grass and soil. Temporal analysis of ARGs and MGEs associated with manure treatment indicated ARGs patterns were similar across the different manure types in the manure treated soil and grass phyllosphere. Manure treatment resulted in the enrichment in members of the indigenous microbiota and the introduction of manure associated bacteria, with this impact extending past the recommended six-week exclusion period. However, these bacteria were in low relative abundance and manure treatment was not found to significantly impact the overall composition of the microbiome or resistome. This provides evidence that the current guidelines facilitate reduction of biological risk to livestock. Additionally, in soil and grass samples MGEs correlated with ARGs from clinically important antimicrobial classes, indicating the key role MGEs play in horizontal gene transfer in agricultural grassland. These results demonstrate the role of the grass phyllosphere as an under-studied sink of AMR.

Long-term persistence and leaching of Escherichia coli in temperate maritime soils.

Enteropathogen contamination of groundwater, including potable water sources, is a global concern. The spreading on land of animal slurries and manures, which can contain a broad range of pathogenic microorganisms, is considered a major contributor to this contamination. Some of the pathogenic microorganisms applied to soil have been observed to leach through the soil into groundwater, which poses a risk to public health. There is a critical need, therefore, for characterization of pathogen movement through the vadose zone for assessment of the risk to groundwater quality due to agricultural activities. A lysimeter experiment was performed to investigate the effect of soil type and condition on the fate and transport of potential bacterial pathogens, using Escherichia coli as a marker, in four Irish soils (n = 9). Cattle slurry (34 tonnes per ha) was spread on intact soil monoliths (depth, 1 m; diameter, 0.6 m) in the spring and summer. No effect of treatment or the initial soil moisture on the E. coli that leached from the soil was observed. Leaching of E. coli was observed predominantly from one soil type (average, 1.11 +/- 0.77 CFU ml(-1)), a poorly drained Luvic Stagnosol, under natural rainfall conditions, and preferential flow was an important transport mechanism. E. coli was found to have persisted in control soils for more than 9 years, indicating that autochthonous E. coli populations are capable of becoming naturalized in the low-temperature environments of temperate maritime soils and that they can move through soil. This may compromise the use of E. coli as an indicator of fecal pollution of waters in these regions.

Characterization of environmentally persistent Escherichia coli isolates leached from an Irish soil.

Soils are typically considered to be suboptimal environments for enteric organisms, but there is increasing evidence that Escherichia coli populations can become resident in soil under favorable conditions. Previous work reported the growth of autochthonous E. coli in a maritime temperate Luvic Stagnosol soil, and this study aimed to characterize, by molecular and physiological means, the genetic diversity and physiology of environmentally persistent E. coli isolates leached from the soil. Molecular analysis (16S rRNA sequencing, enterobacterial repetitive intergenic consensus PCR, pulsed-field gel electrophoresis, and a multiplex PCR method) established the genetic diversity of the isolates (n = 7), while physiological methods determined the metabolic capability and environmental fitness of the isolates, relative to those of laboratory strains, under the conditions tested. Genotypic analysis indicated that the leached isolates do not form a single genetic grouping but that multiple genotypic groups are capable of surviving and proliferating in this environment. In physiological studies, environmental isolates grew well across a broad range of temperatures and media, in comparison with the growth of laboratory strains. These findings suggest that certain E. coli strains may have the ability to colonize and adapt to soil conditions. The resulting lack of fecal specificity has implications for the use of E. coli as an indicator of fecal pollution in the environment.

Clay mineral type effect on bacterial enteropathogen survival in soil.

Enteropathogens released into the environment can represent a serious risk to public health. Soil clay content has long been known to have an important effect on enteropathogen survival in soil, generally enhancing survival. However, clay mineral composition in soils varies, and different clay minerals have specific physiochemical properties that would be expected to impact differentially on survival. This work investigated the effect of clay materials, with a predominance of a particular mineral type (montmorillonite, kaolinite, or illite), on the survival in soil microcosms over 96 days of Listeria monocytogenes, Salmonella Dublin, and Escherichia coli O157. Clay mineral addition was found to alter a number of physicochemical parameters in soil, including cation exchange capacity and surface area, and this was specific to the mineral type. Clay mineral addition enhanced enteropathogen survival in soil. The type of clay mineral was found to differentially affect enteropathogen survival and the effect was enteropathogen-specific.

Insights into the low-temperature adaptation and nutritional flexibility of a soil-persistent Escherichia coli.

An understanding of the survival capacity of Escherichia coli in soil is critical for the evaluation of its role as a faecal indicator. Recent reports that E. coli can become long-term residents in maritime temperate soils have raised the question of how the organism survives and competes for niche space in the suboptimal soil environment. The ability of an environmental isolate to utilize 380 substrates was assessed together with that of a reference laboratory strain (E. coli K12) at both 15 and 37 °C. At 15 °C, the environmental strain could utilize 161 substrates, with only 67 utilizable by the reference strain, while at 37 °C, 239 and 223 substrates could be utilized by each strain respectively. An investigation into the cold response of the strains revealed that E. coli K12 was found to reduce the expression of biosynthetic proteins at 15 °C, while the environmental isolate seemed to switch on proteins involved in stress response, suggesting low-temperature adaptation in the latter. Taken together, the results indicate that the environmentally persistent E. coli strain is well adapted to use a wide range of nutrient sources at 15 °C and to direct its protein expression to maintain a relatively fast growth rate at low temperature.