Life at the borderlands: microbiomes of interfaces critical to One Health.

Microbiomes are foundational components of the environment that provide essential services relating to food security, carbon sequestration, human health, and the overall well-being of ecosystems. Microbiota exert their effects primarily through complex interactions at interfaces with their plant, animal, and human hosts, as well as within the soil environment. This review aims to explore the ecological, evolutionary, and molecular processes governing the establishment and function of microbiome-host relationships, specifically at interfaces critical to One Health-a transdisciplinary framework that recognizes that the health outcomes of people, animals, plants, and the environment are tightly interconnected. Within the context of One Health, the core principles underpinning microbiome assembly will be discussed in detail, including biofilm formation, microbial recruitment strategies, mechanisms of microbial attachment, community succession, and the effect these processes have on host function and health. Finally, this review will catalogue recent advances in microbiology and microbial ecology methods that can be used to profile microbial interfaces, with particular attention to multi-omic, advanced imaging, and modelling approaches. These technologies are essential for delineating the general and specific principles governing microbiome assembly and functions, mapping microbial interconnectivity across varying spatial and temporal scales, and for the establishment of predictive frameworks that will guide the development of targeted microbiome-interventions to deliver One Health outcomes.

Do small RNAs unlock the below ground microbiome-plant interaction mystery?

Over the past few decades, regulatory RNAs, such as small RNAs (sRNAs), have received increasing attention in the context of host-microbe interactions due to their diverse roles in controlling various biological processes in eukaryotes. In addition, studies have identified an increasing number of sRNAs with novel functions across a wide range of bacteria. What is not well understood is why cells regulate gene expression through post-transcriptional mechanisms rather than at the initiation of transcription. The finding of a multitude of sRNAs and their identified associated targets has allowed further investigation into the role of sRNAs in mediating gene regulation. These foundational data allow for further development of hypotheses concerning how a precise control of gene activity is accomplished through the combination of transcriptional and post-transcriptional regulation. Recently, sRNAs have been reported to participate in interkingdom communication and signalling where sRNAs originating from one kingdom are able to target or control gene expression in another kingdom. For example, small RNAs of fungal pathogens that silence plant genes and vice-versa plant sRNAs that mediate bacterial gene expression. However, there is currently a lack of evidence regarding sRNA-based inter-kingdom signalling across more than two interacting organisms. A habitat that provides an excellent opportunity to investigate interconnectivity is the plant rhizosphere, a multifaceted ecosystem where plants and associated soil microbes are known to interact. In this paper, we discuss how the interconnectivity of bacteria, fungi, and plants within the rhizosphere may be mediated by bacterial sRNAs with a particular focus on disease suppressive and non-suppressive soils. We discuss the potential roles sRNAs may play in the below-ground world and identify potential areas of future research, particularly in reference to the regulation of plant immunity genes by bacterial and fungal communities in disease-suppressive and non-disease-suppressive soils.

The preceding root system drives the composition and function of the rhizosphere microbiome.

BACKGROUND: The soil environment is responsible for sustaining most terrestrial plant life, yet we know surprisingly little about the important functions carried out by diverse microbial communities in soil. Soil microbes that inhabit the channels of decaying root systems, the detritusphere, are likely to be essential for plant growth and health, as these channels are the preferred locations of new root growth. Understanding the microbial metagenome of the detritusphere, and how it responds to agricultural management such as crop rotations and soil tillage, is vital for improving global food production. RESULTS: This study establishes an in-depth soil microbial gene catalogue based on the living-decaying rhizosphere niches in a cropping soil. The detritusphere microbiome regulates the composition and function of the rhizosphere microbiome to a greater extent than plant type: rhizosphere microbiomes of wheat and chickpea were homogenous (65-87% similarity) in the presence of decaying root (DR) systems but were heterogeneous (3-24% similarity) where DR was disrupted by tillage. When the microbiomes of the rhizosphere and the detritusphere interact in the presence of DR, there is significant degradation of plant root exudates by the rhizosphere microbiome, and genes associated with membrane transporters, carbohydrate and amino acid metabolism are enriched. CONCLUSIONS: The study describes the diversity and functional capacity of a high-quality soil microbial metagenome. The results demonstrate the contribution of the detritusphere microbiome in determining the metagenome of developing root systems. Modifications in root microbial function through soil management can ultimately govern plant health, productivity and food security.

Diazotroph Diversity and Nitrogen Fixation in Summer Active Perennial Grasses in a Mediterranean Region Agricultural Soil.

Summer-growing perennial grasses such as Panicum coloratum L. cv. Bambatsi (Bambatsi panic), Chloris gayana Kunth cv. Katambora (Rhodes grass) and Digitaria eriantha Steud. cv. Premier (Premier digit grass) growing in the poor fertility sandy soils in the Mediterranean regions of southern Australia and western Australia mainly depend upon soil N and biological N inputs through diazotrophic (free living or associative) N fixation. We investigated the community composition and diversity (nifH-amplicon sequencing), abundance (qPCR) and functional capacity (15N incubation assay) of the endophytic diazotrophic community in the below and above ground plant parts of field grown and unfertilized grasses. Results showed a diverse and abundant diazotrophic community inside plant both above and below-ground and there was a distinct diazotrophic assemblage in the different plant parts in all the three grasses. There was a limited difference in the diversity between leaves, stems and roots except that Panicum grass roots harbored greater species richness. Nitrogen fixation potentials ranged between 0.24 and 5.9 mg N kg-1 day-1 and N fixation capacity was found in both the above and below ground plant parts. Results confirmed previous reports of plant species-based variation and that Alpha-Proteobacteria were the dominant group of nifH-harboring taxa both in the belowground and aboveground parts of the three grass species. Results also showed a well-structured nifH-harboring community in all plant parts, an example for a functional endophytic community. Overall, the variation in the number and identity of module hubs and connectors among the different plant parts suggests that co-occurrence patterns within the nifH-harboring community specific to individual compartments and local environments of the niches within each plant part may dictate the overall composition of diazotrophs within a plant.

Vineyard Soil Microbiome Composition Related to Rotundone Concentration in Australian Cool Climate 'Peppery' Shiraz Grapes.

Soil microbial communities have an integral association with plants and play an important role in shaping plant nutrition, health, crop productivity and product quality. The influence of bacteria and fungi on wine fermentation is well known. However, little is known about the role of soil microbes, other than microbial pathogens, on grape composition or their role in vintage or site (terroir) impacts on grape composition. In this study, we used an amplicon sequencing approach to investigate the potential relationships between soil microbes and inherent spatial variation in grape metabolite composition - specifically, the concentration of the 'impact aroma compound' rotundone in Shiraz grapes (Vitis vinifera L.) grown in a 6.1 ha vineyard in the Grampians region of Victoria, Australia. Previous work had demonstrated temporal stability in patterns of within-vineyard spatial variation in rotundone concentration, enabling identification of defined 'zones' of inherently 'low' or 'high' concentration of this grape metabolite. 16S rRNA and ITS region-amplicon sequencing analysis of microbial communities in the surface soils collected from these zones indicated marked differences between zones in the genetic diversity and composition of the soil bacterial and fungal microbiome. Soils in the High rotundone zone exhibited higher diversity of bacteria, but lower diversity of fungi, compared to the soils in the Low rotundone zone. In addition, the network analysis of the microbial community in the High rotundone zone soils appeared well structured, especially with respect to the bacterial community, compared to that in the Low rotundone zone soils. The key differences in the microbial community structure between the rotundone zones are obvious for taxa/groups of both bacteria and fungi, particularly for bacteria belonging to Acidobacteria-GP4 and GP7, Rhizobiales, Gaiellaceae, Alphaproteobacteria and the Nectriaceae and Tremellaceae families of fungi. Although mulching in some parts of the vineyard caused changes in bacterial and fungal composition and overall microbial catabolic diversity and activity, its effects did not mask the rotundone zone-based variation. This finding of a systematic rotundone zone-based variation in soil microbiomes suggests an opportunity to bring together understanding of microbial ecology, plant biochemistry, and viticultural management for improved management of grape metabolism, composition and wine flavor.

Technologies for the Selection, Culture and Metabolic Profiling of Unique Rhizosphere Microorganisms for Natural Product Discovery.

Small molecule discovery has benefitted from the development of technologies that have aided in the culture and identification of soil microorganisms and the subsequent analysis of their respective metabolomes. We report herein on the use of both culture dependent and independent approaches for evaluation of soil microbial diversity in the rhizosphere of canola, a crop known to support a diverse microbiome, including plant growth promoting rhizobacteria. Initial screening of rhizosphere soils showed that microbial diversity, particularly bacterial, was greatest at crop maturity; therefore organismal recovery was attempted with soil collected at canola harvest. Two standard media (Mueller Hinton and gellan gum) were evaluated following inoculation with soil aqueous suspensions and compared with a novel "rhizochip" prototype buried in a living canola crop rhizosphere for microbial culture in situ. Following successful recovery and identification of 375 rhizosphere microbiota of interest from all culture methods, isolates were identified by Sanger sequencing and/or characterization using morphological and biochemical traits. Three bacterial isolates of interest were randomly selected as case studies for intensive metabolic profiling. After successful culture in liquid media and solvent extraction, individual extracts were subjected to evaluation by UHPLC-DAD-QToF-MS, resulting in the rapid characterization of metabolites of interest from cultures of two isolates. After evaluation of key molecular features, unique or unusual bacterial metabolites were annotated and are reported herein.

Evaluation of ACC-deaminase-producing rhizobacteria to alleviate water-stress impacts in wheat (Triticum aestivum L.) plants.

Application of plant-growth-promoting rhizobacteria (PGPR) is an environmentally sustainable option to reduce the effects of abiotic and biotic stresses on plant growth and productivity. Bacteria isolated from rain-fed agriculture field soils in the Central Himalaya Kumaun region, India, were evaluated for the production of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase. Those producing ACC deaminase in high amounts were evaluated for their potential to improve wheat (Triticum aestivum L.) plant growth under irrigated and water-stress conditions in two glasshouse experiments. Some of the isolates also showed other plant-growth-promoting (PGP) traits, e.g., N2 fixation, siderophore production, and phosphate solubilization; however, strains with higher ACC deaminase activity showed the greatest effects. These were Variovorax paradoxus RAA3; Pseudomonas spp. DPC12, DPB13, DPB15, DPB16; Achromobacter spp. PSA7, PSB8; and Ochrobactrum anthropi DPC9. In both simulated irrigated and water-stress conditions, a single inoculation of RAA3 and a consortium of DPC9 + DPB13 + DPB15 + DPB16 significantly improved wheat plant growth and foliar nutrient concentrations and caused significant positive changes in antioxidant properties compared with noninoculated plants especially under water stress. These findings imply that PGPB having ACC deaminase activity together with other PGP traits could potentially be effective inoculants to improve the growth of wheat plants in water-stressed rain-fed environments.

Comparative Metatranscriptomics of Wheat Rhizosphere Microbiomes in Disease Suppressive and Non-suppressive Soils for Rhizoctonia solani AG8.

The soilborne fungus Rhizoctonia solani anastomosis group (AG) 8 is a major pathogen of grain crops resulting in substantial production losses. In the absence of resistant cultivars of wheat or barley, a sustainable and enduring method for disease control may lie in the enhancement of biological disease suppression. Evidence of effective biological control of R. solani AG8 through disease suppression has been well documented at our study site in Avon, South Australia. A comparative metatranscriptomic approach was applied to assess the taxonomic and functional characteristics of the rhizosphere microbiome of wheat plants grown in adjacent fields which are suppressive and non-suppressive to the plant pathogen R. solani AG8. Analysis of 12 rhizosphere metatranscriptomes (six per field) was undertaken using two bioinformatic approaches involving unassembled and assembled reads. Differential expression analysis showed the dominant taxa in the rhizosphere based on mRNA annotation were Arthrobacter spp. and Pseudomonas spp. for non-suppressive samples and Stenotrophomonas spp. and Buttiauxella spp. for the suppressive samples. The assembled metatranscriptome analysis identified more differentially expressed genes than the unassembled analysis in the comparison of suppressive and non-suppressive samples. Suppressive samples showed greater expression of a polyketide cyclase, a terpenoid biosynthesis backbone gene (dxs) and many cold shock proteins (csp). Non-suppressive samples were characterised by greater expression of antibiotic genes such as non-heme chloroperoxidase (cpo) which is involved in pyrrolnitrin synthesis, and phenazine biosynthesis family protein F (phzF) and its transcriptional activator protein (phzR). A large number of genes involved in detoxifying reactive oxygen species (ROS) and superoxide radicals (sod, cat, ahp, bcp, gpx1, trx) were also expressed in the non-suppressive rhizosphere samples most likely in response to the infection of wheat roots by R. solani AG8. Together these results provide new insight into microbial gene expression in the rhizosphere of wheat in soils suppressive and non-suppressive to R. solani AG8. The approach taken and the genes involved in these functions provide direction for future studies to determine more precisely the molecular interplay of plant-microbe-pathogen interactions with the ultimate goal of the development of management options that promote beneficial rhizosphere microflora to reduce R. solani AG8 infection of crops.

Sulfur and Zinc Availability from Co-granulated Zn-Enriched Elemental Sulfur Fertilizers.

Acidification by oxidation of elemental sulfur (ES) can solubilize ZnO, providing slow release of both sulfur (S) and zinc (Zn) in soil. For this study, a new granular fertilizer with ES and ZnO was produced and evaluated. The effect of incorporating microorganisms or a carbon source in the granule was also evaluated. Four granulated ES-Zn fertilizers with and without S-oxidizing microorganisms, a commercial ES pastille, ZnSO4, and ZnO were applied to the center of Petri dishes containing two contrasting pH soils. Soil pH, CaCl2-extractable S and Zn, and remaining ES were evaluated at 30 and 60 days in two soil sections (0-5 and 5-9 mm from the fertilizer application site). A visualization test was performed to evaluate Zn diffusion over time. A significant pH decrease was observed in the acidic soil for all ES-Zn fertilizer treatments and in the alkaline soil for the Acidithiobacillus thiooxidans-inoculated treatment only. In agreement with Zn visualization tests, extractable-Zn concentrations were higher from the point of application in the acidic (62.9 mg dm-3) compared to the alkaline soil (5.5 mg dm-3). Elemental S oxidation was greater in the acidic soil (20.9%) than slightly alkaline soil (12%). The ES-Zn granular fertilizers increased S and Zn concentrations in soil and can provide a strategically slow release of nutrients to the soil.

Temperature dependency of virus and nanoparticle transport and retention in saturated porous media.

The influence of temperature on virus (PRD1 and ΦX174) and carboxyl-modified latex nanoparticle (50 and 100nm) attachment was examined in sand-packed columns under various physiochemical conditions. When the solution ionic strength (IS) equaled 10 and 30mM, the attachment rate coefficient (katt) increased up to 109% (p<0.0002) and the percentage of the sand surface area that contributed to attachment (Sf) increased up to 160% (p<0.002) when the temperature was increased from 4 to 20°C. Temperature effects at IS=10 and 30mM were also dependent on the system hydrodynamics; i.e., enhanced retention at a lower pore water velocity (0.1m/day). Conversely, this same temperature increase had a negligible influence on katt and Sf values when IS was 1mM or >50mM. An explanation for these observations was obtained from extended interaction energy calculations that considered nanoscale roughness and chemical heterogeneity on the sand surface. Interaction energy calculations demonstrated that the energy barrier to attachment in the primary minimum (∆Φa) decreased with increasing IS, chemical heterogeneity, and temperature, especially in the presence of small amounts of nanoscale roughness (e.g., roughness fraction of 0.05 and height of 20nm in the zone of influence). Temperature had a negligible effect on katt and Sf when the IS=1mM because of the large energy barrier, and at IS=50mM because of the absence of an energy barrier. Conversely, temperature had a large influence on katt and Sf when the IS was 10 and 30mM because of the presence of a small ∆Φa on sand with nanoscale roughness and a chemical (positive zeta potential) heterogeneity. This has large implications for setting parameters for the accurate modeling and transport prediction of virus and nanoparticle contaminants in ground water systems.

Quantifying the Sensitivity of Soil Microbial Communities to Silver Sulfide Nanoparticles Using Metagenome Sequencing.

Soils are a sink for sulfidised-silver nanoparticles (Ag2S-NPs), yet there are limited ecotoxicity data for their effects on microbial communities. Conventional toxicity tests typically target a single test species or function, which does not reflect the broader community response. Using a combination of quantitative PCR, 16S rRNA amplicon sequencing and species sensitivity distribution (SSD) methods, we have developed a new approach to calculate silver-based NP toxicity thresholds (HCx, hazardous concentrations) that are protective of specific members (operational taxonomic units, OTUs) of the soil microbial community. At the HC20 (80% of species protected), soil OTUs were significantly less sensitive to Ag2S-NPs compared to AgNPs and Ag+ (5.9, 1.4 and 1.4 mg Ag kg-1, respectively). However at more conservative HC values, there were no significant differences. These trends in OTU responses matched with those seen in a specific microbial function (rate of nitrification) and amoA-bacteria gene abundance. This study provides a novel molecular-based framework for quantifying the effect of a toxicant on whole soil microbial communities while still determining sensitive genera/species. Methods and results described here provide a benchmark for microbial community ecotoxicological studies and we recommend that future revisions of Soil Quality Guidelines for AgNPs and other such toxicants consider this approach.

Size Matters: Assessing Optimum Soil Sample Size for Fungal and Bacterial Community Structure Analyses Using High Throughput Sequencing of rRNA Gene Amplicons.

We examined the effect of different soil sample sizes obtained from an agricultural field, under a single cropping system uniform in soil properties and aboveground crop responses, on bacterial and fungal community structure and microbial diversity indices. DNA extracted from soil sample sizes of 0.25, 1, 5, and 10 g using MoBIO kits and from 10 and 100 g sizes using a bead-beating method (SARDI) were used as templates for high-throughput sequencing of 16S and 28S rRNA gene amplicons for bacteria and fungi, respectively, on the Illumina MiSeq and Roche 454 platforms. Sample size significantly affected overall bacterial and fungal community structure, replicate dispersion and the number of operational taxonomic units (OTUs) retrieved. Richness, evenness and diversity were also significantly affected. The largest diversity estimates were always associated with the 10 g MoBIO extractions with a corresponding reduction in replicate dispersion. For the fungal data, smaller MoBIO extractions identified more unclassified Eukaryota incertae sedis and unclassified glomeromycota while the SARDI method retrieved more abundant OTUs containing unclassified Pleosporales and the fungal genera Alternaria and Cercophora. Overall, these findings indicate that a 10 g soil DNA extraction is most suitable for both soil bacterial and fungal communities for retrieving optimal diversity while still capturing rarer taxa in concert with decreasing replicate variation.

Introducing BASE: the Biomes of Australian Soil Environments soil microbial diversity database.

BACKGROUND: Microbial inhabitants of soils are important to ecosystem and planetary functions, yet there are large gaps in our knowledge of their diversity and ecology. The 'Biomes of Australian Soil Environments' (BASE) project has generated a database of microbial diversity with associated metadata across extensive environmental gradients at continental scale. As the characterisation of microbes rapidly expands, the BASE database provides an evolving platform for interrogating and integrating microbial diversity and function. FINDINGS: BASE currently provides amplicon sequences and associated contextual data for over 900 sites encompassing all Australian states and territories, a wide variety of bioregions, vegetation and land-use types. Amplicons target bacteria, archaea and general and fungal-specific eukaryotes. The growing database will soon include metagenomics data. Data are provided in both raw sequence (FASTQ) and analysed OTU table formats and are accessed via the project's data portal, which provides a user-friendly search tool to quickly identify samples of interest. Processed data can be visually interrogated and intersected with other Australian diversity and environmental data using tools developed by the 'Atlas of Living Australia'. CONCLUSIONS: Developed within an open data framework, the BASE project is the first Australian soil microbial diversity database. The database will grow and link to other global efforts to explore microbial, plant, animal, and marine biodiversity. Its design and open access nature ensures that BASE will evolve as a valuable tool for documenting an often overlooked component of biodiversity and the many microbe-driven processes that are essential to sustain soil function and ecosystem services.

Capitalizing on deliberate, accidental, and GM-driven environmental change caused by crop modification.

The transgenic traits associated with the majority of commercial genetically modified crops are focused on improving herbicide and insecticide management practices. The use of the transgenic technology in these crops and the associated chemistry has been the basis of studies that provide evidence for occasional improvement in environmental benefits due to the use of less residual herbicides, more targeted pesticides, and reduced field traffic. This is nicely exemplified through studies using Environmental Impact Quotient (EIQ) assessments. Whilst EIQ evaluations may sometimes illustrate environmental benefits they have their limitations. EIQ evaluations are not a surrogate for Environmental Risk Assessments and may not reflect real environmental interactions between crops and the environment. Addressing the impact cultivated plants have on the environment generally attracts little public attention and research funding, but the introduction of GM has facilitated an expansion of research to address potential environmental concerns from government, NGOs, industry, consumers, and growers. In this commentary, some evidence from our own research and several key papers that highlight EIQ assessments of the impact crops are having on the environment are presented. This information may be useful as an education tool on the potential benefits of GM and conventional farming. In addition, other deliberate, accidental, and GM-driven benefits derived from the examination of GM cropping systems is briefly discussed.