Priority establishment of soil bacteria in rhizosphere limited the spread of tetracycline resistance genes from pig manure to soil-plant systems based on synthetic communities approach.

The spread of antibiotic resistance genes (ARGs) in agroecosystems through the application of animal manure is a global threat to human and environmental health. However, the adaptability and colonization ability of animal manure-derived bacteria determine the spread pathways of ARG in agroecosystems, which have rarely been studied. Here, we performed an invasion experiment by creating a synthetic communities (SynCom) with ten isolates from pig manure and followed its assembly during gnotobiotic cultivation of a soil-Arabidopsis thaliana (A. thaliana) system. We found that Firmicutes in the SynCom were efficiently filtered out in the rhizosphere, thereby limiting the entry of tetracycline resistance genes (TRGs) into the plant. However, Proteobacteria and Actinobacteria in the SynCom were able to establish in all compartments of the soil-plant system thereby spreading TRGs from manure to soil and plant. The presence of native soil bacteria prevented the establishment of manure-borne bacteria and effectively reduced the spread of TRGs. Achromobacter mucicolens and Pantoea septica were the main vectors for the entry of tetA into plants. Furthermore, doxycycline stress promoted the horizontal gene transfer (HGT) of the conjugative resistance plasmid RP4 within the SynCom in A. thaliana by upregulating the expression of HGT-related mRNAs. Therefore, this study provides evidence for the dissemination pathways of ARGs in agricultural systems through the invasion of manure-derived bacteria and HGT by conjugative resistance plasmids and demonstrates that the priority establishment of soil bacteria in the rhizosphere limited the spread of TRGs from pig manure to soil-plant systems.

Community coalescence and plant host filtering determine the spread of tetracycline resistance genes from pig manure into the microbiome continuum of the soil-plant system.

The spread of livestock manure-borne antibiotic resistance genes (ARGs) into agroecosystems through manure application poses a potential threat to human health. However, there is still a knowledge gap concerning ARG dissemination in coalescing manure, soil and plant microbiomes. Here, we examined the fate of tetracycline resistance genes (TRGs) originating from pig manure microbiomes and spread in the soil-A thaliana system and explored the effects of microbial functions on TRGs spread at different interfaces. Our results indicate that the TRGs abundances in all microbiome continuum of the soil-A. thaliana system were significantly increased with the application of a living manure microbiome, although the addition of manure with both an active and inactive microbiome caused a shift in the microbial community composition. This was attributed to the increasing relative abundances of tetA, tetL, tetM, tetO, tetW and tolC in the system. The application of living manure with DOX residues resulted in the highest relative abundance of total TRGs (3.30×10-3 copies/16S rRNA gene copies) in the rhizosphere soil samples. Community coalescence of the manure and soil microbiomes increased the abundance of Firmicutes in the soil and root microbiome, which directly explains the increase in TRG abundance observed in these interfaces. In contrast, the leaf microbiome differed markedly from that of the remaining samples, indicating strong plant host filtering effects on Firmicutes and TRGs from pig manure. The random forest machine learning model revealed microbial functions and their significant positive correlation with TRG abundance in the microbiome continuum of the system. Our findings revealed that community coalescence is the main driver of TRG spread from manure to the soil and root microbiomes. Plant host filtering effects play a crucial role in allowing certain microbial groups to occupy ecological niches in the leaves, thereby limiting the establishment of manure-borne TRGs in aboveground plant tissues.

Removal of antibiotic resistance genes during swine manure composting is strongly impaired by high levels of doxycycline residues.

Antibiotic resistance genes (ARGs) are emerging pollutants that enter the farm and surrounding environment via the manure of antibiotic-treated animals. Pretreatment of livestock manure by composting decreases ARGs abundance, but how antibiotic residues affect ARGs removal efficiency remains poorly understood. Here, we explored the fate of the resistome under different doxycycline residue levels during aerobic swine manure composting. Metagenomic sequencing showed that the presence of high levels of doxycycline generally had a higher abundance of tetracycline ARGs, and their dominant host bacteria of Firmicutes, especially Clostridium and Streptococcus, also had limited elimination in composting under high levels of doxycycline stress. Moreover, high levels of doxycycline impaired the removal of the total ARGs number in finished composts, with a removal rate of 51.74 % compared to 63.70 % and 71.52 % for the control and low-level doxycycline manure, respectively. Horizontal gene transfer and strengthened correlations among the bacterial community fostered ARGs preservation at high doxycycline levels during composting. In addition, ARGs carried by both plasmids and chromosomes, such as multidrug ARGs, showed wide host characteristics and rebound during compost maturation. Compared with chromosomes, a greater variety of ARGs on plasmids suggested that the majority of ARGs were characterized by horizontal mobility during composting, and the cross-host characteristics of ARGs during composting deserve further attention. This study provided deep insight into the fate of ARGs under residual antibiotic stress during manure composting and reminded the associated risk for environmental and public health.

From gut to mud: dissemination of antimicrobial resistance between animal and agricultural niches.

With increasing reports on antimicrobial resistance (AMR) in humans, animals and the environment, we are at risk of returning to a pre-antibiotic era. Therefore, AMR is recognized as one of the major global health threats of this century. Antibiotics are used extensively in farming systems to treat and prevent infections in food animals or to increase their growth. Besides the risk of a transfer of AMR between the human and the animal sector, there is another yet largely overlooked sector in the One Health triad. Human-dominated ecosystems such as agricultural soils are a major sink for antibiotics and AMR originating from livestock farming. This review summarizes current knowledge on the prevalence of AMR at the interface of animal and agricultural production and discusses the potential implications for human health. Soil resistomes are augmented by the application of manure from treated livestock. Subsequent transfer of AMR into plant microbiomes may likely play a critical role in human exposure to antibiotic resistance in the environment. Based on the knowledge that is currently available we advocate that more attention should be paid to the role of environmental resistomes in the AMR crisis.

Mining Synergistic Microbial Interactions: A Roadmap on How to Integrate Multi-Omics Data.

Mining interspecies interactions remain a challenge due to the complex nature of microbial communities and the need for computational power to handle big data. Our meta-analysis indicates that genetic potential alone does not resolve all issues involving mining of microbial interactions. Nevertheless, it can be used as the starting point to infer synergistic interspecies interactions and to limit the search space (i.e., number of species and metabolic reactions) to a manageable size. A reduced search space decreases the number of additional experiments necessary to validate the inferred putative interactions. As validation experiments, we examine how multi-omics and state of the art imaging techniques may further improve our understanding of species interactions' role in ecosystem processes. Finally, we analyze pros and cons from the current methods to infer microbial interactions from genetic potential and propose a new theoretical framework based on: (i) genomic information of key members of a community; (ii) information of ecosystem processes involved with a specific hypothesis or research question; (iii) the ability to identify putative species' contributions to ecosystem processes of interest; and, (iv) validation of putative microbial interactions through integration of other data sources.

Associational effects in the microbial neighborhood.

Even though "perfect" metagenomes or metatranscriptomes are close at hand, the implicit assumption of spatial homogeneity in the "omic" approaches makes it difficult if not impossible to relate those data to ecological processes occurring in natural and man-made ecosystems. In fact, the distribution of microbes in their habitats is far from being uniform and random. Microbial communities show a high degree of spatial organization that stems from environmental gradients and local interactions. These interactions can be very complex and may involve multiple species. Several studies highlighted the importance of indirect interactions for community stability, but the absence of a theoretical framework for microbial ecology restricts the possibilities to strike a balance between the investigation of simple communities with purely pairwise interactions and the attempts to understand interaction patterns in whole communities based on meta-omics studies. Here we suggest adapting the concept of Associational Effects (AE) from plant ecology, to better understand the link between ecological interactions, spatial arrangement, and stability in microbial communities. By bringing together a conceptual framework developed for plants and observations made for microbes, this perspective article fosters synthesis of related disciplines to yield novel insights into the advancing field of spatial microbial ecology. To promote the integration into microbial ecology, we (i) outline the theoretical background of AE, (ii) collect underlying mechanisms by literature synthesis, (iii) propose a three-point roadmap for the investigation of AE in microbial communities, and (iv) discuss its implications for microbial ecology research.

Combining QTL mapping with transcriptome and metabolome profiling reveals a possible role for ABA signaling in resistance against the cabbage whitefly in cabbage.

Whiteflies are among the world's most significant agricultural pests and chemical insecticides are extensively used to reduce crop damage to acceptable levels. However, nearly all insecticides pose a threat to the environment and alternative control methods, such as breeding of crop varieties that are inherently insect-resistant, are needed. Previously, a strong source of plant-age dependent resistance to the cabbage whitefly (Aleyrodes proletella) has been identified in the modern white cabbage (Brassica oleracea var. capitata) variety Rivera. However, nothing is known about the molecular mechanisms or the genes involved in this resistance. In the present study, a multidisciplinary approach combining transcriptome and metabolome profiling with genetic mapping was used to identify the molecular players of whitefly resistance in cabbage. Transcriptome profiles of young (susceptible) and older (resistant) Rivera plants were analyzed using RNA sequencing. While many genes involved in general processes were differentially expressed between both ages, several defense-related processes were overrepresented in the transcriptome profile of older plants. Hormone measurements revealed that jasmonic acid (JA) levels decreased upon whitefly infestation at both plant ages. Interestingly, abscisic acid (ABA) levels showed contrasting effects in response to whitefly infestation: ABA levels were reduced in young plants but induced in older plants upon whitefly feeding. Auxin levels were significantly lower in older plants compared with young plants, independent of whitefly presence, while glucosinolate levels were higher. Additionally, whitefly performance was monitored in an F2 population derived from a cross between Rivera and the susceptible white cabbage variety Christmas Drumhead. Significant QTL intervals were mapped on chromosome 2 and 9 for oviposition rate and whitefly adult survival, respectively. Several genes that were higher expressed in older plants and located in the identified QTL intervals were orthologous to Arabidopsis genes that have been related to ABA signaling, suggesting a role for ABA in the regulation of resistance towards whiteflies. Our results show that combining different omics approaches is a useful strategy to identify candidate genes underlying insect resistance.

Ecology of Contaminant Biotransformation in the Mycosphere: Role of Transport Processes.

Fungi and bacteria often share common microhabitats. Their co-occurrence and coevolution give rise to manifold ecological interactions in the mycosphere, here defined as the microhabitats surrounding and affected by hyphae and mycelia. The extensive structure of mycelia provides ideal "logistic networks" for transport of bacteria and matter in structurally and chemically heterogeneous soil ecosystems. We describe the characteristics of the mycosphere as a unique and highly dynamic bacterial habitat and a hot spot for contaminant biotransformation. In particular, we emphasize the role of the mycosphere for (i) bacterial dispersal and colonization of subsurface interfaces and new habitats, (ii) matter transport processes and contaminant bioaccessibility, and (iii) the functional stability of microbial ecosystems when exposed to environmental fluctuations such as stress or disturbances. Adopting concepts from ecological theory, the chapter disentangles bacterial-fungal impacts on contaminant biotransformation in a systemic approach that interlinks empirical data from microbial ecosystems with simulation data from computational models. This approach provides generic information on key factors, processes, and ecological principles that drive microbial contaminant biotransformation in soil. We highlight that the transport processes create favorable habitat conditions for efficient bacterial contaminant degradation in the mycosphere. In-depth observation, understanding, and prediction of the role of mycosphere transport processes will support the use of bacterial-fungal interactions in nature-based solutions for contaminant biotransformation in natural and man-made ecosystems, respectively.

Spatiotemporal disturbance characteristics determine functional stability and collapse risk of simulated microbial ecosystems.

Terrestrial microbial ecosystems are exposed to many types of disturbances varying in their spatial and temporal characteristics. The ability to cope with these disturbances is crucial for maintaining microbial ecosystem functions, especially if disturbances recur regularly. Thus, understanding microbial ecosystem dynamics under recurrent disturbances and identifying drivers of functional stability and thresholds for functional collapse is important. Using a spatially explicit ecological model of bacterial growth, dispersal, and substrate consumption, we simulated spatially heterogeneous recurrent disturbances and investigated the dynamic response of pollutant biodegradation - exemplarily for an important ecosystem function. We found that thresholds for functional collapse are controlled by the combination of disturbance frequency and spatial configuration (spatiotemporal disturbance regime). For rare disturbances, the occurrence of functional collapse is promoted by low spatial disturbance fragmentation. For frequent disturbances, functional collapse is almost inevitable. Moreover, the relevance of bacterial growth and dispersal for functional stability also depends on the spatiotemporal disturbance regime. Under disturbance regimes with moderate severity, microbial properties can strongly affect functional stability and shift the threshold for functional collapse. Similarly, networks facilitating bacterial dispersal can delay functional collapse. Consequently, measures to enhance or sustain bacterial growth/dispersal are promising strategies to prevent functional collapses under moderate disturbance regimes.

Current Challenges in Plant Eco-Metabolomics.

The relatively new research discipline of Eco-Metabolomics is the application of metabolomics techniques to ecology with the aim to characterise biochemical interactions of organisms across different spatial and temporal scales. Metabolomics is an untargeted biochemical approach to measure many thousands of metabolites in different species, including plants and animals. Changes in metabolite concentrations can provide mechanistic evidence for biochemical processes that are relevant at ecological scales. These include physiological, phenotypic and morphological responses of plants and communities to environmental changes and also interactions with other organisms. Traditionally, research in biochemistry and ecology comes from two different directions and is performed at distinct spatiotemporal scales. Biochemical studies most often focus on intrinsic processes in individuals at physiological and cellular scales. Generally, they take a bottom-up approach scaling up cellular processes from spatiotemporally fine to coarser scales. Ecological studies usually focus on extrinsic processes acting upon organisms at population and community scales and typically study top-down and bottom-up processes in combination. Eco-Metabolomics is a transdisciplinary research discipline that links biochemistry and ecology and connects the distinct spatiotemporal scales. In this review, we focus on approaches to study chemical and biochemical interactions of plants at various ecological levels, mainly plant⁻organismal interactions, and discuss related examples from other domains. We present recent developments and highlight advancements in Eco-Metabolomics over the last decade from various angles. We further address the five key challenges: (1) complex experimental designs and large variation of metabolite profiles; (2) feature extraction; (3) metabolite identification; (4) statistical analyses; and (5) bioinformatics software tools and workflows. The presented solutions to these challenges will advance connecting the distinct spatiotemporal scales and bridging biochemistry and ecology.

Functional Resistance to Recurrent Spatially Heterogeneous Disturbances Is Facilitated by Increased Activity of Surviving Bacteria in a Virtual Ecosystem.

Bacterial degradation of organic compounds is an important ecosystem function with relevance to, e.g., the cycling of elements or the degradation of organic contaminants. It remains an open question, however, to which extent ecosystems are able to maintain such biodegradation function under recurrent disturbances (functional resistance) and how this is related to the bacterial biomass abundance. In this paper, we use a numerical simulation approach to systematically analyze the dynamic response of a microbial population to recurrent disturbances of different spatial distribution. The spatially explicit model considers microbial degradation, growth, dispersal, and spatial networks that facilitate bacterial dispersal mimicking effects of mycelial networks in nature. We find: (i) There is a certain capacity for high resistance of biodegradation performance to recurrent disturbances. (ii) If this resistance capacity is exceeded, spatial zones of different biodegradation performance develop, ranging from no or reduced to even increased performance. (iii) Bacterial biomass and biodegradation dynamics respond inversely to the spatial fragmentation of disturbances: overall biodegradation performance improves with increasing fragmentation, but bacterial biomass declines. (iv) Bacterial dispersal networks can enhance functional resistance against recurrent disturbances, mainly by reactivating zones in the core of disturbed areas, even though this leads to an overall reduction of bacterial biomass.

Mycelium-mediated transfer of water and nutrients stimulates bacterial activity in dry and oligotrophic environments.

Fungal-bacterial interactions are highly diverse and contribute to many ecosystem processes. Their emergence under common environmental stress scenarios however, remains elusive. Here we use a synthetic microbial ecosystem based on the germination of Bacillus subtilis spores to examine whether fungal and fungal-like (oomycete) mycelia reduce bacterial water and nutrient stress in an otherwise dry and nutrient-poor microhabitat. We find that the presence of mycelia enables the germination and subsequent growth of bacterial spores near the hyphae. Using a combination of time of flight- and nanoscale secondary ion mass spectrometry (ToF- and nanoSIMS) coupled with stable isotope labelling, we link spore germination to hyphal transfer of water, carbon and nitrogen. Our study provides direct experimental evidence for the stimulation of bacterial activity by mycelial supply of scarce resources in dry and nutrient-free environments. We propose that mycelia may stimulate bacterial activity and thus contribute to sustaining ecosystem functioning in stressed habitats.

Bacterial Dispersal Promotes Biodegradation in Heterogeneous Systems Exposed to Osmotic Stress.

Contaminant biodegradation in soils is hampered by the heterogeneous distribution of degrading communities colonizing isolated microenvironments as a result of the soil architecture. Over the last years, soil salinization was recognized as an additional problem especially in arid and semiarid ecosystems as it drastically reduces the activity and motility of bacteria. Here, we studied the importance of different spatial processes for benzoate biodegradation at an environmentally relevant range of osmotic potentials (ΔΨo) using model ecosystems exhibiting a heterogeneous distribution of the soil-borne bacterium Pseudomonas putida KT2440. Three systematically manipulated scenarios allowed us to cover the effects of (i) substrate diffusion, (ii) substrate diffusion and autonomous bacterial dispersal, and (iii) substrate diffusion and autonomous as well as mediated bacterial dispersal along glass fiber networks mimicking fungal hyphae. To quantify the relative importance of the different spatial processes, we compared these heterogeneous scenarios to a reference value obtained for each ΔΨo by means of a quasi-optimal scenario in which degraders were ab initio homogeneously distributed. Substrate diffusion as the sole spatial process was insufficient to counteract the disadvantage due to spatial degrader heterogeneity at ΔΨo ranging from 0 to -1 MPa. In this scenario, only 13.8-21.3% of the quasi-optimal biodegradation performance could be achieved. In the same range of ΔΨo values, substrate diffusion in combination with bacterial dispersal allowed between 68.6 and 36.2% of the performance showing a clear downwards trend with decreasing ΔΨo. At -1.5 MPa, however, this scenario performed worse than the diffusion scenario, possibly as a result of energetic disadvantages associated with flagellum synthesis and emerging requirements to exceed a critical population density to resist osmotic stress. Network-mediated bacterial dispersal kept biodegradation almost consistently high with an average of 70.7 ± 7.8%, regardless of the strength of the osmotic stress. We propose that especially fungal network-mediated bacterial dispersal is a key process to achieve high functionality of heterogeneous microbial ecosystems also at reduced osmotic potentials. Thus, mechanical stress by, for example, soil homogenization should be kept low in order to preserve fungal network integrity.

Mycelium-Like Networks Increase Bacterial Dispersal, Growth, and Biodegradation in a Model Ecosystem at Various Water Potentials.

Fungal mycelia serve as effective dispersal networks for bacteria in water-unsaturated environments, thereby allowing bacteria to maintain important functions, such as biodegradation. However, poor knowledge exists on the effects of dispersal networks at various osmotic (Ψo) and matric (Ψm) potentials, which contribute to the water potential mainly in terrestrial soil environments. Here we studied the effects of artificial mycelium-like dispersal networks on bacterial dispersal dynamics and subsequent effects on growth and benzoate biodegradation at ΔΨo and ΔΨm values between 0 and -1.5 MPa. In a multiple-microcosm approach, we used a green fluorescent protein (GFP)-tagged derivative of the soil bacterium Pseudomonas putida KT2440 as a model organism and sodium benzoate as a representative of polar aromatic contaminants. We found that decreasing ΔΨo and ΔΨm values slowed bacterial dispersal in the system, leading to decelerated growth and benzoate degradation. In contrast, dispersal networks facilitated bacterial movement at ΔΨo and ΔΨm values between 0 and -0.5 MPa and thus improved the absolute biodegradation performance by up to 52 and 119% for ΔΨo and ΔΨm, respectively. This strong functional interrelationship was further emphasized by a high positive correlation between population dispersal, population growth, and degradation. We propose that dispersal networks may sustain the functionality of microbial ecosystems at low osmotic and matric potentials.

Substrate oxidation by dye-decolorizing peroxidases (DyPs) from wood- and litter-degrading agaricomycetes compared to other fungal and plant heme-peroxidases.

Catalytic and physicochemical properties of representative fungal dye-decolorizing peroxidases (DyPs) of wood- (WRF) and litter-decomposing white-rot fungi (LDF) are summarized and compared, including one recombinant Mycetinis scorodonius DyP (rMscDyP; LDF), the wild-type Auricularia auricula-judae DyP (AauDyP; WRF), and two new DyPs secreted by the jelly fungi Exidia glandulosa (EglDyP; WRF) and Mycena epipterygia (MepDyP; LDF). Homogeneous preparations of these DyPs were obtained after different steps of fast protein liquid chromatography, and they increase the total number of characterized fungal DyP proteins to eight. The peptide sequences of AauDyP, MepDyP, and EglDyP showed highest homologies (52-56%) to the DyPs of M. scorodonius. Five out of the eight characterized fungal DyPs were used to evaluate their catalytic properties compared to classic fungal and plant heme peroxidases, namely lignin peroxidase of Phanerochaete chrysosporium (PchLiP; WRF), versatile peroxidase of Bjerkandera adusta (BadVP; WRF), and generic peroxidases of Coprinopsis cinerea (CiP) and Glycine max (soybean peroxidase=SBP). All DyPs tested possess unique properties regarding the stability at low pH values: 50-90% enzymatic activity remained after 4-h exposition at pH 2.5, and the oxidation of nonphenolic aromatic substrates (lignin model compounds) was optimal below pH 3. Furthermore, all DyPs efficiently oxidized recalcitrant dyes (e.g., Azure B) as well as the phenolic substrate 2,6-dimethoxyphenol. Thus, DyPs combine features of different peroxidases on the functional level and may be part of the biocatalytic system secreted by fungi for the oxidation of lignin and/or toxic aromatic compounds.