Concepts and criteria defining emerging microbiome applications.

In recent years, microbiomes and their potential applications for human, animal or plant health, food production and environmental management came into the spotlight of major national and international policies and strategies. This has been accompanied by substantial R&D investments in both public and private sectors, with an increasing number of products entering the market. Despite widespread agreement on the potential of microbiomes and their uses across disciplines, stakeholders and countries, there is no consensus on what defines a microbiome application. This often results in non-comprehensive communication or insufficient documentation making commercialisation and acceptance of the novel products challenging. To showcase the complexity of this issue we discuss two selected, well-established applications and propose criteria defining a microbiome application and their conditions of use for clear communication, facilitating suitable regulatory frameworks and building trust among stakeholders.

Harnessing the plant microbiome for sustainable crop production.

Global research on the plant microbiome has enhanced our understanding of the complex interactions between plants and microorganisms. The structure and functions of plant-associated microorganisms, as well as the genetic, biochemical, physical and metabolic factors that influence the beneficial traits of plant microbiota have also been intensively studied. Harnessing the plant microbiome has led to the development of various microbial applications to improve crop productivity in the face of a range of challenges, for example, climate change, abiotic and biotic stresses, and declining soil properties. Microorganisms, particularly nitrogen-fixing rhizobia as well as mycorrhizae and biocontrol agents, have been applied for decades to improve plant nutrition and health. Still, there are limitations regarding efficacy and consistency under field conditions. Also, the wealth of expanding knowledge on microbiome diversity, functions and interactions represents a huge source of information to exploit for new types of application. In this Review, we explore plant microbiome functions, mechanisms, assembly and types of interaction, and discuss current applications and their pitfalls. Furthermore, we elaborate on how the latest findings in plant microbiome research may lead to the development of new or more advanced applications. Finally, we discuss research gaps to fully leverage microbiome functions for sustainable plant production.

Soil pH, developmental stages and geographical origin differently influence the root metabolomic diversity and root-related microbial diversity of Echium vulgare from native habitats.

Improved understanding of the complex interaction between plant metabolism, environmental conditions and the plant-associated microbiome requires an interdisciplinary approach: Our hypothesis in our multiomics study posited that several environmental and biotic factors have modulating effects on the microbiome and metabolome of the roots of wild Echium vulgare plants. Furthermore, we postulated reciprocal interactions between the root metabolome and microbiome. We investigated the metabolic content, the genetic variability, and the prokaryotic microbiome in the root systems of wild E. vulgare plants at rosette and flowering stages across six distinct locations. We incorporated the assessment of soil microbiomes and the measurement of selected soil chemical composition factors. Two distinct genetic clusters were determined based on microsatellite analysis without a consistent alignment with the geographical proximity between the locations. The microbial diversity of both the roots of E. vulgare and the surrounding bulk soil exhibited significant divergence across locations, varying soil pH characteristics, and within the identified plant genetic clusters. Notably, acidophilic bacteria were characteristic inhabitants of both soil and roots under acidic soil conditions, emphasizing the close interconnectedness between these compartments. The metabolome of E. vulgare significantly differed between root samples from different developmental stages, geographical locations, and soil pH levels. The developmental stage was the dominant driver of metabolome changes, with significantly higher concentrations of sugars, pyrrolizidine alkaloids, and some of their precursors in rosette stage plant roots. Our study featured the complex dynamics between soil pH, plant development, geographical locations, plant genetics, plant metabolome and microbiome, shedding light on existing knowledge gaps.

The microbial-driven nitrogen cycle and its relevance for plant nutrition.

Nitrogen (N) is a vital nutrient and an essential component of biological macromolecules, such as nucleic acids and proteins. Microorganisms represent major drivers of N-cycling processes in all ecosystems, including the soil and plant environment. The availability of N is a major growth limiting factor for plants and it is significantly affected by the plant microbiome. Plants and microorganisms form complex interaction networks resulting in molecular signaling, nutrient exchange and other distinct metabolic responses. In these networks, microbial partners influence growth and N use efficiency of plants either positively or negatively. Harnessing the beneficial effects of specific players within crop microbiomes is a promising strategy to counteract the emerging threats for human and planetary health due to the overuse of industrial N fertilizers. However, in addition to N-providing activities (e.g. the well-known symbiosis of legumes and Rhizobium bacteria), other plant-microorganism interactions must be considered to obtain a complete picture of how microbial driven N-transformations might affect plant nutrition. For this, we review recent insights into the tight interplay between plants and N-cycling microorganisms focusing on microbial N-transformation processes representing N sources and sinks that ultimately shape the plant N acquisition.

From Microbes to Microbiomes: Applications for Plant Health and Sustainable Agriculture.

Plant-microbe interaction research has had a transformative trajectory, from individual microbial isolate studies to comprehensive analyses of plant microbiomes within the broader phytobiome framework. Acknowledging the indispensable role of plant microbiomes in shaping plant health, agriculture, and ecosystem resilience, we underscore the urgent need for sustainable crop production strategies in the face of contemporary challenges. We discuss how the synergies between advancements in 'omics technologies and artificial intelligence can help advance the profound potential of plant microbiomes. Furthermore, we propose a multifaceted approach encompassing translational considerations, transdisciplinary research initiatives, public-private partnerships, regulatory policy development, and pragmatic expectations for the practical application of plant microbiome knowledge across diverse agricultural landscapes. We advocate for strategic collaboration and intentional transdisciplinary efforts to unlock the benefits offered by plant microbiomes and address pressing global issues in food security. By emphasizing a nuanced understanding of plant microbiome complexities and fostering realistic expectations, we encourage the scientific community to navigate the transformative journey from discoveries in the laboratory to field applications. As companies specializing in agricultural microbes and microbiomes undergo shifts, we highlight the necessity of understanding how to approach sustainable agriculture with site-specific management solutions. While cautioning against overpromising, we underscore the excitement of exploring the many impacts of microbiome-plant interactions. We emphasize the importance of collaborative endeavors with societal partners to accelerate our collective capacity to harness the diverse and yet-to-be-discovered beneficial activities of plant microbiomes.

Variovorax sp. strain P1R9 applied individually or as part of bacterial consortia enhances wheat germination under salt stress conditions.

Endophytes isolated from extremophile plants are interesting microbes for improving the stress tolerance of agricultural plants. Here, we isolated and characterized endophytic bacteria showing plant growth-promoting (PGP) traits from plants in two extreme Chilean biomes (Atacama Desert and Chilean Patagonia). Forty-two isolates were characterized as both halotolerant auxin producers (2-51 mg L-1) and 1-aminocyclopropane-1-carboxylate (ACC)-degrading bacteria (15-28 µmol αKB mg protein-1 h-1). The most efficient isolates were tested as single strains, in dual and triple consortia, or in combination with previously reported PGP rhizobacteria (Klebsiella sp. 27IJA and 8LJA) for their impact on the germination of salt-exposed (0.15 M and 0.25 M NaCl) wheat seeds. Interestingly, strain P1R9, identified as Variovorax sp., enhanced wheat germination under salt stress conditions when applied individually or as part of bacterial consortia. Under salt stress, plants inoculated with dual consortia containing the strain Variovorax sp. P1R9 showed higher biomass (41%) and reduced lipid peroxidation (33-56%) than uninoculated plants. Although the underlying mechanisms remain elusive, our data suggest that the application of Variovorax sp. P1R9, alone or as a member of PGP consortia, may improve the salt stress tolerance of wheat plants.

Endophytic bacterial communities in ungerminated and germinated seeds of commercial vegetables.

Chile is a prominent seed exporter globally, but the seed microbiome of vegetables (46% of seeds) and its role in the early stages of plant growth have remained largely unexplored. Here, we employed DNA metabarcoding analysis to investigate the composition and putative functions of endophytic bacterial communities in ungerminated and germinated seeds of the commercial vegetables Apiaceae (parsley and carrot), Asteraceae (lettuce), Brassicaceae (cabbage and broccoli), and Solanaceae (tomato). Bacterial quantification showed 104 to 108 copies of the 16S rRNA gene per gram of ungerminated and germinated seeds. Alpha diversity analysis (e.g., Chao1, Shannon, and Simpson indices) did not indicate significant differences (Kruskal-Wallis test) between ungerminated and germinated seeds, except for Solanaceae. However, beta diversity (PCoA) analysis showed distinctions (Adonis test) between ungerminated and germinated seeds, except Apiaceae. Pseudomonadota and Bacillota were identified as the dominant and specialist taxa in both ungerminated and germinated seed samples. Chemoheterotrophy and fermentation were predicted as the main microbial functional groups in the endophytic bacterial community. Notably, a considerable number of the 143 isolated endophytic strains displayed plant growth-promoting traits (10 to 64%) and biocontrol activity (74% to 82%) against plant pathogens (Xanthomonas and Pseudomonas). This study revealed the high variability in the abundance, diversity, composition, and functionality of endophytic bacteria between ungerminated and germinated seeds in globally commercialized vegetables. Furthermore, potential beneficial endophytic bacteria contained in their seed microbiomes that may contribute to the microbiome of the early stages, development, growth and progeny of vegetables were found.

Impact of conservation tillage on wheat performance and its microbiome.

Winter wheat is an important cereal consumed worldwide. However, current management practices involving chemical fertilizers, irrigation, and intensive tillage may have negative impacts on the environment. Conservation agriculture is often presented as a sustainable alternative to maintain wheat production, favoring the beneficial microbiome. Here, we evaluated the impact of different water regimes (rainfed and irrigated), fertilization levels (half and full fertilization), and tillage practices (occasional tillage and no-tillage) on wheat performance, microbial activity, and rhizosphere- and root-associated microbial communities of four winter wheat genotypes (Antequera, Allez-y, Apache, and Cellule) grown in a field experiment. Wheat performance (i.e., yield, plant nitrogen concentrations, and total nitrogen uptake) was mainly affected by irrigation, fertilization, and genotype, whereas microbial activity (i.e., protease and alkaline phosphatase activities) was affected by irrigation. Amplicon sequencing data revealed that habitat (rhizosphere vs. root) was the main factor shaping microbial communities and confirmed that the selection of endophytic microbial communities takes place thanks to specific plant-microbiome interactions. Among the experimental factors applied, the interaction of irrigation and tillage influenced rhizosphere- and root-associated microbiomes. The findings presented in this work make it possible to link agricultural practices to microbial communities, paving the way for better monitoring of these microorganisms in the context of agroecosystem sustainability.

Food systems microbiome-related educational needs.

Within the European-funded Coordination and Support Action MicrobiomeSupport (https://www.microbiomesupport.eu/), the Workshop 'Education in Food Systems Microbiome Related Sciences: Needs for Universities, Industry and Public Health Systems' brought together over 70 researchers, public health and industry partners from all over the world to work on elaborating microbiome-related educational needs in food systems. This publication provides a summary of discussions held during and after the workshop and the resulting recommendations.

Microbiome Interconnectedness throughout Environments with Major Consequences for Healthy People and a Healthy Planet.

Microbiomes have highly important roles for ecosystem functioning and carry out key functions that support planetary health, including nutrient cycling, climate regulation, and water filtration. Microbiomes are also intimately associated with complex multicellular organisms such as humans, other animals, plants, and insects and perform crucial roles for the health of their hosts. Although we are starting to understand that microbiomes in different systems are interconnected, there is still a poor understanding of microbiome transfer and connectivity. In this review we show how microbiomes are connected within and transferred between different habitats and discuss the functional consequences of these connections. Microbiome transfer occurs between and within abiotic (e.g., air, soil, and water) and biotic environments, and can either be mediated through different vectors (e.g., insects or food) or direct interactions. Such transfer processes may also include the transmission of pathogens or antibiotic resistance genes. However, here, we highlight the fact that microbiome transmission can have positive effects on planetary and human health, where transmitted microorganisms potentially providing novel functions may be important for the adaptation of ecosystems.

Diverse bacteria colonizing leaves and the rhizosphere of lettuce degrade azoxystrobin.

Concerns about the possible effects of pesticide residues on both the environment and human health have increased worldwide. Bioremediation by the use of microorganisms to degrade or remove these residues has emerged as a powerful technology. However, the knowledge about the potential of different microorganisms for pesticide degradation is limited. This study focused on the isolation and characterisation of bacterial strains with the potential to degrade the active fungicide ingredient azoxystrobin. Potential degrading bacteria were tested in vitro and in the greenhouse, and the genomes of the best degrading strains were sequenced and analysed. We identified and characterised 59 unique bacterial strains, which were further tested in vitro and in greenhouse trials for their degradation activity. The best degraders from a foliar application trial in the greenhouse were identified as Bacillus subtilis strain MK101, Pseudomonas kermanshahensis strain MK113 and Rhodococcus fascians strain MK144 and analysed by whole genome sequencing. Genome analysis revealed that these three bacterial strains encode several genes predicted to be involved in the degradation of pesticides e.g., benC, pcaG, pcaH, however we could not find any specific gene previously reported to be involved in azoxystrobin degradation e.g., strH. Genome analysis pinpointed to some potential activities involved in plant growth promotion.

Potato root-associated microbiomes adapt to combined water and nutrient limitation and have a plant genotype-specific role for plant stress mitigation.

BACKGROUND: Due to climate change and reduced use of fertilizers combined stress scenarios are becoming increasingly frequent in crop production. In a field experiment we tested the effect of combined water and phosphorus limitation on the growth performance and plant traits of eight tetraploid and two diploid potato varieties as well as on root-associated microbiome diversity and functional potential. Microbiome and metagenome analysis targeted the diversity and potential functions of prokaryotes, fungi, plasmids, and bacteriophages and was linked to plant traits like tuber yield or timing of canopy closure. RESULTS: The different potato genotypes responded differently to the combined stress and hosted distinct microbiota in the rhizosphere and the root endosphere. Proximity to the root, stress and potato genotype had significant effects on bacteria, whereas fungi were only mildly affected. To address the involvement of microbial functions, we investigated well and poorly performing potato genotypes (Stirling and Desirée, respectively) under stress conditions and executed a metagenome analysis of rhizosphere microbiomes subjected to stress and no stress conditions. Functions like ROS detoxification, aromatic amino acid and terpene metabolism were enriched and in synchrony with the metabolism of stressed plants. In Desirée, Pseudonocardiales had the genetic potential to take up assimilates produced in the fast-growing canopy and to reduce plant stress-sensing by degrading ethylene, but overall yield losses were high. In Stirling, Xanthomonadales had the genetic potential to reduce oxidative stress and to produce biofilms, potentially around roots. Biofilm formation could be involved in drought resilience and nutrient accessibility of Stirling and explain the recorded low yield losses. In the rhizosphere exposed to combined stress, the relative abundance of plasmids was reduced, and the diversity of phages was enriched. Moreover, mobile elements like plasmids and phages were affected by combined stresses in a genotype-specific manner. CONCLUSION: Our study gives new insights into the interconnectedness of root-associated microbiota and plant stress responses in the field. Functional genes in the metagenome, phylogenetic composition and mobile elements play a role in potato stress adaption. In a poor and a well performing potato genotype grown under stress conditions, distinct functional genes pinpoint to a distinct stress sensing, water availability and compounds in the rhizospheres.

The need for an integrated multi-OMICs approach in microbiome science in the food system.

Microbiome science as an interdisciplinary research field has evolved rapidly over the past two decades, becoming a popular topic not only in the scientific community and among the general public, but also in the food industry due to the growing demand for microbiome-based technologies that provide added-value solutions. Microbiome research has expanded in the context of food systems, strongly driven by methodological advances in different -omics fields that leverage our understanding of microbial diversity and function. However, managing and integrating different complex -omics layers are still challenging. Within the Coordinated Support Action MicrobiomeSupport (https://www.microbiomesupport.eu/), a project supported by the European Commission, the workshop "Metagenomics, Metaproteomics and Metabolomics: the need for data integration in microbiome research" gathered 70 participants from different microbiome research fields relevant to food systems, to discuss challenges in microbiome research and to promote a switch from microbiome-based descriptive studies to functional studies, elucidating the biology and interactive roles of microbiomes in food systems. A combination of technologies is proposed. This will reduce the biases resulting from each individual technology and result in a more comprehensive view of the biological system as a whole. Although combinations of different datasets are still rare, advanced bioinformatics tools and artificial intelligence approaches can contribute to understanding, prediction, and management of the microbiome, thereby providing the basis for the improvement of food quality and safety.

Metabolite Production in Alkanna tinctoria Links Plant Development with the Recruitment of Individual Members of Microbiome Thriving at the Root-Soil Interface.

Plants are naturally associated with diverse microbial communities, which play significant roles in plant performance, such as growth promotion or fending off pathogens. The roots of Alkanna tinctoria L. are rich in naphthoquinones, particularly the medicinally used enantiomers alkannin and shikonin and their derivatives. Former studies already have shown that microorganisms may modulate plant metabolism. To further investigate the potential interaction between A. tinctoria and associated microorganisms, we performed a greenhouse experiment in which A. tinctoria plants were grown in the presence of three distinct soil microbiomes. At four defined plant developmental stages, we made an in-depth assessment of bacterial and fungal root-associated microbiomes as well as all extracted primary and secondary metabolite content of root material. Our results showed that the plant developmental stage was the most important driver influencing the plant metabolite content, revealing peak contents of alkannin/shikonin derivatives at the fruiting stage. Plant root microbial diversity was influenced both by bulk soil origin and to a small extent by the developmental stage. The performed correlation analyses and cooccurrence networks on the measured metabolite content and the abundance of individual bacterial and fungal taxa suggested a dynamic and at times positive or negative relationship between root-associated microorganisms and root metabolism. In particular, the bacterial genera Labrys and Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium as well as four species of the fungal genus Penicillium were found to be positively correlated with higher content of alkannins. IMPORTANCE Previous studies have shown that individual, isolated microorganisms may influence secondary metabolism of plants and induce or stimulate the production of medicinally relevant secondary metabolism. Here, we analyzed the microbiome-metabolome linkage of the medicinal plant Alkanna tinctoria, which is known to produce valuable compounds, particularly the naphthoquinones alkannin and shikonin and their derivatives. A detailed bacterial and fungal microbiome and metabolome analysis of A. tinctoria roots revealed that the plant developmental stage influenced root metabolite production, whereas soil inoculants from three different geographical origins in which plants were grown shaped root-associated microbiota. Metabolomes of plant roots of the same developmental stage across different soils were highly similar, pinpointing to plant maturity as the primary driver of secondary metabolite production. Correlation and network analyses identified bacterial and fungal taxa showing a positive relationship between root-associated microorganisms and root metabolism. In particular, the bacterial genera Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium and Labrys as well as the fungal species of genus Penicillium were found to be positively correlated with higher content of alkannins.

Microbiome ethics, guiding principles for microbiome research, use and knowledge management.

The overarching biological impact of microbiomes on their hosts, and more generally their environment, reflects the co-evolution of a mutualistic symbiosis, generating fitness for both. Knowledge of microbiomes, their systemic role, interactions, and impact grows exponentially. When a research field of importance for planetary health evolves so rapidly, it is essential to consider it from an ethical holistic perspective. However, to date, the topic of microbiome ethics has received relatively little attention considering its importance. Here, ethical analysis of microbiome research, innovation, use, and potential impact is structured around the four cornerstone principles of ethics: Do Good; Don't Harm; Respect; Act Justly. This simple, but not simplistic approach allows ethical issues to be communicative and operational. The essence of the paper is captured in a set of eleven microbiome ethics recommendations, e.g., proposing gut microbiome status as common global heritage, similar to the internationally agreed status of major food crops.

Microbiome Research as an Effective Driver of Success Stories in Agrifood Systems - A Selection of Case Studies.

Increasing knowledge of the microbiome has led to significant advancements in the agrifood system. Case studies based on microbiome applications have been reported worldwide and, in this review, we have selected 14 success stories that showcase the importance of microbiome research in advancing the agrifood system. The selected case studies describe products, methodologies, applications, tools, and processes that created an economic and societal impact. Additionally, they cover a broad range of fields within the agrifood chain: the management of diseases and putative pathogens; the use of microorganism as soil fertilizers and plant strengtheners; the investigation of the microbial dynamics occurring during food fermentation; the presence of microorganisms and/or genes associated with hazards for animal and human health (e.g., mycotoxins, spoilage agents, or pathogens) in feeds, foods, and their processing environments; applications to improve HACCP systems; and the identification of novel probiotics and prebiotics to improve the animal gut microbiome or to prevent chronic non-communicable diseases in humans (e.g., obesity complications). The microbiomes of soil, plants, and animals are pivotal for ensuring human and environmental health and this review highlights the impact that microbiome applications have with this regard.

Metadata harmonization-Standards are the key for a better usage of omics data for integrative microbiome analysis.

BACKGROUND: Tremendous amounts of data generated from microbiome research studies during the last decades require not only standards for sampling and preparation of omics data but also clear concepts of how the metadata is prepared to ensure re-use for integrative and interdisciplinary microbiome analysis. RESULTS: In this Commentary, we present our views on the key issues related to the current system for metadata submission in omics research, and propose the development of a global metadata system. Such a system should be easy to use, clearly structured in a hierarchical way, and should be compatible with all existing microbiome data repositories, following common standards for minimal required information and common ontology. Although minimum metadata requirements are essential for microbiome datasets, the immense technological progress requires a flexible system, which will have to be constantly improved and re-thought. While FAIR principles (Findable, Accessible, Interoperable, and Reusable) are already considered, international legal issues on genetic resource and sequence sharing provided by the Convention on Biological Diversity need more awareness and engagement of the scientific community. CONCLUSIONS: The suggested approach for metadata entries would strongly improve retrieving and re-using data as demonstrated in several representative use cases. These integrative analyses, in turn, would further advance the potential of microbiome research for novel scientific discoveries and the development of microbiome-derived products.

In-depth genome analysis of Bacillus sp. BH32, a salt stress-tolerant endophyte obtained from a halophyte in a semiarid region.

Endophytic strains belonging to the Bacillus cereus group were isolated from the halophytes Atriplex halimus L. (Amaranthaceae) and Tamarix aphylla L. (Tamaricaceae) from costal and continental regions in Algeria. Based on their salt tolerance (up to 5%), the strains were tested for their ability to alleviate salt stress in tomato and wheat. Bacillus sp. strain BH32 showed the highest potential to reduce salinity stress (up to + 50% and + 58% of dry weight improvement, in tomato and wheat, respectively, compared to the control). To determine putative mechanisms involved in salt tolerance and plant growth promotion, the whole genome of Bacillus sp. BH32 was sequenced, annotated, and used for comparative genomics against the genomes of closely related strains. The pangenome of Bacillus sp. BH32 and its closest relative was further analyzed. The phylogenomic analyses confirmed its taxonomic position, a member of the Bacillus cereus group, with intergenomic distances (GBDP analysis) pinpointing to a new taxon (digital DNA-DNA hybridization, dDDH < 70%). Genome mining unveiled several genes involved in stress tolerance, production of anti-oxidants and genes involved in plant growth promotion as well as in the production of secondary metabolites. KEY POINTS : • Bacillus sp. BH32 and other bacterial endophytes were isolated from halophytes, to be tested on tomato and wheat and to limit salt stress adverse effects. • The strain with the highest potential was then studied at the genomic level to highlight numerous genes linked to plant growth promotion and stress tolerance. • Pangenome approaches suggest that the strain belongs to a new taxon within the Bacillus cereus group.

Calling for a systems approach in microbiome research and innovation.

Microbiomes are all around us in natural and cultivated ecosystems, for example, soils, plants, animals and our own body. Microbiomes are essential players of biotechnological applications, and their functions drive human, animal, plant and environmental health. The rapidly developing microbiome research landscape was studied by a global mapping excercise and bibliometric analysis. Although microbiome research is performed in many different science fields, using similar concepts within and across fields, microbiomes are mostly investigated one ecosystem at-a-time. In order to fully understand microbiome impacts and leverage microbial functions, research needs to adopt a systems approach connecting microbiomes and research initiatives in divergent fields to create understanding on how microbiomes can be modulated for desirable functions as a basis of sustainable, circular bioeconomy.