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.

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.

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.

Modulating Drought Stress Response of Maize by a Synthetic Bacterial Community.

Plant perception and responses to environmental stresses are known to encompass a complex set of mechanisms in which the microbiome is involved. Knowledge about plant physiological responses is therefore critical for understanding the contribution of the microbiome to plant resilience. However, as plant growth is a dynamic process, a major hurdle is to find appropriate tools to effectively measure temporal variations of different plant physiological parameters. Here, we used a non-invasive real-time phenotyping platform in a one-to-one (plant-sensors) set up to investigate the impact of a synthetic community (SynCom) harboring plant-beneficial bacteria on the physiology and response of three commercial maize hybrids to drought stress (DS). SynCom inoculation significantly reduced yield loss and modulated vital physiological traits. SynCom-inoculated plants displayed lower leaf temperature, reduced turgor loss under severe DS and a faster recovery upon rehydration, likely as a result of sap flow modulation and better water usage. Microbiome profiling revealed that SynCom bacterial members were able to robustly colonize mature plants and recruit soil/seed-borne beneficial microbes. The high-resolution temporal data allowed us to record instant plant responses to daily environmental fluctuations, thus revealing the impact of the microbiome in modulating maize physiology, resilience to drought, and crop productivity.

Farming systems influence the compositional, structural, and functional characteristics of the sugarcane-associated microbiome.

Sugarcane (Saccharum spp.) has been produced worldwide as a relevant source of food and sustainable energy. However, the constant need to increase crop yield has led to excessive use of synthetic agrochemical inputs such as inorganic fertilizers, herbicides, and pesticides in plant cultures. It is known that these conventional practices can lead to deleterious effects on health and the environment. Organic farming emerges as a sustainable alternative to conventional systems; however, farm management influences in plant-associated microbiomes remain unclear. Here, the aim is to identify the effects of farming systems on the sugarcane microbiota. To address this issue, we sampled the microbiota from soils and plants under organic and conventional farming from two crop fields in Brazil. Then, we evaluated their compositional, structural, and functional traits through amplification and sequencing of phylogenetic markers of bacteria (16S rRNA gene, V3-V4 region) and fungi (Internal Transcribed Spacer - ITS2). The data processing and analyses by the DADA2 pipeline revealed 12,839 bacterial and 3,222 fungal sequence variants. Moreover, differences between analogous niches were detected considering the contrasting farming systems, with samples from the conventional system showing a slightly greater richness and diversity of microorganisms. The composition is also different between the farming systems, with 389 and 401 differentially abundant taxa for bacteria and fungi, respectively, including taxa capable of promoting plant growth. The microbial co-occurrence networks showed structural changes in microbial communities, where organic networks were more cohesive since they had closer taxa and less modularity by niches. Finally, the functional prediction revealed enriched metabolic pathways, including the increased presence of antimicrobial resistance in the conventional farming system. Taken together, our findings reveal functional, structural, and compositional adaptations of the microbial communities associated with sugarcane plants in the field, according to farming management. With this, we point out the need to unravel the mechanisms driving these adaptations.

From Microbiome to Traits: Designing Synthetic Microbial Communities for Improved Crop Resiliency.

Plants teem with microorganisms, whose tremendous diversity and role in plant-microbe interactions are being increasingly explored. Microbial communities create a functional bond with their hosts and express beneficial traits capable of enhancing plant performance. Therefore, a significant task of microbiome research has been identifying novel beneficial microbial traits that can contribute to crop productivity, particularly under adverse environmental conditions. However, although knowledge has exponentially accumulated in recent years, few novel methods regarding the process of designing inoculants for agriculture have been presented. A recently introduced approach is the use of synthetic microbial communities (SynComs), which involves applying concepts from both microbial ecology and genetics to design inoculants. Here, we discuss how to translate this rationale for delivering stable and effective inoculants for agriculture by tailoring SynComs with microorganisms possessing traits for robust colonization, prevalence throughout plant development and specific beneficial functions for plants. Computational methods, including machine learning and artificial intelligence, will leverage the approaches of screening and identifying beneficial microbes while improving the process of determining the best combination of microbes for a desired plant phenotype. We focus on recent advances that deepen our knowledge of plant-microbe interactions and critically discuss the prospect of using microbes to create SynComs capable of enhancing crop resiliency against stressful conditions.

Correction to: Microbiome definition re-visited: old concepts and new challenges.

An amendment to this paper has been published and can be accessed via the original article.

Genome Sequences of a Plant Beneficial Synthetic Bacterial Community Reveal Genetic Features for Successful Plant Colonization.

Despite the availability of data on the functional and phylogenetic diversity of plant-associated microbiota, the molecular mechanisms governing the successful establishment of plant bacterial communities remain mostly elusive. To investigate bacterial traits associated with successful colonization of plants, we sequenced the genome of 26 bacteria of a synthetic microbial community (SynCom), 12 of which displayed robust and 14 displayed non-robust colonization lifestyles when inoculated in maize plants. We examined the colonization profile of individual bacteria in inoculated plants and inspected their genomes for traits correlated to the colonization lifestyle. Comparative genomic analysis between robust and non-robust bacteria revealed that commonly investigated plant growth-promoting features such as auxin production, nitrogen (N) fixation, phosphate acquisition, and ACC deaminase are not deterministic for robust colonization. Functions related to carbon (C) and N acquisition, including transporters of carbohydrates and amino acids, and kinases involved in signaling mechanisms associated with C and N uptake, were enriched in robust colonizers. While enrichment of carbohydrate transporters was linked to a wide range of metabolites, amino acid transporters were primarily related to the uptake of branched-chain amino acids. Our findings identify diversification of nutrient uptake phenotypes in bacteria as determinants for successful bacterial colonization of plants.

Microbiomes of Velloziaceae from phosphorus-impoverished soils of the campos rupestres, a biodiversity hotspot.

The rocky, seasonally-dry and nutrient-impoverished soils of the Brazilian campos rupestres impose severe growth-limiting conditions on plants. Species of a dominant plant family, Velloziaceae, are highly specialized to low-nutrient conditions and seasonal water availability of this environment, where phosphorus (P) is the key limiting nutrient. Despite plant-microbe associations playing critical roles in stressful ecosystems, the contribution of these interactions in the campos rupestres remains poorly studied. Here we present the first microbiome data of Velloziaceae spp. thriving in contrasting substrates of campos rupestres. We assessed the microbiomes of Vellozia epidendroides, which occupies shallow patches of soil, and Barbacenia macrantha, growing on exposed rocks. The prokaryotic and fungal profiles were assessed by rRNA barcode sequencing of epiphytic and endophytic compartments of roots, stems, leaves and surrounding soil/rocks. We also generated root and substrate (rock/soil)-associated metagenomes of each plant species. We foresee that these data will contribute to decipher how the microbiome contributes to plant functioning in the campos rupestres, and to unravel new strategies for improved crop productivity in stressful environments.

A Community-Based Culture Collection for Targeting Novel Plant Growth-Promoting Bacteria from the Sugarcane Microbiome.

The soil-plant ecosystem harbors an immense microbial diversity that challenges investigative approaches to study traits underlying plant-microbe association. Studies solely based on culture-dependent techniques have overlooked most microbial diversity. Here we describe the concomitant use of culture-dependent and -independent techniques to target plant-beneficial microbial groups from the sugarcane microbiome. The community-based culture collection (CBC) approach was used to access microbes from roots and stalks. The CBC recovered 399 unique bacteria representing 15.9% of the rhizosphere core microbiome and 61.6-65.3% of the endophytic core microbiomes of stalks. By cross-referencing the CBC (culture-dependent) with the sugarcane microbiome profile (culture-independent), we designed a synthetic community comprised of naturally occurring highly abundant bacterial groups from roots and stalks, most of which has been poorly explored so far. We then used maize as a model to probe the abundance-based synthetic inoculant. We show that when inoculated in maize plants, members of the synthetic community efficiently colonize plant organs, displace the natural microbiota and dominate at 53.9% of the rhizosphere microbial abundance. As a result, inoculated plants increased biomass by 3.4-fold as compared to uninoculated plants. The results demonstrate that abundance-based synthetic inoculants can be successfully applied to recover beneficial plant microbes from plant microbiota.

Multiplex amplicon sequencing for microbe identification in community-based culture collections.

Microbiome analysis using metagenomic sequencing has revealed a vast microbial diversity associated with plants. Identifying the molecular functions associated with microbiome-plant interaction is a significant challenge concerning the development of microbiome-derived technologies applied to agriculture. An alternative to accelerate the discovery of the microbiome benefits to plants is to construct microbial culture collections concomitant with accessing microbial community structure and abundance. However, traditional methods of isolation, cultivation, and identification of microbes are time-consuming and expensive. Here we describe a method for identification of microbes in culture collections constructed by picking colonies from primary platings that may contain single or multiple microorganisms, which we named community-based culture collections (CBC). A multiplexing 16S rRNA gene amplicon sequencing based on two-step PCR amplifications with tagged primers for plates, rows, and columns allowed the identification of the microbial composition regardless if the well contains single or multiple microorganisms. The multiplexing system enables pooling amplicons into a single tube. The sequencing performed on the PacBio platform led to recovery near-full-length 16S rRNA gene sequences allowing accurate identification of microorganism composition in each plate well. Cross-referencing with plant microbiome structure and abundance allowed the estimation of diversity and abundance representation of microorganism in the CBC.

Unlocking the bacterial and fungal communities assemblages of sugarcane microbiome.

Plant microbiome and its manipulation herald a new era for plant biotechnology with the potential to benefit sustainable crop production. However, studies evaluating the diversity, structure and impact of the microbiota in economic important crops are still rare. Here we describe a comprehensive inventory of the structure and assemblage of the bacterial and fungal communities associated with sugarcane. Our analysis identified 23,811 bacterial OTUs and an unexpected 11,727 fungal OTUs inhabiting the endophytic and exophytic compartments of roots, shoots, and leaves. These communities originate primarily from native soil around plants and colonize plant organs in distinct patterns. The sample type is the primary driver of fungal community assemblage, and the organ compartment plays a major role in bacterial community assemblage. We identified core bacterial and fungal communities composed of less than 20% of the total microbial richness but accounting for over 90% of the total microbial relative abundance. The roots showed 89 core bacterial families, 19 of which accounted for 44% of the total relative abundance. Stalks are dominated by groups of yeasts that represent over 12% of total relative abundance. The core microbiome described here comprise groups whose biological role underlies important traits in plant growth and fermentative processes.

Structure, Organization, and Expression of the Alpha Prolamin Multigenic Family Bring New Insights into the Evolutionary Relationships among Grasses.

Prolamins are the major seed storage proteins of grasses. In maize and related species, prolamins are classified into α-, ß-, γ-, and δ-subclasses by their solubility properties. α-prolamins are encoded by multigene families and have a secondary structure that consists of tandem α-helix repeats. Maize has two α-prolamin subclasses, namely the 19 and 22 kDa subclasses that contain nine and 10 α-helix repeats, respectively. Here, we present an evolutionary study based on the structure, organization, and expression of α-prolamins in maize, sugarcane, sorghum, and coix. True 22 kDa subclasses containing 10 repeats are conserved in all four species, but true 19 kDa subclasses containing nine repeats are found only in maize and sugarcane. We discovered a 19 kDa-like α-coixin that, as in sorghum, is encoded by few genes. These data suggest that a 19 kDa progenitor present in the ancestor common to maize, coix, sorghum, and sugarcane was preserved at low copy number in coix and sorghum, while amplified into multigene family architecture in maize and sugarcane. The expression profiling of α-prolamins, verified by two-dimensional gels, showed highly conserved multispot composition for the 19 kDa α-prolamins in maize and sugarcane. Coix and sorghum did not present true 19 kDa α-prolamin spots. Our data show remarkable similarity between maize and sugarcane 19 kDa α-prolamins regarding both gene structure and expression. Since the multigene architecture of 19 kDa α-canein appeared after sugarcane diverged from sorghum, our data suggest that maize and sugarcane might have acquired the multigene family encoding these storage proteins from a common ancestor.