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In the recent years, the holobiont concept has emerged as a theoretical and experimental framework to study the interactions between hosts and their associated microbial communities in all types of ecosystems. The spread of this concept in many branches of biology results from the fairly recent realization of the ubiquitous nature of host-associated microbes and their central role in host biology, ecology, and evolution. Through this special series “Host-microbiota interactions: from holobiont theory to analysis,” we wanted to promote this field of research which has considerable implications for human health, food production, and ecosystem protection. In this preface, we highlight a collection of articles selected for this special issue that show, use, or debate the concept of holobiont to approach taxonomically and ecologically diverse organisms, from humans and plants to sponges and insects. We also identify some theoretical and methodological challenges and propose directions for future research on holobionts.

Soils are the product of the activities of plants, which supply organic matter and play a pivotal role in weathering rocks and minerals. Many plant species have a distinct ecological amplitude that shows restriction to specific soil types. In the numerous interactions between plants and soil, micro-organisms also play a key role. Here we review the existing literature on interactions between plants, microorganisms and soils, and include considerations of evolutionary time scales, where possible. Some of these interactions involve intricate systems of communication, which in the case of symbioses such as the arbuscular mycorrhizal symbiosis are several hundreds of millions years old; others involve the release of exudates from roots, and other products of rhizodeposition that are used as substrates for soil microorganisms. The possible reasons for the survival value of this loss of carbon over tens or hundreds of millions of years of evolution of higher plants are discussed, taking a cost-benefit approach. Coevolution of plants and rhizosphere microorganisms is discussed, in the light of known ecological interactions between various partners in terrestrial ecosystems. Finally, the role of higher plants, especially deep-rooted plants and associated microorganisms in the weathering of rocks and minerals, ultimately contributing to pedogenesis, is addressed. We show that rhizosphere processes in the long run are central to biogeochemical cycles, soil formation and Earth history. Major anticipated discoveries will enhance our basic understanding and allow applications of new knowledge to deal with nutrient deficiencies, pests and diseases, and the challenges of increasing global food production and agroecosystem productivity in an environmentally responsible manner.

Plant species are important drivers of soil microbial communities. However, how plant functional traits are shaping these communities has received less attention though linking plant and microbial traits is crucial for better understanding plant-microbe interactions. Our objective was to determine how plant-microbe interactions were affected by plant traits. Specifically we analyzed how interactions between plant species and microbes involved in nitrogen cycling were affected by plant traits related to nitrogen nutrition in interaction with soil nitrogen availability. Eleven plant species, selected along an oligotrophic-nitrophilic gradient, were grown individually in a nitrogen-poor soil with two levels of nitrate availability. Plant traits for both carbon and nitrogen nutrition were measured and the genetic structure and abundance of rhizosphere microbial communities, in particular the ammonia oxidizer and nitrate reducer guilds, were analyzed. The structure of the bacterial community in the rhizosphere differed significantly between plant species and these differences depended on nitrogen availability. The results suggest that the rate of nitrogen uptake per unit of root biomass and per day is a key plant trait, explaining why the effect of nitrogen availability on the structure of the bacterial community depends on the plant species. We also showed that the abundance of nitrate reducing bacteria always decreased with increasing nitrogen uptake per unit of root biomass per day, indicating that there was competition for nitrate between plants and nitrate reducing bacteria. This study demonstrates that nitrate-reducing microorganisms may be adversely affected by plants with a high nitrogen uptake rate. Our work puts forward the role of traits related to nitrogen in plant-microbe interactions, whereas carbon is commonly considered as the main driver. It also suggests that plant traits related to ecophysiological processes, such as nitrogen uptake rates, are more relevant for understanding plant-microbe interactions than composite traits, such as nitrophily, which are related to a number of ecophysiological processes.

The turnover of organic matter in soil depends on the activity of microbial decomposers. However, little is known about how modifications of the diversity of soil microbial communities induced by fresh organic matter (FOM) inputs can regulate carbon cycling. Here, we investigated the decomposition of two 13 C labeled crop residues (wheat and alfalfa) and the dynamics of the genetic structure and taxonomic composition of the soil bacterial communities decomposing 13 C labeled FOM and native unlabeled soil organic matter (SOM), respectively. It was achieved by combining the stable isotope probing method with molecular tools (DNA genotyping and pyrosequencing of 16S rDNA). Although a priming effect (PE) was always induced by residue addition, its intensity increased with the degradability of the plant residue. The input of both wheat and alfalfa residues induced a rapid dynamics of FOM-degrading communities, corresponding to the stimulation of bacterial phyla which have been previously described as copiotrophic organisms. However, the dynamics and the identity of the bacterial groups stimulated depended on the residue added, with Firmicutes dominating in the wheat treatment and Proteobacteria dominating in the alfalfa treatment after 3 days of incubation. In both treatments, SOM-degrading communities were dominated by Acidobacteria, Verrucomicrobia, and Gemmatimonadetes phyla which have been previously described as oligotrophic organisms. An early stimulation of SOM-degrading populations mainly belonging to Firmicutes and Bacteroidetes groups was observed in the alfalfa treatment whereas no change occurred in the wheat treatment. Our findings support the hypothesis that the succession of bacterial taxonomic groups occurring in SOM- and FOM-degrading communities during the degradation process may be an important driver of the PE, and consequently of carbon dynamics in soil.

Fungi constitute an important group in soil biological diversity and functioning. However, characterization and knowledge of fungal communities is hampered because few primer sets are available to quantify fungal abundance by real-time quantitative PCR (real-time Q-PCR). The aim in this study was to quantify fungal abundance in soils by incorporating, into a real-time Q-PCR using the SYBRGreen® method, a primer set already used to study the genetic structure of soil fungal communities. To satisfy the real-time Q-PCR requirements to enhance the accuracy and reproducibility of the detection technique, this study focused on the 18S rRNA gene conserved regions. These regions are little affected by length polymorphism and may provide sufficiently small targets, a crucial criterion for enhancing accuracy and reproducibility of the detection technique. An in silico analysis of 33 primer sets targeting the 18S rRNA gene was performed to select the primer set with the best potential for real-time Q-PCR: short amplicon length; good fungal specificity and coverage. The best consensus between specificity, coverage and amplicon length among the 33 sets tested was the primer set FR1/FF390. This in silico analysis of the specificity of FR1/FF390 also provided additional information to the previously published analysis on this primer set. The specificity of the primer set FR1/FF390 for Fungi was validated in vitro by cloning--sequencing the amplicons obtained from a real time Q-PCR assay performed on five independent soil samples. This assay was also used to evaluate the sensitivity and reproducibility of the method. Finally, fungal abundance in samples from 24 soils with contrasting physico-chemical and environmental characteristics was examined and ranked to determine the importance of soil texture, organic carbon content, C∶N ratio and land use in determining fungal abundance in soils.

The temporal dynamics of rhizosphere and root microbiota composition was compared between healthy and infected Chinese cabbage plants by the pathogen Plasmodiophora brassicae. When inoculated with P. brassicae, disease was measured at five sampling dates from early root hair infection to late gall development. The first symptoms of clubroot disease appeared 14 days after inoculation (DAI) and increased drastically between 14 and 35 DAI. The structure of microbial communities associated to rhizosphere soil and root from healthy and inoculated plants was characterized through high-throughput DNA sequencing of bacterial (16S) and fungal (18S) molecular markers and compared at each sampling date. In healthy plants, Proteobacteria and Bacteroidetes bacterial phyla dominated the rhizosphere and root microbiota of Chinese cabbage. Rhizosphere bacterial communities contained higher abundances of Actinobacteria and Firmicutes compared to the roots. Moreover, a drastic shift of fungal communities of healthy plants occurred between the two last sampling dates, especially in plant roots, where most of Ascomycota fungi dominated until they were replaced by a fungus assigned to the Chytridiomycota phylum. Parasitic invasion by P. brassicae disrupted the rhizosphere and root-associated community assembly at a late step during the root secondary cortical infection stage of clubroot disease. At this stage, Flavisolibacter and Streptomyces in the rhizosphere, and Bacillus in the roots, were drastically less abundant upon parasite invasion. Rhizosphere of plants colonized by P. brassicae was significantly more invaded by the Chytridiomycota fungus, which could reflect a mutualistic relationship in this compartment between these two microorganisms.

Background Most metazoans are involved in durable relationships with microbes which can take several forms, from mutualism to parasitism. The advances of NGS technologies and bioinformatics tools have opened opportunities to shed light on the diversity of microbial communities and to give some insights into the functions they perform in a broad array of hosts. The pea aphid is a model system for the study of insect-bacteria symbiosis. It is organized in a complex of biotypes, each adapted to specific host plants. It harbors both an obligatory symbiont supplying key nutrients and several facultative symbionts bringing additional functions to the host, such as protection against biotic and abiotic stresses. However, little is known on how the symbiont genomic diversity is structured at different scales: across host biotypes, among individuals of the same biotype, or within individual aphids, which limits our understanding on how these multi-partner symbioses evolve and interact. Results We present a framework well adapted to the study of genomic diversity and evolutionary dynamics of the pea aphid holobiont from metagenomic read sets, based on mapping to reference genomes and whole genome variant calling. Our results revealed that the pea aphid microbiota is dominated by a few heritable bacterial symbionts reported in earlier works, with no discovery of new microbial associates. However, we detected a large and heterogeneous genotypic diversity associated with the different symbionts of the pea aphid. Partitioning analysis showed that this fine resolution diversity is distributed across the three considered scales. Phylogenetic analyses highlighted frequent horizontal transfers of facultative symbionts between host lineages, indicative of flexible associations between the pea aphid and its microbiota. However, the evolutionary dynamics of symbiotic associations strongly varied depending on the symbiont, reflecting different histories and possible constraints. In addition, at the intra-host scale, we showed that different symbiont strains may coexist inside the same aphid host. Conclusions We present a methodological framework for the detailed analysis of NGS data from microbial communities of moderate complexity and gave major insights into the extent of diversity in pea aphid-symbiont associations and the range of evolutionary trajectories they could take.

Soil DNA extraction has become a critical step in describing microbial biodiversity. Historically, ascertaining overarching microbial ecological theories has been hindered as independent studies have used numerous custom and commercial DNA extraction procedures. For that reason, a standardized soil DNA extraction method (ISO-11063) was previously published. However, although this ISO method is suited for molecular tools such as quantitative PCR and community fingerprinting techniques, it has only been optimized for examining soil bacteria. Therefore, the aim of this study was to assess an appropriate soil DNA extraction procedure for examining bacterial, archaeal and fungal diversity in soils of contrasting land-use and physico-chemical properties. Three different procedures were tested: the ISO-11063 standard; a custom procedure (GnS-GII); and a modified ISO procedure (ISOm) which includes a different mechanical lysis step (a FastPrep ®-24 lysis step instead of the recommended bead-beating). The efficacy of each method was first assessed by estimating microbial biomass through total DNA quantification. Then, the abundances and community structure of bacteria, archaea and fungi were determined using real-time PCR and terminal restriction fragment length polymorphism approaches. Results showed that DNA yield was improved with the GnS-GII and ISOm procedures, and fungal community patterns were found to be strongly dependent on the extraction method. The main methodological factor responsible for differences between extraction procedure efficiencies was found to be the soil homogenization step. For integrative studies which aim to examine bacteria, archaea and fungi simultaneously, the ISOm procedure results in higher DNA recovery and better represents microbial communities.

The objective was to further the understanding of the complex interaction between the soil microbiota, a plant pathogen and the host plant. The effect of different soil microbial diversities on the level of disease and on the transcriptomes of the interacting pathogen and host plant was studied. Summary The contribution of surrounding plant microbiota to disease development has led to the ‘pathobiome’ concept, which represents the interaction between the pathogen, the host plant and the associated biotic microbial community, resulting or not in plant disease. The aim herein is to understand how the soil microbial environment may influence the functions of a pathogen and its pathogenesis, and the molecular response of the plant to the infection, with a dual‐RNAseq transcriptomics approach. We address this question using Brassica napus and Plasmodiophora brassicae, the pathogen responsible for clubroot. A time‐course experiment was conducted to study interactions between P. brassicae, two B. napus genotypes and three soils harbouring high, medium or low microbiota diversities and levels of richness. The soil microbial diversity levels had an impact on disease development (symptom levels and pathogen quantity). The P. brassicae and B. napus transcriptional patterns were modulated by these microbial diversities, these modulations being dependent on the host genotype plant and the kinetic time. The functional analysis of gene expressions allowed the identification of pathogen and plant host functions potentially involved in the change of plant disease level, such as pathogenicity‐related genes (NUDIX effector) in P. brassicae and plant defence‐related genes (glucosinolate metabolism) in B. napus.

In the postgenomic era, there is a clear recognition of the limitations of nucleic acid-based methods for getting information on functions expressed by microbial communities in situ. In this context, the large-scale study of proteins expressed by indigenous microbial communities (metaproteome) should provide information to gain insights into the functioning of the microbial component in ecosystems. Characterization of the metaproteome is expected to provide data linking genetic and functional diversity of microbial communities. Studies on the metaproteome together with those on the metagenome and the metatranscriptome will contribute to progress in our knowledge of microbial communities and their contribution in ecosystem functioning. Effectiveness of the metaproteomic approach will be improved as increasing metagenomic information is made available thanks to the environmental sequencing projects currently running. More specifically, analysis of metaproteome in contrasted environmental situations should allow (1) tracking new functional genes and metabolic pathways and (2) identifying proteins preferentially associated with specific stresses. These proteins considered as functional bioindicators should contribute, in the future, to help policy makers in defining strategies for sustainable management of our environment.