Plant-associated microorganisms have been shown to critically affect host physiology and performance, suggesting that evolution and ecology of plants and animals can only be understood in a holobiont (host and its associated organisms) context. Host-associated microbial community structures are affected by abiotic and host factors, and increased attention is given to the role of the microbiome in interactions such as pathogen inhibition. However, little is known about how these factors act on the microbial community, and especially what role microbe–microbe interaction dynamics play. We have begun to address this knowledge gap for phyllosphere microbiomes of plants by simultaneously studying three major groups of Arabidopsis thaliana symbionts (bacteria, fungi and oomycetes) using a systems biology approach. We evaluated multiple potential factors of microbial community control: we sampled various wild A. thaliana populations at different times, performed field plantings with different host genotypes, and implemented successive host colonization experiments under lab conditions where abiotic factors, host genotype, and pathogen colonization was manipulated. Our results indicate that both abiotic factors and host genotype interact to affect plant colonization by all three groups of microbes. Considering microbe–microbe interactions, however, uncovered a network of interkingdom interactions with significant contributions to community structure. As in other scale-free networks, a small number of taxa, which we call microbial “hubs,” are strongly interconnected and have a severe effect on communities. By documenting these microbe–microbe interactions, we uncover an important mechanism explaining how abiotic factors and host genotypic signatures control microbial communities. In short, they act directly on “hub” microbes, which, via microbe–microbe interactions, transmit the effects to the microbial community. We analyzed two “hub” microbes (the obligate biotrophic oomycete pathogen Albugo and the basidiomycete yeast fungus Dioszegia) more closely. Albugo had strong effects on epiphytic and endophytic bacterial colonization. Specifically, alpha diversity decreased and beta diversity stabilized in the presence of Albugo infection, whereas they otherwise varied between plants. Dioszegia, on the other hand, provided evidence for direct hub interaction with phyllosphere bacteria. The identification of microbial “hubs” and their importance in phyllosphere microbiome structuring has crucial implications for plant–pathogen and microbe–microbe research and opens new entry points for ecosystem management and future targeted biocontrol. The revelation that effects can cascade through communities via “hub” microbes is important to understand community structure perturbations in parallel fields including human microbiomes and bioprocesses. In particular, parallels to human microbiome “keystone” pathogens and microbes open new avenues of interdisciplinary research that promise to better our understanding of functions of host-assoc...

SummaryRoots of healthy plants are inhabited by soil-derived bacteria, fungi, and oomycetes that have evolved independently in distinct kingdoms of life. How these microorganisms interact and to what extent those interactions affect plant health are poorly understood. We examined root-associated microbial communities from three Arabidopsis thaliana populations and detected mostly negative correlations between bacteria and filamentous microbial eukaryotes. We established microbial culture collections for reconstitution experiments using germ-free A. thaliana. In plants inoculated with mono- or multi-kingdom synthetic microbial consortia, we observed a profound impact of the bacterial root microbiota on fungal and oomycetal community structure and diversity. We demonstrate that the bacterial microbiota is essential for plant survival and protection against root-derived filamentous eukaryotes. Deconvolution of 2,862 binary bacterial-fungal interactions ex situ, combined with community perturbation experiments in planta, indicate that biocontrol activity of bacterial root commensals is a redundant trait that maintains microbial interkingdom balance for plant health.

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17Roots of healthy plants are inhabited by soil-derived bacteria, fungi, and oomycetes that have 18 evolved independently in distinct kingdoms of life. How these microorganisms interact and to 19 what extent those interactions affect plant health are poorly understood. We examined root-20 associated microbial communities from three Arabidopsis thaliana populations and detected 21 mostly negative correlations between bacteria and filamentous microbial eukaryotes. We 22 established microbial culture collections for reconstitution experiments using germ-free A. 23 thaliana. In plants inoculated with mono-or multi-kingdom synthetic microbial consortia, we 24 observed a profound impact of the bacterial root microbiota on fungal and oomycetal 25 2 community structure and diversity. We demonstrate that the bacterial microbiota is essential 26 for plant survival and protection against root-derived filamentous eukaryotes. Deconvolution 27 of 2,862 binary bacterial-fungal interactions ex situ, combined with community perturbation 28 experiments in planta, indicate that biocontrol activity of bacterial root commensals is a 29 redundant trait that maintains microbial interkingdom balance for plant health. 30 31 reconstitution experiments, we provide community-level evidence that negative interactions 51 between prokaryotic and eukaryotic root microbiota members are critical for plant host 52 survival and maintenance of host-microbiota balance. 53 54 Results 55Root-associated microbial assemblages. We collected A. thaliana plants from natural 56 populations at two neighbouring sites in Germany (Geyen and Pulheim; 5 km apart) and a 57 more distant location in France (Saint-Dié; ~300 km away) ( Figure S1; Table S1). For each 58 population, four replicates, each consisting of four pooled A. thaliana individuals were 59 prepared, together with corresponding bulk soils. Root samples were fractionated into 60 episphere and endosphere compartments, enriching for microbes residing on the root surface 61 or inside roots, respectively ( Figure S2). We characterized the multi-kingdom microbial 62 consortia along the soil-root continuum by simultaneous DNA amplicon sequencing of the 63 bacterial 16S rRNA gene and fungal as well as oomycetal Internal Transcribed Spacer (ITS) 64 regions (Agler et al. 2016) ( Table S2). Alpha diversity indices (within-sample diversity) 65 indicated a gradual decrease of microbial diversity from bulk soil to the root endosphere 66 (Kruskal-Wallis test, p<0.01; Figure S3). Profiles of microbial class abundance between 67 sample-types ( Figure 1A) and Operational Taxonomic Unit (OTU) enrichment tests 68 conducted using a linear model between soil, root episphere and root endosphere samples 69 (p<0.05, Figure 1B) identified 96 bacterial, 24 fungal and one oomycetal OTU that are 70 consistently enriched in plant roots across all three sites. This, together with the reduced alpha 71 diversity, points to a gating role of the root surface for entry into the root interior for each of 72 the three microbial kingdoms ...

Recent field and laboratory experiments with perennial Boechera stricta and annual Arabidopsis thaliana suggest that the root microbiota influences flowering time. Here we examined in long-term time-course experiments the bacterial root microbiota of the arctic-alpine perennial Arabis alpina in natural and controlled environments by 16S rRNA gene profiling. We identified soil type and residence time of plants in soil as major determinants explaining up to 15% of root microbiota variation, whereas environmental conditions and host genotype explain maximally 11% of variation. When grown in the same soil, the root microbiota composition of perennial A. alpina is largely similar to those of its annual relatives A. thaliana and Cardamine hirsuta. Non-flowering wild-type A. alpina and flowering pep1 mutant plants assemble an essentially indistinguishable root microbiota, thereby uncoupling flowering time from plant residence time-dependent microbiota changes. This reveals the robustness of the root microbiota against the onset and perpetual flowering of A. alpina. Together with previous studies, this implies a model in which parts of the root microbiota modulate flowering time, whereas, after microbiota acquisition during vegetative growth, the established root-associated bacterial assemblage is structurally robust to perturbations caused by flowering and drastic changes in plant stature.

High productivity and specificity in anaerobic digesters arise because complex microbiomes organize into a metabolic cascade to maximize energy recovery and to utilize the advantage that the gaseous end product methane freely bubbles out of the system. These lessons were applied to ascertain whether a reactor microbiome could be shaped to produce a different end product. The liquid product n-caproic acid was chosen, which is a 6-carbon-chain carboxylic acid that is valuable and that has a relatively low maximum solubility concentration for product recovery. Acetoclastic methanogenesis was inhibited by pH control and a route was provided for n-caproic acid extraction by implementing selective, in-line recovery. Next, ethanol was supplemented to promote chain elongation, which is a pathway in which short-chain carboxylic acids are elongated sequentially into medium-chain carboxylic acids with two-carbon units derived from ethanol. The reactor microbiome developed accordingly with the terminal process catalyzed by chain-elongating bacteria. As a result, n-caproic acid production rates increased to levels comparable to anaerobic digestion systems for solid waste treatment.