Ecology and Evolution of Plant Microbiomes.

Microorganisms colonizing plant surfaces and internal tissues provide a number of life-support functions for their host. Despite increasing recognition of the vast functional capabilities of the plant microbiome, our understanding of the ecology and evolution of the taxonomically hyperdiverse microbial communities is limited. Here, we review current knowledge of plant genotypic and phenotypic traits as well as allogenic and autogenic factors that shape microbiome composition and functions. We give specific emphasis to the impact of plant domestication on microbiome assembly and how insights into microbiomes of wild plant relatives and native habitats can contribute to reinstate or enrich for microorganisms with beneficial effects on plant growth, development, and health. Finally, we introduce new concepts and perspectives in plant microbiome research, in particular how community ecology theory can provide a mechanistic framework to unravel the interplay of distinct ecological processes-i.e., selection, dispersal, drift, diversification-that structure the plant microbiome.

The plant stress hormone jasmonic acid evokes defensive responses in streptomycetes.

IMPORTANCE: Microorganisms that live on or inside plants can influence plant growth and health. Among the plant-associated bacteria, streptomycetes play an important role in defense against plant diseases, but the underlying mechanisms are not well understood. Here, we demonstrate that the plant hormones jasmonic acid (JA) and methyl jasmonate directly affect the life cycle of streptomycetes by modulating antibiotic synthesis and promoting faster development. Moreover, the plant hormones specifically stimulate the synthesis of the polyketide antibiotic actinorhodin in Streptomyces coelicolor. JA is then modified in the cell by amino acid conjugation, thereby quenching toxicity. Collectively, these results provide new insight into the impact of a key plant hormone on diverse phenotypic responses of streptomycetes.

Plant neighbours can make or break the disease transmission chain of a fungal root pathogen.

Biodiversity can reduce or increase disease transmission. These divergent effects suggest that community composition rather than diversity per se determines disease transmission. In natural plant communities, little is known about the functional roles of neighbouring plant species in belowground disease transmission. Here, we experimentally investigated disease transmission of a fungal root pathogen (Rhizoctonia solani) in two focal plant species in combinations with four neighbour species of two ages. We developed stochastic models to test the relative importance of two transmission-modifying mechanisms: (1) infected hosts serve as nutrient supply to increase hyphal growth, so that successful disease transmission is self-reinforcing; and (2) plant resistance increases during plant development. Neighbouring plants either reduced or increased disease transmission in the focal plants. These effects depended on neighbour age, but could not be explained by a simple dichotomy between hosts and nonhost neighbours. Model selection revealed that both transmission-modifying mechanisms are relevant and that focal host-neighbour interactions changed which mechanisms steered disease transmission rate. Our work shows that neighbour-induced shifts in the importance of these mechanisms across root networks either make or break disease transmission chains. Understanding how diversity affects disease transmission thus requires integrating interactions between focal and neighbour species and their pathogens.

Metabolic signatures of rhizobacteria-induced plant growth promotion.

Various root-colonizing bacterial species can promote plant growth and trigger systemic resistance against aboveground leaf pathogens and herbivore insects. To date, the underlying metabolic signatures of these rhizobacteria-induced plant phenotypes are poorly understood. To identify core metabolic pathways that are targeted by growth-promoting rhizobacteria, we used combinations of three plant species and three rhizobacterial species and interrogated plant shoot chemistry by untargeted metabolomics. A substantial part (50%-64%) of the metabolites detected in plant shoot tissue was differentially affected by the rhizobacteria. Among others, the phenylpropanoid pathway was targeted by the rhizobacteria in each of the three plant species. Differential regulation of the various branches of the phenylpropanoid pathways showed an association with either plant growth promotion or growth reduction. Overall, suppression of flavonoid biosynthesis was associated with growth promotion, while growth reduction showed elevated levels of flavonoids. Subsequent assays with 12 Arabidopsis flavonoid biosynthetic mutants revealed that the proanthocyanidin branch plays an essential role in rhizobacteria-mediated growth promotion. Our study also showed that a number of pharmaceutically and nutritionally relevant metabolites in the plant shoot were significantly increased by rhizobacterial treatment, providing new avenues to use rhizobacteria to tilt plant metabolism towards the biosynthesis of valuable natural plant products.

Exploring the Volatiles Released from Roots of Wild and Domesticated Tomato Plants under Insect Attack.

Plants produce volatile organic compounds that are important in communication and defense. While studies have largely focused on volatiles emitted from aboveground plant parts upon exposure to biotic or abiotic stresses, volatile emissions from roots upon aboveground stress are less studied. Here, we investigated if tomato plants under insect herbivore attack exhibited a different root volatilome than non-stressed plants, and whether this was influenced by the plant's genetic background. To this end, we analyzed one domesticated and one wild tomato species, i.e., Solanum lycopersicum cv Moneymaker and Solanum pimpinellifolium, respectively, exposed to leaf herbivory by the insect Spodoptera exigua. Root volatiles were trapped with two sorbent materials, HiSorb and PDMS, at 24 h after exposure to insect stress. Our results revealed that differences in root volatilome were species-, stress-, and material-dependent. Upon leaf herbivory, the domesticated and wild tomato species showed different root volatile profiles. The wild species presented the largest change in root volatile compounds with an overall reduction in monoterpene emission under stress. Similarly, the domesticated species presented a slight reduction in monoterpene emission and an increased production of fatty-acid-derived volatiles under stress. Volatile profiles differed between the two sorbent materials, and both were required to obtain a more comprehensive characterization of the root volatilome. Collectively, these results provide a strong basis to further unravel the impact of herbivory stress on systemic volatile emissions.

Volatiles from soil-borne fungi affect directional growth of roots.

Volatiles play major roles in mediating ecological interactions between soil (micro)organisms and plants. It is well-established that microbial volatiles can increase root biomass and lateral root formation. To date, however, it is unknown whether microbial volatiles can affect directional root growth. Here, we present a novel method to study belowground volatile-mediated interactions. As proof-of-concept, we designed a root Y-tube olfactometer, and tested the effects of volatiles from four different soil-borne fungi on directional growth of Brassica rapa roots in soil. Subsequently, we compared the fungal volatile organic compounds (VOCs) previously profiled with Gas Chromatography-Mass Spectrometry (GC-MS). Using our newly designed setup, we show that directional root growth in soil is differentially affected by fungal volatiles. Roots grew more frequently toward volatiles from the root pathogen Rhizoctonia solani, whereas volatiles from the other three saprophytic fungi did not impact directional root growth. GC-MS profiling showed that six VOCs were exclusively emitted by R. solani. These findings verify that this novel method is suitable to unravel the intriguing chemical cross-talk between roots and soil-borne fungi and its impact on root growth.

Discovery of Thanafactin A, a Linear, Proline-Containing Octalipopeptide from Pseudomonas sp. SH-C52, Motivated by Genome Mining.

Genome mining of the bacterial strains Pseudomonas sp. SH-C52 and Pseudomonas fluorescens DSM 11579 showed that both strains contained a highly similar gene cluster encoding an octamodular nonribosomal peptide synthetase (NRPS) system which was not associated with a known secondary metabolite. Insertional mutagenesis of an NRPS component followed by comparative profiling led to the discovery of the corresponding novel linear octalipopeptide thanafactin A, which was subsequently isolated and its structure determined by two-dimensional NMR and further spectroscopic and chromatographic methods. In bioassays, thanafactin A exhibited weak protease inhibitory activity and was found to modulate swarming motility in a strain-specific manner.

Microbial and volatile profiling of soils suppressive to Fusarium culmorum of wheat.

In disease-suppressive soils, microbiota protect plants from root infections. Bacterial members of this microbiota have been shown to produce specific molecules that mediate this phenotype. To date, however, studies have focused on individual suppressive soils and the degree of natural variability of soil suppressiveness remains unclear. Here, we screened a large collection of field soils for suppressiveness to Fusarium culmorum using wheat (Triticum aestivum) as a model host plant. A high variation of disease suppressiveness was observed, with 14% showing a clear suppressive phenotype. The microbiological basis of suppressiveness to F. culmorum was confirmed by gamma sterilization and soil transplantation. Amplicon sequencing revealed diverse bacterial taxonomic compositions and no specific taxa were found exclusively enriched in all suppressive soils. Nonetheless, co-occurrence network analysis revealed that two suppressive soils shared an overrepresented bacterial guild dominated by various Acidobacteria. In addition, our study revealed that volatile emission may contribute to suppression, but not for all suppressive soils. Our study raises new questions regarding the possible mechanistic variability of disease-suppressive phenotypes across physico-chemically different soils. Accordingly, we anticipate that larger-scale soil profiling, along with functional studies, will enable a deeper understanding of disease-suppressive microbiomes.

Volatiles of pathogenic and non-pathogenic soil-borne fungi affect plant development and resistance to insects.

Plants are ubiquitously exposed to a wide diversity of (micro)organisms, including mutualists and antagonists. Prior to direct contact, plants can perceive microbial organic and inorganic volatile compounds (hereafter: volatiles) from a distance that, in turn, may affect plant development and resistance. To date, however, the specificity of plant responses to volatiles emitted by pathogenic and non-pathogenic fungi and the ecological consequences of such responses remain largely elusive. We investigated whether Arabidopsis thaliana plants can differentiate between volatiles of pathogenic and non-pathogenic soil-borne fungi. We profiled volatile organic compounds (VOCs) and measured CO2 emission of 11 fungi. We assessed the main effects of fungal volatiles on plant development and insect resistance. Despite distinct differences in VOC profiles between the pathogenic and non-pathogenic fungi, plants did not discriminate, based on plant phenotypic responses, between pathogenic and non-pathogenic fungi. Overall, plant growth was promoted and flowering was accelerated upon exposure to fungal volatiles, irrespectively of fungal CO2 emission levels. In addition, plants became significantly more susceptible to a generalist insect leaf-chewing herbivore upon exposure to the volatiles of some of the fungi, demonstrating that a prior fungal volatile exposure can negatively affect plant resistance. These data indicate that plant development and resistance can be modulated in response to exposure to fungal volatiles.

Modulation of plant chemistry by beneficial root microbiota.

Covering: 1981-2017Plants are colonized by an astounding number of microorganisms that can reach cell densities much greater than the number of plant cells. Various plant-associated microorganisms can have profound beneficial effects on plant growth, development, physiology and tolerance to (a)biotic stress. In return, plants release metabolites into their direct surroundings, thereby feeding the microbial community and influencing their composition, gene expression and the production of secondary metabolites. Similarly, microbes living on and in plant tissue may induce known and yet unknown biosynthetic pathways in plants leading to diverse alterations in the plant metabolome. Here, we provide an overview of the impact of beneficial microbiota on plant chemistry, with an emphasis on bacteria living on or inside root tissues. We will also provide new perspectives on deciphering the yet untapped potential of microbe-mediated alteration of plant chemistry as an alternative platform to discover new pathways, genes and enzymes involved the biosynthesis of high value natural plant products.

Priming of Plant Growth Promotion by Volatiles of Root-Associated Microbacterium spp.

Volatile compounds produced by plant-associated microorganisms represent a diverse resource to promote plant growth and health. Here, we investigated the effect of volatiles from root-associated Microbacterium species on plant growth and development. Volatiles of eight strains induced significant increases in shoot and root biomass of Arabidopsis but differed in their effects on root architecture. Microbacterium strain EC8 also enhanced root and shoot biomass of lettuce and tomato. Biomass increases were also observed for plants exposed only briefly to volatiles from EC8 prior to transplantation of the seedlings to soil. These results indicate that volatiles from EC8 can prime plants for growth promotion without direct and prolonged contact. We further showed that the induction of plant growth promotion is tissue specific; that is, exposure of roots to volatiles from EC8 led to an increase in plant biomass, whereas shoot exposure resulted in no or less growth promotion. Gas chromatography-quadrupole time of flight mass spectometry (GC-QTOF-MS) analysis revealed that EC8 produces a wide array of sulfur-containing compounds, as well as ketones. Bioassays with synthetic sulfur volatile compounds revealed that the plant growth response to dimethyl trisulfide was concentration-dependent, with a significant increase in shoot weight at 1 µM and negative effects on plant biomass at concentrations higher than 1 mM. Genome-wide transcriptome analysis of volatile-exposed Arabidopsis seedlings showed upregulation of genes involved in assimilation and transport of sulfate and nitrate. Collectively, these results show that root-associated Microbacterium primes plants, via the roots, for growth promotion, most likely via modulation of sulfur and nitrogen metabolism.IMPORTANCE In the past decade, various studies have described the effects of microbial volatiles on other (micro)organisms in vitro, but their broad-spectrum activity in vivo and the mechanisms underlying volatile-mediated plant growth promotion have not been addressed in detail. Here, we revealed that volatiles from root-associated bacteria of the genus Microbacterium can enhance the growth of different plant species and can prime plants for growth promotion without direct and prolonged contact between the bacterium and the plant. Collectively, these results provide new opportunities for sustainable agriculture and horticulture by exposing roots of plants only briefly to a specific blend of microbial volatile compounds prior to transplantation of the seedlings to the greenhouse or field. This strategy has no need for large-scale introduction or root colonization and survival of the microbial inoculant.

Lost in diversity: the interactions between soil-borne fungi, biodiversity and plant productivity.

There is consensus that plant species richness enhances plant productivity within natural grasslands, but the underlying drivers remain debated. Recently, differential accumulation of soil-borne fungal pathogens across the plant diversity gradient has been proposed as a cause of this pattern. However, the below-ground environment has generally been treated as a 'black box' in biodiversity experiments, leaving these fungi unidentified. Using next generation sequencing and pathogenicity assays, we analysed the community composition of root-associated fungi from a biodiversity experiment to examine if evidence exists for host specificity and negative density dependence in the interplay between soil-borne fungi, plant diversity and productivity. Plant species were colonised by distinct (pathogenic) fungal communities and isolated fungal species showed negative, species-specific effects on plant growth. Moreover, 57% of the pathogenic fungal operational taxonomic units (OTUs) recorded in plant monocultures were not detected in eight plant species plots, suggesting a loss of pathogenic OTUs with plant diversity. Our work provides strong evidence for host specificity and negative density-dependent effects of root-associated fungi on plant species in grasslands. Our work substantiates the hypothesis that fungal root pathogens are an important driver of biodiversity-ecosystem functioning relationships.

Inter- and intracellular colonization of Arabidopsis roots by endophytic actinobacteria and the impact of plant hormones on their antimicrobial activity.

Many actinobacteria live in close association with eukaryotes such as fungi, insects, animals and plants. Plant-associated actinobacteria display (endo)symbiotic, saprophytic or pathogenic life styles, and can make up a substantial part of the endophytic community. Here, we characterised endophytic actinobacteria isolated from root tissue of Arabidopsis thaliana (Arabidopsis) plants grown in soil from a natural ecosystem. Many of these actinobacteria belong to the family of Streptomycetaceae with Streptomyces olivochromogenes and Streptomyces clavifer as well represented species. When seeds of Arabidopsis were inoculated with spores of Streptomyces strain coa1, which shows high similarity to S. olivochromogenes, roots were colonised intercellularly and, unexpectedly, also intracellularly. Subsequent exposure of endophytic isolates to plant hormones typically found in root and shoot tissues of Arabidopsis led to altered antibiotic production against Escherichia coli and Bacillus subtilis. Taken together, our work reveals remarkable colonization patterns of endophytic streptomycetes with specific traits that may allow a competitive advantage inside root tissue.

Membrane Interactions of Natural Cyclic Lipodepsipeptides of the Viscosin Group.

Many Pseudomonas spp. produce cyclic lipodepsipeptides (CLPs), which, besides their role in biological functions such as motility, biofilm formation and interspecies interactions, are antimicrobial. It has been established that interaction with the cellular membrane is central to the mode of action of CLPs. In this work, we focus on the CLPs of the so-called viscosin group, aiming to assess the impact of the main structural variations observed within this group on both the antimicrobial activity and the interaction with model membranes. The antimicrobial activity of viscosin, viscosinamide A, WLIP and pseudodesmin A were all tested on a broad panel of mainly Gram-positive bacteria. Their capacity to permeabilize or fuse PG/PE/cardiolipin model membrane vesicles is assessed using fluorescent probes. We find that the Glu2/Gln2 structural variation within the viscosin group is the main factor that influences both the membrane permeabilization properties and the minimum inhibitory concentration of bacterial growth, while the configuration of the Leu5 residue has no apparent effect. The CLP-membrane interactions were further evaluated using CD and FT-IR spectroscopy on model membranes consisting of PG/PE/cardiolipin or POPC with or without cholesterol. In contrast to previous studies, we observe no conformational change upon membrane insertion. The CLPs interact both with the polar heads and aliphatic tails of model membrane systems, altering bilayer fluidity, while cholesterol reduces CLP insertion depth.

Genome-wide analysis of bacterial determinants of plant growth promotion and induced systemic resistance by Pseudomonas fluorescens.

Pseudomonas fluorescens strain SS101 (Pf.SS101) promotes growth of Arabidopsis thaliana, enhances greening and lateral root formation, and induces systemic resistance (ISR) against the bacterial pathogen Pseudomonas syringae pv. tomato (Pst). Here, targeted and untargeted approaches were adopted to identify bacterial determinants and underlying mechanisms involved in plant growth promotion and ISR by Pf.SS101. Based on targeted analyses, no evidence was found for volatiles, lipopeptides and siderophores in plant growth promotion by Pf.SS101. Untargeted, genome-wide analyses of 7488 random transposon mutants of Pf.SS101 led to the identification of 21 mutants defective in both plant growth promotion and ISR. Many of these mutants, however, were auxotrophic and impaired in root colonization. Genetic analysis of three mutants followed by site-directed mutagenesis, genetic complementation and plant bioassays revealed the involvement of the phosphogluconate dehydratase gene edd, the response regulator gene colR and the adenylsulfate reductase gene cysH in both plant growth promotion and ISR. Subsequent comparative plant transcriptomics analyses strongly suggest that modulation of sulfur assimilation, auxin biosynthesis and transport, steroid biosynthesis and carbohydrate metabolism in Arabidopsis are key mechanisms linked to growth promotion and ISR by Pf.SS101.

Impact of soil heat on reassembly of bacterial communities in the rhizosphere microbiome and plant disease suppression.

The rhizosphere microbiome offers a range of ecosystem services to the plant, including nutrient acquisition and tolerance to (a)biotic stress. Here, analysing the data by Mendes et al. (2011), we show that short heat disturbances (50 or 80 °C, 1 h) of a soil suppressive to the root pathogenic fungus Rhizoctonia solani caused significant increase in alpha diversity of the rhizobacterial community and led to partial or complete loss of disease protection. A reassembly model is proposed where bacterial families that are heat tolerant and have high growth rates significantly increase in relative abundance after heat disturbance, while temperature-sensitive and slow-growing bacteria have a disadvantage. The results also pointed to a potential role of slow-growing, heat-tolerant bacterial families from Actinobacteria and Acidobacteria phyla in plant disease protection. In conclusion, short heat disturbance of soil results in rearrangement of rhizobacterial communities and this is correlated with changes in the ecosystem service disease suppression.

Impact of plant domestication on rhizosphere microbiome assembly and functions.

The rhizosphere microbiome is pivotal for plant health and growth, providing defence against pests and diseases, facilitating nutrient acquisition and helping plants to withstand abiotic stresses. Plants can actively recruit members of the soil microbial community for positive feedbacks, but the underlying mechanisms and plant traits that drive microbiome assembly and functions are largely unknown. Domestication of plant species has substantially contributed to human civilization, but also caused a strong decrease in the genetic diversity of modern crop cultivars that may have affected the ability of plants to establish beneficial associations with rhizosphere microbes. Here, we review how plants shape the rhizosphere microbiome and how domestication may have impacted rhizosphere microbiome assembly and functions via habitat expansion and via changes in crop management practices, root exudation, root architecture, and plant litter quality. We also propose a "back to the roots" framework that comprises the exploration of the microbiome of indigenous plants and their native habitats for the identification of plant and microbial traits with the ultimate goal to reinstate beneficial associations that may have been undermined during plant domestication.

Living on the edge: emergence of spontaneous gac mutations in Pseudomonas protegens during swarming motility.

Swarming motility is a flagella-driven multicellular behaviour that allows bacteria to colonize new niches and escape competition. Here, we investigated the evolution of specific mutations in the GacS/GacA two-component regulatory system in swarming colonies of Pseudomonas protegens Pf-5. Experimental evolution assays showed that repeated rounds of swarming by wildtype Pf-5 drives the accumulation of gacS/gacA spontaneous mutants on the swarming edge. These mutants cannot swarm on their own because they lack production of the biosurfactant orfamide A, but they do co-swarm with orfamide-producing wildtype Pf-5. These co-swarming assays further demonstrated that ΔgacA mutant cells indeed predominate on the edge and that initial ΔgacA:wildtype Pf-5 ratios of at least 2:1 lead to a collapse of the swarming colony. Subsequent whole-genome transcriptome analyses revealed that genes associated with motility, resource acquisition, chemotaxis and efflux were significantly upregulated in ΔgacA mutant on swarming medium. Moreover, transmission electron microscopy showed that ΔgacA mutant cells were longer and more flagellated than wildtype cells, which may explain their predominance on the swarming edge. We postulate that adaptive evolution through point mutations is a common feature of range-expanding microbial populations and that the putative fitness benefits of these mutations during dispersal of bacteria into new territories are frequency-dependent.

Elucidating the Diversity of Aquatic Microdochium and Trichoderma Species and Their Activity against the Fish Pathogen Saprolegnia diclina.

Animals and plants are increasingly threatened by emerging fungal and oomycete diseases. Amongst oomycetes, Saprolegnia species cause population declines in aquatic animals, especially fish and amphibians, resulting in significant perturbation in biodiversity, ecological balance and food security. Due to the prohibition of several chemical control agents, novel sustainable measures are required to control Saprolegnia infections in aquaculture. Previously, fungal community analysis by terminal restriction fragment length polymorphism (T-RFLP) revealed that the Ascomycota, specifically the genus Microdochium, was an abundant fungal phylum associated with salmon eggs from a commercial fish farm. Here, phylogenetic analyses showed that most fungal isolates obtained from salmon eggs were closely related to Microdochium lycopodinum/Microdochium phragmitis and Trichoderma viride species. Phylogenetic and quantitative PCR analyses showed both a quantitative and qualitative difference in Trichoderma population between diseased and healthy salmon eggs, which was not the case for the Microdochium population. In vitro antagonistic activity of the fungi against Saprolegnia diclina was isolate-dependent; for most Trichoderma isolates, the typical mycoparasitic coiling around and/or formation of papilla-like structures on S. diclina hyphae were observed. These results suggest that among the fungal community associated with salmon eggs, Trichoderma species may play a role in Saprolegnia suppression in aquaculture.