Unearthing the plant-microbe quid pro quo in root associations with beneficial fungi.

Mutualistic symbiotic associations between multicellular eukaryotes and their microbiota are driven by the exchange of nutrients in a quid pro quo manner. In the widespread arbuscular mycorrhizal (AM) symbiosis involving plant roots and Glomeromycotina fungi, the mycobiont is supplied with carbon through photosynthesis, which in return supplies the host plant with essential minerals such as phosphorus (P). Most terrestrial plants are largely dependent on AM fungi for nutrients, which raises the question of how plants that are unable to form a functional AM sustain their P nutrition. AM nonhost plants can form alternative, evolutionarily younger, mycorrhizal associations such as the ectomycorrhiza, ericoid and orchid mycorrhiza. However, it is unclear how plants such as the Brassicaceae species Arabidopsis thaliana, which do not form known mycorrhizal symbioses, have adapted to the loss of these essential mycorrhizal traits. Isotope tracing experiments with root-colonizing fungi have revealed the existence of new 'mycorrhizal-like' fungi capable of transferring nutrients such as nitrogen (N) and P to plants, including Brassicaceae. Here, we provide an overview of the biology of trophic relationships between roots and fungi and how these associations might support plant adaptation to climate change.

Root-associated fungal microbiota of nonmycorrhizal Arabis alpina and its contribution to plant phosphorus nutrition.

Most land plants live in association with arbuscular mycorrhizal (AM) fungi and rely on this symbiosis to scavenge phosphorus (P) from soil. The ability to establish this partnership has been lost in some plant lineages like the Brassicaceae, which raises the question of what alternative nutrition strategies such plants have to grow in P-impoverished soils. To understand the contribution of plant-microbiota interactions, we studied the root-associated fungal microbiome of Arabis alpina (Brassicaceae) with the hypothesis that some of its components can promote plant P acquisition. Using amplicon sequencing of the fungal internal transcribed spacer 2, we studied the root and rhizosphere fungal communities of A. alpina growing under natural and controlled conditions including low-P soils and identified a set of 15 fungal taxa consistently detected in its roots. This cohort included a Helotiales taxon exhibiting high abundance in roots of wild A. alpina growing in an extremely P-limited soil. Consequently, we isolated and subsequently reintroduced a specimen from this taxon into its native P-poor soil in which it improved plant growth and P uptake. The fungus exhibited mycorrhiza-like traits including colonization of the root endosphere and P transfer to the plant. Genome analysis revealed a link between its endophytic lifestyle and the expansion of its repertoire of carbohydrate-active enzymes. We report the discovery of a plant-fungus interaction facilitating the growth of a nonmycorrhizal plant under native P-limited conditions, thus uncovering a previously underestimated role of root fungal microbiota in P cycling.

Dysfunction in the arbuscular mycorrhizal symbiosis has consistent but small effects on the establishment of the fungal microbiota in Lotus japonicus.

Most land plants establish mutualistic interactions with arbuscular mycorrhizal (AM) fungi. Intracellular accommodation of AM fungal symbionts remodels important host traits like root morphology and nutrient acquisition. How mycorrhizal colonization impacts plant microbiota is unclear. To understand the impact of AM symbiosis on fungal microbiota, ten Lotus japonicus mutants impaired at different stages of AM formation were grown in non-sterile natural soil and their root-associated fungal communities were studied. Plant mutants lacking the capacity to form mature arbuscules (arb- ) exhibited limited growth performance associated with altered phosphorus (P) acquisition and reduction-oxidation (redox) processes. Furthermore, arb- plants assembled moderately but consistently different root-associated fungal microbiota, characterized by the depletion of Glomeromycota and the concomitant enrichment of Ascomycota, including Dactylonectria torresensis. Single and co-inoculation experiments showed a strong reduction of root colonization by D. torresensis in the presence of AM fungus Rhizophagus irregularis, particularly in arbuscule-forming plants. Our results suggest that impairment of central symbiotic functions in AM host plants leads to specific changes in root microbiomes and in tripartite interactions between the host plant, AM and non-AM fungi. This lays the foundation for mechanistic studies on microbe-microbe and microbe-host interactions in AM symbiosis of the model L. japonicus.

Plant-mediated effects of soil phosphorus on the root-associated fungal microbiota in Arabidopsis thaliana.

Plants respond to phosphorus (P) limitation through an array of morphological, physiological and metabolic changes which are part of the phosphate (Pi) starvation response (PSR). This response influences the establishment of the arbuscular mycorrhizal (AM) symbiosis in most land plants. It is, however, unknown to what extent available P and the PSR redefine plant interactions with the fungal microbiota in soil. Using amplicon sequencing of the fungal taxonomic marker ITS2, we examined the changes in root-associated fungal communities in the AM nonhost species Arabidopsis thaliana in response to soil amendment with P and to genetic perturbations in the plant PSR. We observed robust shifts in root-associated fungal communities of P-replete plants in comparison with their P-deprived counterparts, while bulk soil communities remained unaltered. Moreover, plants carrying mutations in the phosphate signaling network genes, phr1, phl1 and pho2, exhibited similarly altered root fungal communities characterized by the depletion of the chytridiomycete taxon Olpidium brassicae specifically under P-replete conditions. This study highlights the nutritional status and the underlying nutrient signaling network of an AM nonhost plant as previously unrecognized factors influencing the assembly of the plant fungal microbiota in response to P in nonsterile soil.

Rhizosphere ecology and phytoprotection in soils naturally suppressive to Thielaviopsis black root rot of tobacco.

Soil suppressiveness to disease is an intriguing emerging property in agroecosystems, with important implications because it enables significant protection of susceptible plants from soil-borne pathogens. Unlike many soils where disease suppressiveness requires crop monoculture to establish, certain soils are naturally suppressive to disease, and this type of specific disease suppressiveness is maintained despite crop rotation. Soils naturally suppressive to Thielaviopsis basicola-mediated black root rot of tobacco and other crops occur in Morens region (Switzerland) and have been studied for over 30 years. In Morens, vermiculite-rich suppressive soils formed on morainic deposits while illite-rich conducive soils developed on sandstone, but suppressiveness is of microbial origin. Antagonistic pseudomonads play a role in black root rot suppressiveness, including Pseudomonas protegens (formerly P. fluorescens) CHA0, a major model strain for research. However, other types of rhizobacterial taxa may differ in prevalence between suppressive and conducive soils, suggesting that the microbial basis of black root rot suppressiveness could be far more complex than solely a Pseudomonas property. This first review on black root rot suppressive soils covers early findings on these soils, the significance of recent results, and compares them with other types of suppressive soils in terms of rhizosphere ecology and plant protection mechanisms.

Effect of clay mineralogy on iron bioavailability and rhizosphere transcription of 2,4-diacetylphloroglucinol biosynthetic genes in biocontrol Pseudomonas protegens.

Pseudomonas strains producing 2,4-diacetylphloroglucinol (DAPG) can protect plants from soilborne phytopathogens and are considered the primary reason for suppressiveness of morainic Swiss soils to Thielaviopsis basicola-mediated black root-rot disease of tobacco, even though they also occur nearby in conducive sandstone soils. The underlying molecular mechanisms accounting for this discrepancy are not understood. In this study, we assessed the hypothesis that the presence of iron-rich vermiculite clay (dominant in suppressive soils) instead of illite (dominant in neighboring conducive soils) translates into higher levels of iron bioavailability and transcription of Pseudomonas DAPG synthetic genes in the tobacco rhizosphere. Rhizosphere monitoring of reporter gene systems pvd-inaZ and phlA-gfp in Pseudomonas protegens indicated that the level of iron bioavailability and the number of cells expressing phl genes (DAPG synthesis), respectively, were higher in vermiculitic than in illitic artificial soils. This was in accordance with the effect of iron on phlA-gfp expression in vitro and, indeed, iron addition to the illitic soil increased the number of cells expressing phlA-gfp. Similar findings were made in the presence of the pathogen T. basicola. Altogether, results substantiate the hypothesis that iron-releasing minerals may confer disease suppressiveness by modulating iron bioavailability in the rhizosphere and expression of biocontrol-relevant genes in antagonistic P. protegens.

The Leaf Microbiome of Arabidopsis Displays Reproducible Dynamics and Patterns throughout the Growing Season.

Leaves are primarily responsible for the plant's photosynthetic activity. Thus, changes in the leaf microbiota, which includes deleterious and beneficial microbes, can have far-reaching effects on plant fitness and productivity. Identifying the processes and microorganisms that drive these changes over a plant's lifetime is, therefore, crucial. In this study, we analyzed the temporal dynamics in the leaf microbiome of Arabidopsis thaliana, integrating changes in both composition and microbe-microbe interactions via the study of microbial networks. Field-grown Arabidopsis were used to monitor leaf bacterial, fungal and oomycete communities throughout the plant's natural growing season (extending from November to March) over three consecutive years. Our results revealed the existence of conserved temporal patterns, with microbial communities and networks going through a stabilization phase of decreased diversity and variability at the beginning of the plant's growing season. Despite a high turnover in these communities, we identified 19 "core" taxa persisting on Arabidopsis leaves across time and plant generations. With the hypothesis these microbes could be playing key roles in the structuring of leaf microbial communities, we conducted a time-informed microbial network analysis which showed core taxa are not necessarily highly connected network "hubs," and "hubs" alternate with time. Our study shows that leaf microbial communities exhibit reproducible dynamics and patterns, suggesting the potential of using our understanding of temporal trajectories in microbial community composition to design experiments aimed at driving these communities toward desired states. IMPORTANCE Utilizing plant microbiota to promote plant growth and plant health is key to more environmentally friendly agriculture. A major bottleneck in the engineering of plant-beneficial microbial communities is the low persistence of applied microbes under filed conditions, especially considering plant leaves. Indeed, although many leaf-associated microorganisms have the potential to promote plant growth and protect plants from pathogens, few of them are able to survive and thrive over time. In our study, we could show that leaf microbial communities are very variable at the beginning of the plant growing season but become more and more similar and less variable as the season progresses. We further identify a cohort of 19 "core" microbes, systematically present on plant leaves that would make these microbes exceptional candidates for future agricultural applications.

Obtaining deeper insights into microbiome diversity using a simple method to block host and nontargets in amplicon sequencing.

Profiling diverse microbiomes is revolutionizing our understanding of biological mechanisms and ecologically relevant problems, including metaorganism (host + microbiome) assembly, functions and adaptation. Amplicon sequencing of multiple conserved, phylogenetically informative loci has therefore become an instrumental tool for many researchers. Investigations in many systems are hindered, however, since essential sequencing depth can be lost by amplification of nontarget DNA from hosts or overabundant microorganisms. Here, we introduce "blocking oligos", a low-cost and flexible method using standard oligonucleotides to block amplification of diverse nontargets and software to aid their design. We apply them primarily in leaves, where exceptional challenges with host amplification prevail. A. thaliana-specific blocking oligos applied in eight different target loci reduce undesirable host amplification by up to 90%. To expand applicability, we designed universal 16S and 18S rRNA gene plant blocking oligos for targets that are conserved in diverse plant species and demonstrate that they efficiently block five plant species from five orders spanning monocots and dicots (Bromus erectus, Plantago lanceolata, Lotus corniculatus, Amaranth sp., Arabidopsis thaliana). These can increase alpha diversity discovery without biasing beta diversity patterns and do not compromise microbial load information inherent to plant-derived 16S rRNA gene amplicon sequencing data. Finally, we designed and tested blocking oligos to avoid amplification of 18S rRNA genes of a sporulating oomycete pathogen, demonstrating their effectiveness in applications well beyond plants. Using these tools, we generated a survey of the A. thaliana leaf microbiome based on eight loci targeting bacterial, fungal, oomycete and other eukaryotic microorganisms and discuss complementarity of commonly used amplicon sequencing regions for describing leaf microbiota. This approach has potential to make questions in a variety of study systems more tractable by making amplicon sequencing more targeted, leading to deeper, systems-based insights into microbial discovery. For fast and easy design for blocking oligos for any nontarget DNA in other study systems, we developed a publicly available R package.

Arabidopsis thaliana and Pseudomonas Pathogens Exhibit Stable Associations over Evolutionary Timescales.

Crop disease outbreaks are often associated with clonal expansions of single pathogenic lineages. To determine whether similar boom-and-bust scenarios hold for wild pathosystems, we carried out a multi-year, multi-site survey of Pseudomonas in its natural host Arabidopsis thaliana. The most common Pseudomonas lineage corresponded to a ubiquitous pathogenic clade. Sequencing of 1,524 genomes revealed this lineage to have diversified approximately 300,000 years ago, containing dozens of genetically identifiable pathogenic sublineages. There is differentiation at the level of both gene content and disease phenotype, although the differentiation may not provide fitness advantages to specific sublineages. The coexistence of sublineages indicates that in contrast to crop systems, no single strain has been able to overtake the studied A. thaliana populations in the recent past. Our results suggest that selective pressures acting on a plant pathogen in wild hosts are likely to be much more complex than those in agricultural systems.

Distribution of 2,4-Diacetylphloroglucinol Biosynthetic Genes among the Pseudomonas spp. Reveals Unexpected Polyphyletism.

Fluorescent pseudomonads protecting plant roots from phytopathogens by producing 2,4-diacetylphloroglucinol (DAPG) are considered to form a monophyletic lineage comprised of DAPG+Pseudomonas strains in the "P. corrugata" and "P. protegens" subgroups of the "Pseudomonas fluorescens" group. However, DAPG production ability has not been investigated for many species of these two subgroups, and whether or not the DAPG+Pseudomonas are truly monophyletic remained to be verified. Thus, the distribution of the DAPG biosynthetic operon (phlACBD genes) in the Pseudomonas spp. was investigated in sequenced genomes and type strains. Results showed that the DAPG+Pseudomonas include species of the "P. fluorescens" group, i.e., P. protegens, P. brassicacearum, P. kilonensis, and P. thivervalensis, as expected, as well as P. gingeri in which it had not been documented. Surprisingly, they also include bacteria outside the "P. fluorescens" group, as exemplified by Pseudomonas sp. OT69, and even two Betaproteobacteria genera. The phl operon-based phylogenetic tree was substantially congruent with the one inferred from concatenated housekeeping genes rpoB, gyrB, and rrs. Contrariwise to current supposition, ancestral character reconstructions favored multiple independent acquisitions rather that one ancestral event followed by vertical inheritance. Indeed, based on synteny analyses, these acquisitions appeared to vary according to the Pseudomonas subgroup and even the phylogenetic groups within the subgroups. In conclusion, our study shows that the phl+Pseudomonas populations form a polyphyletic group and suggests that DAPG biosynthesis might not be restricted to this genus. This is important to consider when assessing the ecological significance of phl+ bacterial populations in rhizosphere ecosystems.

Prevalence of type III secretion system in effective biocontrol pseudomonads.

Functional type III secretion system (T3SS) genes are needed for effective biocontrol of Pythium damping-off of cucumber by Pseudomonas fluorescens KD, but whether biocontrol Pseudomonas strains with T3SS genes display overall a higher plant-protecting activity is unknown. The assessment of 198 biocontrol fluorescent pseudomonads originating from 60 soils worldwide indicated that 32% harbour the ATPase-encoding T3SS gene hrcN, which was most often found in tomato isolates. The hrcN(+) biocontrol strains (and especially those also producing 2,4-diacetylphloroglucinol and displaying 1-aminocyclopropane-1-carboxylate deaminase activity) displayed higher plant-protecting ability in comparison with hrcN(-) biocontrol strains, both in the Pythium/cucumber and Fusarium/cucumber pathosystems.

Evaluation of rhizobacterial indicators of tobacco black root rot suppressiveness in farmers' fields.

Very few soil quality indicators include disease-suppressiveness criteria. We assessed whether 64 16S rRNA microarray probes whose signals correlated with tobacco black root rot suppressiveness in greenhouse analysis could also discriminate suppressive from conducive soils under field conditions. Rhizobacterial communities of tobacco and wheat sampled in 2 years from four farmers' fields of contrasted suppressiveness status were compared. The 64 previously identified indicator probes correctly classified 72% of 29 field samples, with nine probes for Azospirillum, Gluconacetobacter, Sphingomonadaceae, Planctomycetes, Mycoplasma, Lactobacillus crispatus and Thermodesulforhabdus providing the best prediction. The whole probe set (1033 probes) revealed strong effects of plant, field location and year on rhizobacterial community composition, and a smaller (7% variance) but significant effect of soil suppressiveness status. Seventeen additional probes correlating with suppressiveness status in the field (noticeably for Agrobacterium, Methylobacterium, Ochrobactrum) were selected, and combined with the nine others, they improved correct sample classification from 72% to 79% (100% tobacco and 63% wheat samples). Pseudomonas probes were not informative in the field, even those targeting biocontrol pseudomonads producing 2,4-diacetylphloroglucinol, nor was quantitative polymerase chain reaction for 2,4-diacetylphloroglucinol-synthesis gene phlD. This study shows that a subset of 16S rRNA probes targeting diverse rhizobacteria can be useful as suppressiveness indicators under field conditions.