The rhizosphere microbiome is a central determinant of plant performance. Microbiome assembly has traditionally been investigated from a bottom-up perspective, assessing how resources such as root exudates drive microbiome assembly. However, the importance of predation as a driver of microbiome structure has to date largely remained overlooked. Here we review the importance of protists, a paraphyletic group of unicellular eukaryotes, as a key regulator of microbiome assembly. Protists can promote plant-beneficial functions within the microbiome, accelerate nutrient cycling, and remove pathogens. We conclude that protists form an essential component of the rhizosphere microbiome and that accounting for predator-prey interactions would greatly improve our ability to predict and manage microbiome function at the service of plant growth and health.A Multitrophic Perspective on the Rhizosphere Microbiome Plant growth, nutrition, and health are to a large extent determined by the activity of associated microorganisms [1]. In particular, plant roots are associated with an active multispecies community, the rhizosphere microbiome (see Glossary), providing several important services to the plant. Root-associated microbes, for instance, mineralize nutrients, manipulate plant hormonal balance, and suppress potential pathogens [2]. The species composition of the rhizosphere microbiome is now recognized to have direct effects on host plant traits [3]. However, our understanding of the determinants of microbiome community assembly and composition is still lacunar, restricting our ability to predict and harness microbiome dynamics and functionality. To date, most studies seeking to address the mechanisms underlying microbiome composition, species turnover, and function have focused on bottom-up drivers of microbial community composition, such as plant developmental stage, soil type, and host genotype [4][5][6]. While bottom-up control is certainly crucial, it represents only half of the story (Figure 1). Microorganisms in the rhizosphere are subjected to top-down control by a range of bacterial and eukaryotic consumers. Among them, free-living protists (Box 1), a highly diverse group of mostly unicellular eukaryotes [7], in our opinion deserve special attention. Protists are highly abundant and active consumers of bacteria and arguably fungi, impact community structure, and play a key role for nutrient cycling in the rhizosphere [8][9][10][11][12][13]. This review primarily addresses the importance of free-living, heterotrophic protists that feed on other organisms. For the sake of simplicity, we hereafter refer to them as 'protists', deliberately omitting mutualistic or parasitic taxa, including animal parasites or plant pathogens.

Plant health is strongly impacted by beneficial and pathogenic plant microbes, which are themselves structured by resource inputs. Organic fertilizer inputs may thus offer a means of steering soil-borne microbes, thereby affecting plant health. Concurrently, soil microbes are subject to top-down control by predators, particularly protists. However, little is known regarding the impact of microbiome predators on plant health-influencing microbes and the interactive links to plant health. Here, we aimed to decipher the importance of predator-prey interactions in influencing plant health. To achieve this goal, we investigated soil and root-associated microbiomes (bacteria, fungi and protists) over nine years of banana planting under conventional and organic fertilization regimes differing in Fusarium wilt disease incidence. We found that the reduced disease incidence and improved yield associated with organic fertilization could be best explained by higher abundances of protists and pathogen-suppressive bacteria (e.g. Bacillus spp.). The pathogen-suppressive actions of predatory protists and Bacillus spp. were mainly determined by their interactions that increased the relative abundance of secondary metabolite Q genes (e.g. nonribosomal peptide synthetase gene) within the microbiome. In a subsequent microcosm assay, we tested the interactions between predatory protists and pathogen-suppressive Bacillus spp. that showed strong improvements in plant defense. Our study shows how protistan predators stimulate disease-suppressive bacteria in the plant microbiome, ultimately enhancing plant health and yield. Thus, we suggest a new biological model useful for improving sustainable agricultural practices that is based on complex interactions between different domains of life.

Background Microbiomes play vital roles in plant health and performance, and the development of plant beneficial microbiomes can be steered by organic fertilizer inputs. Especially well-studied are fertilizer-induced changes on bacteria and fungi and how changes in these groups alter plant performance. However, impacts on protist communities, including their trophic interactions within the microbiome and consequences on plant performance remain largely unknown. Here, we tracked the entire microbiome, including bacteria, fungi, and protists, over six growing seasons of cucumber under different fertilization regimes (conventional, organic, and Trichoderma bio-organic fertilization) and linked microbial data to plant yield to identify plant growth-promoting microbes. Results Yields were higher in the (bio-)organic fertilization treatments. Soil abiotic conditions were altered by the fertilization regime, with the prominent effects coming from the (bio-)organic fertilization treatments. Those treatments also led to the pronounced shifts in protistan communities, especially microbivorous cercozoan protists. We found positive correlations of these protists with plant yield and the density of potentially plant-beneficial microorganisms. We further explored the mechanistic ramifications of these relationships via greenhouse experiments, showing that cercozoan protists can positively impact plant growth, potentially via interactions with plant-beneficial microorganisms including Trichoderma, the biological agent delivered by the bio-fertilizer. Conclusions We show that protists may play central roles in stimulating plant performance through microbiome interactions. Future agricultural practices might aim to specifically enhance plant beneficial protists or apply those protists as novel, sustainable biofertilizers.

Root-colonizing bacteria can support plant growth and help fend off pathogens. It is clear that such bacteria benefit from plant-derived carbon, but it remains ambiguous why they invest in plant-beneficial traits. We suggest that selection via protist predation contributes to recruitment of plant-beneficial traits in rhizosphere bacteria. To this end, we examined the extent to which bacterial traits associated with pathogen inhibition coincide with resistance to protist predation. We investigated the resistance to predation of a collection of Pseudomonas spp. against a range of representative soil protists covering three eukaryotic supergroups. We then examined whether patterns of resistance to predation could be explained by functional traits related to plant growth promotion, disease suppression and root colonization success. We observed a strong correlation between resistance to predation and phytopathogen inhibition. In addition, our analysis highlighted an important contribution of lytic enzymes and motility traits to resist predation by protists. We conclude that the widespread occurrence of plant-protective traits in the rhizosphere microbiome may be driven by the evolutionary pressure for resistance against predation by protists. Protists may therefore act as microbiome regulators promoting native bacteria involved in plant protection against diseases.

Predatory protists are major consumers of soil micro-organisms. By selectively feeding on their prey, they can shape soil microbiome composition and functions. While different protists are known to show diverging impacts, it remains impossible to predict a priori the effect of a given species. Various protist traits including phylogenetic distance, growth rate and volume have been previously linked to the predatory impact of protists. Closely-related protists,however, also showed distinct prey choices which could mirror specificity in their dietary niche. We, therefore, aimed to estimate the dietary niche breadth and overlap of eight protist isolates on 20 bacterial species in plate assays. To assess the informative value of previously suggested and newly proposed (feeding-related) protist traits, we related them to the impacts of predation of each protist on a protist-free soil bacterial community in a soil microcosm via 16S rRNA gene amplicon sequencing. We could demonstrate that each protist showed a distinct feeding pattern in vitro. Further, the assayed protist feeding patterns and growth rates correlated well with the observed predatory impacts on the structure of soil bacterial communities. We thus conclude that in vitro screening has the potential to inform on the specific predatory impact of selected protists.