Identity and timing of protist inoculation affect plant performance largely irrespective of changes in the rhizosphere microbial community.

Bacterivorous soil protists can have positive impacts on plant performance, making them attractive targets for novel strategies to improve crop production. However, we generally lack the knowledge required to make informed choices regarding the protist species to be used or the optimal conditions for such amendments. Here, we examined how identity, diversity, and timing of inoculation of well-described protists impacted plant development and rhizosphere microbiome assembly. We first studied the impact of individual inoculation of six well-characterized protists on lettuce growth, with Cercomonas sp. S24D2 emerging as the strain with the largest impact on plant growth. In a second step, we created a three-protist species mixture inoculant by adding two protist species (Acanthamoeba sp. C13D2 and a heterolobosean isolate S18D10), based on differences in their feeding patterns. We then inoculated Cercomonas sp. either individually or in the protist mixture to lettuce plants 1 week before, simultaneously with, or 1 week after seedling transfer. We monitored plant growth and nutrient content, as well as impacts on the resident soil and rhizosphere microbiome composition. We found that early protist inoculation provided the greatest increase in aboveground biomass compared to the non-inoculated control. Single- and mixed-species inoculations had similar impacts on plant development and only minor impacts on prokaryotic community composition. While early inoculation seems to be the most promising timing for eliciting the positive effects of protist amendments, further, more systematic studies will be necessary to determine the conditions and ecological interactions that yield consistent and predictable improvements in plant performance. IMPORTANCE: The application of microorganisms, including bacterivorous soil protists, has been increasingly suggested as a sustainable agricultural approach. While positive impacts of the presence of predatory protists have been generally reported, the effects of the selected species and amendment conditions are largely unknown. Here, we examined how identity, diversity, and timing of inoculation of well-described protists impacted plant development and rhizosphere microbiome assembly. One species emerged as the one having the strongest impact in our specific system. This result highlights the importance of species selection for optimal outcome, but also suggests a huge potential in the barely investigated protist diversity for targeted application. Furthermore, the application of the inoculants before plant transfer showed the strongest effects on plants, providing some useful and new insights on the optimal time for such amendments.