Enhanced transport of bacteria along root systems by protists can impact plant health.

Soil protists have been shown to contribute to the structure and function of the rhizosphere in a variety of ways. Protists are key contributors to nutrient cycling through the microbial loop, where biomass is digested by protists and otherwise stored nutrients are returned to the environment. Protists have also been shown to feed on plant pathogenic bacteria and alter root microbiomes in ways that may benefit plants. Recently, a mechanism involving bacterial transport, facilitated by protists, has been hypothesized to contribute to the spatial distribution of bacteria in the rhizosphere. Here, we observe the differential abilities of three soil protists: a ciliate (Colpoda sp.), a flagellate (Cercomonas sp.), and a naked amoeba (Acanthamoeba castellanii) to transport nitrogen-fixing Sinorhizobium meliloti to infectible root tips. Co-inoculation of protists plus S. meliloti resulted in the movement of bacteria, as measured by the presence of nitrogen-fixing nodules, up to 15 cm farther down the root systems when compared to plants inoculated with S. meliloti alone. Co-inoculation of the ciliate, Colpoda sp., with S. meliloti, resulted in shoot weights that were similar to plants that grew in nitrogen-replete potting mix. Colpoda sp.-feeding style and motility likely contributed to their success at transporting bacteria through the rhizosphere. We observed that the addition of protists alone without the co-inoculum of S. meliloti resulted in plants with larger shoot weights than control plants. Follow-up experiments showed that protists plus their associated microbiomes were aiding in plant health, likely through means of nutrient cycling.IMPORTANCEProtists represent a significant portion of the rhizosphere microbiome and have been shown to contribute to plant health, yet they are understudied compared to their bacterial and fungal counterparts. This study elucidates their role in the rhizosphere community and suggests a mechanism by which protists can be used to move bacteria along plant roots. We found that the co-inoculation of protists with nitrogen-fixing beneficial bacteria, Sinorhizobium meliloti, resulted in nodules farther down the roots when compared to plants inoculated with S. meliloti alone, and shoot weights similar to plants that received nitrogen fertilizer. These data illustrate the ability of protists to transport viable bacteria to uninhabited regions of the root system.

Soil Protists Can Actively Redistribute Beneficial Bacteria along Medicago truncatula Roots.

The rhizosphere is the region of soil directly influenced by plant roots. The microbial community in the rhizosphere includes fungi, protists, and bacteria: all play significant roles in plant health. The beneficial bacterium Sinorhizobium meliloti infects growing root hairs on nitrogen-starved leguminous plants. Infection leads to the formation of a root nodule, where S. meliloti converts atmospheric nitrogen to ammonia, a bioavailable form. In soil, S. meliloti is often found in biofilms and travels slowly along the roots, leaving developing root hairs at the growing root tips uninfected. Soil protists are an important component of the rhizosphere system, able to travel quickly along roots and water films, who prey on soil bacteria and have been known to egest undigested phagosomes. We show that a soil protist, Colpoda sp., can transport S. meliloti down Medicago truncatula roots. Using model soil microcosms, we directly observed fluorescently labeled S. meliloti along M. truncatula roots and tracked the displacement of the fluorescence signal over time. Two weeks after co-inoculation, this signal extended 52 mm farther down plant roots when Colpoda sp. was also present versus treatments that contained bacteria but not protists. Direct counts also showed protists are required for viable bacteria to reach the deeper sections of our microcosms. Facilitating bacterial transport may be an important mechanism whereby soil protists promote plant health. IMPORTANCE Soil protists are an important part of the microbial community in the rhizosphere. Plants grown with protists fare better than plants grown without protists. Mechanisms through which protists support plant health include nutrient cycling, alteration of the bacterial community through selective feeding, and consumption of plant pathogens. Here, we provide data in support of an additional mechanism: protists act as transport vehicles for bacteria in soil. We show that protist-facilitated transport can deliver plant-beneficial bacteria to the growing tips of roots that may otherwise be sparsely inhabited with bacteria originating from a seed-associated inoculum. By co-inoculating Medicago truncatula roots with both S. meliloti, a nitrogen-fixing legume symbiont, and Colpoda sp., a ciliated protist, we show substantial and statistically significant transport with depth and breadth of bacteria-associated fluorescence as well as transport of viable bacteria. Co-inoculation with shelf-stable encysted soil protists may be employed as a sustainable agriculture biotechnology to better distribute beneficial bacteria and enhance the performance of inoculants.

18S rRNA gene amplicon sequencing combined with culture-based surveys of maize rhizosphere protists reveal dominant, plant-enriched and culturable community members.

Protists play important roles in shaping the microbial community of the rhizosphere and defining these roles will require the study of protist isolates. However, there is still a limited understanding of how well protist isolation efforts can capture the diversity and composition of rhizosphere protistan communities. Here, we report a simultaneous isolation and 18S rRNA gene amplicon sequencing survey describing the protist diversity of maize rhizospheres in two climatically and pedologically distinct sites. We demonstrated that the maize rhizosphere exerted significant and site-dependent effects on the protistan community structure and defined a set of core and rhizosphere-enriched protists. From the same root samples, we generated a library of 103 protist isolates representing 46 18S rRNA gene sequence variants from six eukaryotic supergroups. While cultured isolates represented a small proportion of total protist diversity recovered by sequencing, they included taxa enriched in rhizosphere soils across all samples, encompassing 9% of all core sequence variants. The isolation approach also captured 17 protists not detected through 18S rRNA gene amplicon sequencing. This study demonstrated that maize roots select for distinct protistan communities, and established a diverse protist culture collection that can be used for future research linking protists to rhizosphere status and plant health.