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

Abstract

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.

Keywords: beneficial bacteria; protist; rhizosphere; transport.

Fig 1

Average shoot weight of plants: (A) co-inoculated with a constant number of Sinorhizobium meliloti and differing numbers of three protist types and (B) inoculated with only protists, with only their associated microbiome or with only culture filtrates, except where indicated plants were grown in nitrogen-free potting mix. Box and whisker plots indicate the sample median, with the top and bottom edges of each box representing the upper and lower quartiles, respectively. Whiskers represent the non-outlier minimum and maximum values. Individual red letters indicate treatments that were not significantly different, at P < 0.01, using nested one-way analysis of variance (ANOVA) with Tukey’s correction for multiple comparisons (panels were not cross-compared). For example, treatment 13 (red F) was significantly different than those marked with a red B. In panel A, treatments had 11–27 plants. n = 3 for treatments with 800 and 8,000 protists + Sm and for the Pages-only and Sm-only controls, n = 2 for all treatments with 16,000 protists + Sm, n = 1 for all treatments with 200 protists + Sm. In panel B, n = 3 for all treatments, and each treatment had 6–12 plants. See File S1 for details on n-values and the number of plants per treatment.

Fig 2

Shoot weight in relation to the total number of nodules per plant when co-inoculated with Sinorhizobium meliloti and the differing numbers of three protist types. 95% confidence intervals (CI) overlay linear trendlines for each concentration of protists (16,000, 8,000, 800, or 200 protists per plant). 95% CI for control plants inoculated with S. meliloti only (orange) is also overlayed on each panel in order to facilitate comparisons.

Fig 3

Nodule locations on Medicago truncatula roots when Sinorhizobium meliloti was coinoculated with differing numbers of protists.Measurement starts at the soil surface (0 cm) and extends the length of the pots (20 cm). Individual points represent the furthest nodule on individual plants. Nodules were plotted at the mid-points of each of four root segments (0 to -5, -5 to -10, -10 to -15 and -15 to -20 cm).

Fig 4

Speed of protists and area of influence of UC1. Panel A shows the measured speeds of the three protist types. Whisker plots indicate the sample median, with the top and bottom edges of each box representing the upper and lower quartiles, respectively. Box and whisker plots represent the non-outlier minimum and maximum values. The speed for each protist was significantly different from the other two (P < 0.01). Panel B shows the fluorescent S. meliloti moving toward the stationary UC1 cells resting at the bottom of a culture flask. The elapsed time was 3.1 s. The image was made by color coding each frame (20 frames taken with a 4× objective), then combining the 20 frames via a brightest-pixel Z-projection. Time is ordered: blue-green-red. Measurements show the size of each area of influence. Panel C is similar to B, but the movement of the bacteria is indicated with colored vectors, and the images were taken with a 20 × objective.