Rhizosphere-colonizing bacteria persist in the protist microbiome

Abstract

Soils contain diverse predatory protists that affect the abundance and behavior of rhizosphere bacteria, including bacteria that may benefit plant health. Protists harbor their own bacterial microbiomes, and we previously observed that plants inoculated with protists harbored rhizosphere bacteria similar to those in the protist inoculum. To determine how protist microbiomes affect the rhizosphere, we profiled the bacteria of eight diverse rhizosphere protist isolates after 2 years of laboratory culture. We then compared the protist culture microbiomes to maize rhizosphere communities 6 weeks after protist inoculation. Introduction of protists enriched 13 protist-associated bacterial amplicon sequence variants (ASVs) in the rhizosphere, which comprised ~10% of the rhizosphere bacterial community. Additional bacterial ASVs ranked highly in abundance in both rhizosphere (top 100) and protist (top 20) microbiomes; together, a median 47% of the protist microbiome was enriched or in high rank abundance in the rhizosphere. Inoculation with three out of eight protist cultures positively affected root biomass traits, but a protist mixture had no effect, indicating that the impact of protist-associated bacteria on plant growth is context dependent. Isolates of protist-associated bacteria had both positive and negative effects on protist growth in culture, suggesting that the bacteria use multiple strategies to survive in proximity to predators. This study demonstrates that even after long-term laboratory culture, rhizosphere protist cultures host bacteria that can colonize the rhizosphere of maize. The findings also identify diverse groups of rhizosphere-colonizing bacteria that persist among protist predators, which suggests that these bacteria could associate with or benefit from protists in the soil.

Importance: Understanding the impact of predatory protists on the plant microbiome will be essential to deploy protists in sustainable agriculture. This study shows that eight rhizosphere protist isolates hosted diverse and distinct bacterial communities and that a large proportion of these bacteria could be found colonizing the maize root environment 6 weeks after protists were inoculated onto seedlings. This study demonstrates that certain bacteria from the maize rhizosphere can persist for years in protist cultures and retain the ability to colonize rhizosphere soil, suggesting that protists might support the survival of these rhizosphere bacteria in the absence of the plant.

Keywords: Colpoda; maize; microbiome; protist; rhizosphere.

Fig 1

Bacterial microbiome composition of the protist-free bacterial inocula and the eight protist isolates from the maize rhizosphere. Only the “dominant genera,” i.e., the genera having the 30 highest mean abundances across samples, are shown. Bacterial order is indicated after the colon. The shade corresponds to proportional abundance in the culture sample.

Fig 2

Experimental design showing eight combinations of protist and bacterial treatments. The first five treatments (above the second horizontal line) were designed to compare the impacts of individual protists or the protist mixture on plant rhizosphere communities and plant growth, while the remaining three treatments aimed to examine the impacts of different bacterial inocula or the presence of the plant. Numbers of planted replicates are listed under each circled number. Six plants failed to grow, and 11 16S rhizosphere libraries had fewer than 2,000 reads, so these samples were removed from the study.

Fig 3

Enrichment of bacterial ASVs in the maize rhizosphere 6 weeks after protist inoculation onto germinated seeds. (A) Enrichment by single protists. Diameter of diamonds and dots indicates the mean relative abundance of ASVs in the protist culture microbiome (n = 1) and the rhizosphere samples (n = 9 or 10), respectively. Blue indicates statistical enrichment in the protist treatment relative to protist-uninoculated controls, as determined by DeSeq2. Red stars indicate that the bacteria were enriched when the eight protists were inoculated as a consortium. (B) The proportion of rhizosphere reads representing protist-enriched ASVs after plants were inoculated with single-protist cultures or mixed protists, or uninoculated. Asterisks indicate significant differences from the mixed protist treatment using Dunnett’s multiple comparison test. ***P < 0.0005.

Fig 4

Biomass traits of maize plants inoculated with single protists and an eight-member consortium in a greenhouse study. (A) Root biomass and (B) root-shoot mass ratio 6 weeks after inoculation with zero (n = 29), one (n = 78), or a mixture of eight protists (n = 29) onto germinated maize seeds. (C and D) Root biomass (C) and root-shoot mass ratio (D) after individual protist inoculation in the presence of protist-free soil bacteria (n = 10 for all treatments except UC22 and UC74, which had one plant fail to grow). P values and asterisks indicate differences from the single-protist reference group (A and B) or the uninoculated reference group (C and D) using Dunnett’s multiple comparison test (filled asterisk = P < 0.05; open asterisk = P < 0.1).

Fig 5

Effect of bacterial source (A) and presence of maize plants (B) on protist enrichment of soil bacteria in the rhizosphere. Enrichment of bacteria is shown when protists are added (protist mix “+”) compared to when protists are absent (protist mix “−“). (A) The eight-protist consortium was co-inoculated with bulk soil bacteria from Lockwood Farm (BSC1), Griswold Farm (BSC2), maize rhizosphere soil bacteria from Lockwood Farm (RSC), or no bacteria. (B) Enrichment of bacterial abundance after the eight-protist consortium was inoculated into the center of unplanted pots. Dot diameter indicates the mean relative abundance of the ASV (n = 9 or 10). Blue indicates statistical enrichment in the protist mixture treatment relative to protist-uninoculated controls within each column.

Fig 6

Cell counts of three protist taxa (Allapsa, Cercomonas, and Thaumatomonas) after being fed with heat-killed E. coli alone, or with heat-killed E. coli, in addition to one of four bacterial cultures isolated from protist cultures (n = 3 for each treatment). Cells were counted at 0, 3, and 6 days after the cells were initially fed. Black and red asterisks indicate increased and decreased protist counts after adding the bacterial isolates: black asterisks indicate that there were significantly more protists with the bacterial treatment than with heat-killed E. coli alone, while red asterisks indicate that there were significantly fewer protists (P < 0.05).