Associations with rhizosphere bacteria can confer an adaptive advantage to plants

Abstract

Host-associated microbiomes influence host health. However, it is unclear whether genotypic variations in host organisms influence the microbiome in ways that have adaptive consequences for the host. Here, we show that wild accessions of Arabidopsis thaliana differ in their ability to associate with the root-associated bacterium Pseudomonas fluorescens, with consequences for plant fitness. In a screen of 196 naturally occurring Arabidopsis accessions we identified lines that actively suppress Pseudomonas growth under gnotobiotic conditions. We planted accessions that support disparate levels of fluorescent Pseudomonads in natural soils; 16S ribosomal RNA sequencing revealed that accession-specific differences in the microbial communities were largely limited to a subset of Pseudomonadaceae species. These accession-specific differences in Pseudomonas growth resulted in enhanced or impaired fitness that depended on the host's ability to support Pseudomonas growth, the specific Pseudomonas strains present in the soil and the nature of the stress. We suggest that small host-mediated changes in a microbiome can have large effects on host health.

Figure 1. Natural variation in Arabidopsis affects growth of Pseudomonas in the rhizosphere

a, Using hydroponically grown Arabidopsis plants in 48-well plates, a collection of 196 naturally occurring accessions was screened for P. fluorescens growth in the rhizosphere. n = 6 plants per accession; P < 0.05 by ANOVA and Tukey’s HSD test (compared to Col-0) are shown in orange. b, Five accessions had consistently lower levels of P. fluorescens whether bacterial fluorescence, number of CFUs in the well, or number of CFUs attached to roots were measured; n ≥ 24 plants per treatment. c, Hydroponically grown plants were inoculated with B. subtilis 3610, E. coli OP50, previously sequenced Pseudomonas strains, or Pseudomonas isolates identified in this study. CFUs were either counted directly by plating bacteria in wells, or approximated by measuring bacterial fluorescence; n ≥ 12 plants per treatment. d, The culturable microbiome of Col-0 is enriched for fluorescent Pseudomonads relative to bulk soil and the microbiomes of RRS-10 and Knox-18. e, Fluorescent colonies per g of Cambridge or Carlisle soil or roots grown in these soils. b,c,e, Averages ± s.e.m. are shown; *P < 0.01 by t-test; letters designate P < 0.05 by ANOVA and Tukey’s HSD.

Figure 2. The effect of Arabidopsis genotype is largely limited to OTUs in the family Pseudomonadaceae

a, Abundance of 16S rRNA genes from major bacterial phyla in bulk soil and the rhizospheres of Col-0, RRS-10 and Knox-18. Arrows denote phyla that are significantly enriched or depleted in the rhizosphere of all three accessions in both soils. b,c, Genotype-constrained principal component analysis of bulk soil and rhizosphere samples from Cambridge (b) and Carlisle (c) soils. d, Venn diagrams showing the number of OTUs that are enriched or depleted in the rhizosphere of an individual plant genotype relative to the other two plant genotypes. In Cambridge soil, 60% (15/25) of genotype-dependent differences were Col-0-specific (P = 0.02 by chi-squared test); in Carlisle soil, 55.6% (10/18) of genotype-dependent differences were Col-0 specific (P = 0.03 by chi-squared test). e,f, OTUs in the Pseudomonadaceae that are significantly enriched in the rhizosphere of at least one plant genotype grown in soil from Cambridge (e), or Carlisle (f). *OTUs that are enriched in the Col-0 rhizosphere relative to RRS-10 and Knox-18; P < 0.01 by moderated t-test.

Figure 3. Incompatible Arabidopsis accessions actively inhibit growth of Pseudomonas

a, Knox-18 and RRS-10 plants grown in the same wells as Col-0 can inhibit growth of bacteria in trans; n = 18 plant pairs per treatment. b, Defence gene induction 24 h after treatment with P. fluorescens WCS365 measured by qRT-PCR. Averages ± s.d. of three biological replicates are shown; n = 24 plants per replicate. c, Bacteria grown in 48-well plates with Col-0, RRS-10 or Knox-18 were stained with DAPI (blue) and SYTOX (green); Scale bar: 5 µm. d, Quantification of SYTOX+ positive cells in the Arabidopsis rhizosphere. a,d, *P < 0.001 compared to Col-0 by t-test; Averages ± s.e.m. are shown.

Figure 4. Pseudomonas strains do not promote the growth of incompatible accessions

a, Col-0, RRS-10 and Knox-18 grown on agar plates with no carbon source and inoculated with buffer or the indicated Pseudomonas strains. b, Quantification of fresh plant weight. c, Number of lateral roots on plants shown in a; n = 15 plants per treatment. d, Fresh shoot weight of plants grown in Cambridge soil and treated with 10 mM MgSO₄, bacteria resuspended to an A₆₀₀ of 0.01 in 10 mM MgSO₄ or fertilizer. e, Fresh shoot weight of plants grown in a commercial soil/vermiculite mixture and treated as described in d; in b–d letters designate P < 0.05 by ANOVA and Tukey’s HSD tests; n ≥ 12 plants; averages ± s.e.m. are shown.

Figure 5. Addition of Pseudomonas has a minor but significant effect on soil and rhizosphere community composition

a, Treatment-constrained principal component analysis of bulk soil and rhizosphere samples with or without the addition of Pseudomonas CH267. b, Relative abundance of OTU79 (which probably corresponds to Pseudomonas CH267) in rhizosphere and soil samples with and without CH267 added. c, Abundance (%) of genera in the Firmicutes in Cambridge soil with or without the addition of Pseudomonas CH267. Upon addition of CH267, all genera listed showed significant enrichment in bulk soil and the Col-0 rhizosphere and no significant difference in the rhizospheres of RRS-10 and Knox-18. d, Abundance (%) of the genus Flavobacterium with or without addition of CH267. In b–d *indicates a significant increase; **indicates a significant decrease by ANOVA and moderated t-test (P < 0.01); ns, not significant; averages ± s.e.m. are shown.

Figure 6. Disease outcome depends on host genotype, the Pseudomonas strain present and the pathogen

a, Accessions were grown hydroponically in 48-well plates and treated with buffer, P. fluorescens and/or F. oxysporum (For 815) as indicated. Representative images 14 days post inoculation with WCS365 are shown. b, Percentage survival of plants inoculated with P. fluorescens WCS365, CH229 or CH267 and For 815 after 21 days. a,b, Average ± s.d. of three replicates with n = 6 plants per treatment per rep. c, Growth of Pto DC3000 in the leaves of adult soil-grown plants grown with P. fluorescens in the rhizosphere. n = 12 leaves from six plants; *P < 0.05; **P < 0.01 by Tukey’s HSD test; averages ± s.e.m. are shown.