Disease-induced assemblage of a plant-beneficial bacterial consortium

Abstract

Disease suppressive soils typically develop after a disease outbreak due to the subsequent assembly of protective microbiota in the rhizosphere. The role of the plant immune system in the assemblage of a protective rhizosphere microbiome is largely unknown. In this study, we demonstrate that Arabidopsis thaliana specifically promotes three bacterial species in the rhizosphere upon foliar defense activation by the downy mildew pathogen Hyaloperonospora arabidopsidis. The promoted bacteria were isolated and found to interact synergistically in biofilm formation in vitro. Although separately these bacteria did not affect the plant significantly, together they induced systemic resistance against downy mildew and promoted growth of the plant. Moreover, we show that the soil-mediated legacy of a primary population of downy mildew infected plants confers enhanced protection against this pathogen in a second population of plants growing in the same soil. Together our results indicate that plants can adjust their root microbiome upon pathogen infection and specifically recruit a group of disease resistance-inducing and growth-promoting beneficial microbes, therewith potentially maximizing the chance of survival of their offspring that will grow in the same soil.

Fig. 1

Downy mildew infection promotes growth of specific microbiota in the rhizosphere of Arabidopsis. a Principal component (PC) analysis of microbial communities in unplanted soil and the rhizospheres of pathogen-infected or defense hormone-treated Arabidopsis plants. Microbial communities were isolated from unplanted soil (brown symbols), rhizospheres of untreated control plants (green), rhizospheres of plants of which the leaves were inoculated with the biotroph Hpa (red), or the necrotroph Bc (blue), or rhizospheres of plants of which the leaves were repeatedly treated with SA (yellow), or MeJA (purple). Microbial communities were analyzed 1 week (squares) and 2 weeks (triangles) after the start of the foliar treatments. Eigenvalues of PC1 and PC2 are expressed on the X- and Y-axis, respectively. b Biplot of eOTU correlations (PC scores) to the same PC1 and PC2. Each red dot represents an eOTU. PC1 separates bulk soil eOTUs (left) from rhizosphere eOTUs (right). Three eOTUs that strongly correspond to PC2 are designated with a number. c–e Quantification of eOTU abundance in the different treatments. Boxplots of PhyloChip HybScore per treatment and time point are shown for c Stenotrophomonas sp. eOTU 97, d Xanthomonas sp. eOTU 106 and e Microbacterium sp. eOTU 107. Black dots represent the values of the 4 replicates per treatment. Red plus signs signify the averages. Red asterisks denotes significant differences from control rhizospheres in the same time point (false discovery rate <0.05). U, unplanted soil; C, rhizosphere of control-treated plants; B, rhizosphere of Bc-inoculated plants; H, rhizosphere of Hpa-inoculated plants; S, rhizosphere of SA-treated plants; J, rhizosphere of MeJA-treated plants

Fig. 2

Synergistic interactions between recruited Xanthomonas, Stenotrophomonas, and Microbacterium spp. strains. a Boxplot of biofilm formation by single Xanthomonas sp. WCS2014-23 (X; eOTU 106), Stenotrophomonas sp. WCS2014-113 (S; eOTU 97) and Microbacterium sp. WCS2014-259 (M; eOTU 107) or the double (XS, XM, and SM) and triple combinations (XSM) thereof in Nunc-TSP lid plates. After 24 h of incubation, the biofilm formation was quantified by staining with crystal violet. b Attraction between colonies of X, S, and M grown at increasing proximity on King’s medium B agar

Fig. 3

Synergistic effects of recruited rhizobacteria on systemic immunity against Hpa and plant growth promotion. a Spore production by Hpa on Arabidopsis plants growing on soil pre-inoculated with Pseudomonas simiae WCS417, Xanthomonas sp. WCS2014-23 (X; eOTU 106), Stenotrophomonas sp. WCS2014-113 (S; eOTU 97) and Microbacterium sp. WCS2014-259 (M; eOTU 107or a mixture of X, S and M (XSM). b Boxplot showing shoot fresh weight of 6-week-old Arabidopsis plants grown in soil pre-inoculated with WCS417, X, S, M, or a mixture of X, S, and M. Italic letters depict statistically significant (P < 0.05) differences according to analysis of variance with Tukey’s posthoc test. Black dots represent the respective replicate values. All experiments were repeated at least three times with similar results. c Picture showing 6-week-old Arabidopsis plants grown in control soil or soil pre-inoculated with a mixture of X, S, and M

Fig. 4

Soil-mediated effects of Hpa-infected plants on disease resistance in a subsequent population of plants. a Schematic representation of the experiment. A first population (1°) of Arabidopsis Col-0 seedlings or unplanted soil (U) was inoculated with a Hpa (H) spore suspension or mock treated (M). Seven days after inoculation, aboveground plant parts were removed, after which a second population (2°) of Arabidopsis plants was sown and grown on the remaining soils. After 2 weeks of growth in the pre-conditioned or unconditioned soils, Arabidopsis plants were inoculated or not with Hpa. Disease severity was quantified by counting the number of Hpa spores that were produced at 7 days after inoculation. b Boxplot showing the number of Hpa spores produced on plants growing on the indicated pre-conditioned soils. Different letters indicate statistically significant (P < 0.05) differences according to analysis of variance with Tukey’s posthoc test. Red plus signs signify the averages of the 10 replicates per treatment. Black dots represent the respective replicate values. The experiment was repeated 4 times with similar results [57].