Root-associated fungal microbiota of nonmycorrhizal Arabis alpina and its contribution to plant phosphorus nutrition

Abstract

Most land plants live in association with arbuscular mycorrhizal (AM) fungi and rely on this symbiosis to scavenge phosphorus (P) from soil. The ability to establish this partnership has been lost in some plant lineages like the Brassicaceae, which raises the question of what alternative nutrition strategies such plants have to grow in P-impoverished soils. To understand the contribution of plant-microbiota interactions, we studied the root-associated fungal microbiome of Arabis alpina (Brassicaceae) with the hypothesis that some of its components can promote plant P acquisition. Using amplicon sequencing of the fungal internal transcribed spacer 2, we studied the root and rhizosphere fungal communities of A. alpina growing under natural and controlled conditions including low-P soils and identified a set of 15 fungal taxa consistently detected in its roots. This cohort included a Helotiales taxon exhibiting high abundance in roots of wild A. alpina growing in an extremely P-limited soil. Consequently, we isolated and subsequently reintroduced a specimen from this taxon into its native P-poor soil in which it improved plant growth and P uptake. The fungus exhibited mycorrhiza-like traits including colonization of the root endosphere and P transfer to the plant. Genome analysis revealed a link between its endophytic lifestyle and the expansion of its repertoire of carbohydrate-active enzymes. We report the discovery of a plant-fungus interaction facilitating the growth of a nonmycorrhizal plant under native P-limited conditions, thus uncovering a previously underestimated role of root fungal microbiota in P cycling.

Keywords: Brassicaceae; Helotiales; fungal endophyte; microbiome; nutrient transfer.

Fig. 1.

Comparison of the fungal communities colonizing A. alpina roots and rhizosphere under greenhouse (GrH), common garden (GAR), and natural (WILD) conditions in different soils (RecNK, RecNPK, Lau, and Gal). (A) Experimental setup showing the different plant growing conditions. The geographic origin of the different A. alpina accessions is indicated in parentheses. More information about the soils and the accessions is given in SI Appendix, Tables S1 and S2, respectively. The number of biological replicates per condition (n) is indicated. (B) Principal coordinates analysis on fungal community differences (Bray–Curtis dissimilarities) in the different compartments and conditions. (C) Fungal alpha diversity estimated by Shannon’s diversity index. Letters a–c indicate significant differences between conditions within each compartment (ANOVA and Tukey’s HSD, P < 0.05). (D) Mean relative abundance of the major fungal orders in the different conditions and compartments: bulk soil, rhizosphere, and root. As the four A. alpina accessions studied exhibited similar fungal communities in the garden experiment (SI Appendix, Fig. S2), combined results for the four accessions are shown under the “GAR-Lau” condition.

Fig. 2.

Fungal taxa consistently found in A. alpina roots (>85% prevalence across all root samples). (A) Maximum-likelihood phylogenetic tree of the highly conserved root OTUs. The representative ITS2 sequences from the OTUs were aligned using Muscle (28) and used for tree inference in PhyML (29) with a GTR+I+γ model with optimized parameters. Fungal orders are depicted with different colors; white circles indicate the average relative abundance (Rel. abu.) of the OTU in root samples. Root (Ro)- or rhizosphere (Rz)-enriched OTUs are indicated (comparison root vs. rhizosphere relative abundance, paired t test, P < 0.05). OTUs with 100% prevalence are shown in boldface type. (B) Relative abundance of the 15 highly conserved root OTUs in each root sample. The data are given in Dataset S4.

Fig. 3.

Fungus F229 (OTU00005) increases A. alpina growth and P content under native low-P soil conditions and is capable of hyphal P transfer to the root in vitro. (A) A. alpina F1gal growth in sterile soil microcosm upon water addition (Water), addition of heat-killed fungus (H.K.), and inoculation with F229 (F229) (1.32 ± 0.8 × 10⁴ propagules per microcosm) at 28 dpi. (Scale bars, 1 cm.) (B) Inter- and intracellular fungal root colonization in sterile soil microcosms visualized by confocal microscopy after staining the fungal cell wall with WGA-Alexa (green, a–d), the plant cell wall with propidium iodide (red, a–c), and the cellular membranes with FM4-64 (purple, d). (Scale bars, 30 µm.) (C) Effect of F229 inoculation on shoot fresh weight and shoot P concentration in sterile soil microcosms. The experiment was repeated four times including the Water and F229 treatments and three times including also the H.K. treatment, with three to four microcosms per treatment; similar results were obtained, and compiled results from the four experiments are shown here. Shoot weight was measured on individual plants (n ≥ 56) whereas all of the shoots from one microcosm were pooled to measure shoot P content by ICP-MS (n ≥ 9). Asterisks indicate significant differences between the treatments based on the Mann–Whitney test (P < 0.05). (D) In vitro transfer of ³³P orthophosphate to the plant by F229. The F229 and A. alpina F1gal plants were grown on low-P (100 µM P) or high-P (1,000 µM P) MS medium in a two-compartment system. ³³P was added to the fungal HC, and after 7 (experiment 3), 10 (experiment 2), or 15 (experiment 1) days, ³³P incorporation into the plant shoot growing in the RHC was measured by scintillation counting of individual plants. No fungus was added to the fungal compartment in the mock inoculated treatments. Bars represent individual samples.

Fig. 4.

The endophytic lifestyle of F229 is associated with the expansion of its CAZyme repertoire. (A) Maximum-likelihood phylogenetic tree inferred from five housekeeping genes (28S, 18S, Rpb1, Rpb2, EF1alpha). Bootstrap values >0.75 are indicated with a black dot. Laccaria bicolor sequences were used for tree rooting. Helotiales with plant beneficial, plant pathogenic, or saprophytic lifestyles are indicated; the key is given in B. The full tree is shown in SI Appendix, Fig. S10. (B) Comparative analysis of CAZyme repertoires in the genome of F229 and related Helotiales with plant beneficial, plant pathogenic, or saprophytic lifestyles. Hierarchical clustering on the abundance of CAZyme classes within the Helotiales. AA, auxiliary activities; CBM, carbohydrate-binding module; CE, carbohydrate esterase; GH, glycoside hydrolase; GT, glycosyltransferase; PL, polysaccharide lyase. (C) Hierarchical clustering on the abundance of selected CAZyme families within the Helotiales. Only families showing a significantly higher abundance in plant-beneficial fungi are shown (t tests, P < 0.01). In B and C, the color scale depicts standardized values for each module. Fungal genome sizes are indicated after their name. F229 is shown in boldface type with an asterisk.