Priming of Plant Growth Promotion by Volatiles of Root-Associated Microbacterium spp

Abstract

Volatile compounds produced by plant-associated microorganisms represent a diverse resource to promote plant growth and health. Here, we investigated the effect of volatiles from root-associated Microbacterium species on plant growth and development. Volatiles of eight strains induced significant increases in shoot and root biomass of Arabidopsis but differed in their effects on root architecture. Microbacterium strain EC8 also enhanced root and shoot biomass of lettuce and tomato. Biomass increases were also observed for plants exposed only briefly to volatiles from EC8 prior to transplantation of the seedlings to soil. These results indicate that volatiles from EC8 can prime plants for growth promotion without direct and prolonged contact. We further showed that the induction of plant growth promotion is tissue specific; that is, exposure of roots to volatiles from EC8 led to an increase in plant biomass, whereas shoot exposure resulted in no or less growth promotion. Gas chromatography-quadrupole time of flight mass spectometry (GC-QTOF-MS) analysis revealed that EC8 produces a wide array of sulfur-containing compounds, as well as ketones. Bioassays with synthetic sulfur volatile compounds revealed that the plant growth response to dimethyl trisulfide was concentration-dependent, with a significant increase in shoot weight at 1 μM and negative effects on plant biomass at concentrations higher than 1 mM. Genome-wide transcriptome analysis of volatile-exposed Arabidopsis seedlings showed upregulation of genes involved in assimilation and transport of sulfate and nitrate. Collectively, these results show that root-associated Microbacterium primes plants, via the roots, for growth promotion, most likely via modulation of sulfur and nitrogen metabolism.IMPORTANCE In the past decade, various studies have described the effects of microbial volatiles on other (micro)organisms in vitro, but their broad-spectrum activity in vivo and the mechanisms underlying volatile-mediated plant growth promotion have not been addressed in detail. Here, we revealed that volatiles from root-associated bacteria of the genus Microbacterium can enhance the growth of different plant species and can prime plants for growth promotion without direct and prolonged contact between the bacterium and the plant. Collectively, these results provide new opportunities for sustainable agriculture and horticulture by exposing roots of plants only briefly to a specific blend of microbial volatile compounds prior to transplantation of the seedlings to the greenhouse or field. This strategy has no need for large-scale introduction or root colonization and survival of the microbial inoculant.

Keywords: VOCs; biostimulant; plant-microbe interactions; volatile organic compounds.

FIG 1

Plant growth promotion by volatiles emitted by Microbacterium strains. (A) Phenotypic changes of Arabidopsis seedlings exposed to volatiles from eight Microbacterium strains spot-inoculated on agar medium (10 μl at 10⁹ CFU · ml⁻¹) or from the agar medium only (ctrl). Pictures were taken 14 days after exposure. (B and C) Biomass (mean ± standard error [SE], n = 4 to 5) of shoots (B) and roots (C) of volatile-exposed and control seedlings. Different letters show statistically significant differences (one-way ANOVA, Tukey's HSD post hoc test, P < 0.05). (D) Phenotypic changes of Arabidopsis, lettuce, and tomato seedlings exposed to volatile compounds from Microbacterium strain EC8 or from the agar medium only (ctrl) for 12, 7, and 10 days, respectively. (E to G) Dry biomass (mean ± SE, n = 6 to 8) of shoots (E) and roots (F) and lateral root density (number of lateral roots/length [cm] of primary root) (G) of Arabidopsis, lettuce and tomato seedlings exposed to the volatiles from EC8. ctrl, control seedlings exposed to agar medium only; EC8, seedlings exposed to volatiles from EC8. Asterisks indicate statistically significant differences between volatile-exposed and control seedlings (independent samples t test, P < 0.05).

FIG 2

Priming effects by volatiles from Microbacterium strain EC8 on the growth of Arabidopsis and lettuce seedlings. (A) Shoot dry biomass, (B) flower stem length, and (C) number of flowers of Arabidopsis plants (mean ± SE, n = 9); (D) shoot dry biomass of lettuce plants (mean ± SE, n = 4 to 5). ctrl, control plants exposed to agar medium only; EC8, seedlings exposed to volatiles from EC8. Statistically significant differences between volatile-exposed and control seedlings were determined with an independent samples t test.

FIG 3

Exposure of plant shoots and roots to volatiles from Microbacterium strain EC8. (A) Experimental setup used to expose plant shoots to bacterial VOCs. (B and C) Shoot dry biomass (mean ± SE, n = 6) of volatile-exposed Arabidopsis (B) and lettuce (C) shoots. (D) Experimental setup used to expose plant roots to bacterial volatiles. Bacterial cells were inoculated in soil on the bottom compartment. (E and F) Dry biomass (mean ± SE, n = 9) of Arabidopsis (E) and lettuce (F) shoots and roots. (G) Experimental setup used to expose plant roots to bacterial volatiles. Bacterial cells were inoculated on agar medium on the bottom compartment. (H and I) Dry biomass (mean ± SE, n = 8 or 9) of Arabidopsis (H) and lettuce (I) shoots and roots. ctrl, control plants exposed to agar medium or soil only; EC8, plants exposed to volatiles from EC8; asterisks indicate a statistically significant difference between volatile-exposed and control seedlings; ns, no statistical differences (independent samples t test, P < 0.05).

FIG 4

Volatile organic compounds (VOCs) from Microbacterium strains. (A) Hierarchical cluster and heat map analyses of VOC profiles of Microbacterium. Columns represent three replicate VOC measurements of each of the 8 isolates and the medium alone (control). Rows represent the different VOCs (green, low abundance; red, high abundance). (B) List of VOCs from Microbacterium strain EC8. VOCs displayed were detected only for EC8 or were significantly different (Student's t test, P < 0.05, n = 3) and detected at peak intensities at least twice as high as those in the control (medium only). Compounds were putatively annotated by comparing their mass spectra and calculated linear retention indices (RI) with those of NIST and in-house mass spectral libraries and standard (*).

FIG 5

Effects of synthetic sulfur volatile compounds on shoot and roots of Arabidopsis seedlings. (A) Experimental setup for exposing seedlings to volatile synthetic compounds in vitro. Shoot and dry weight (mean ± SE, n = 5) of Arabidopsis exposed to 20 μl of (B) dimethyl disulfide, (C) dimethyl trisulfide, and (D) mixture (1:1) of dimethyl disulfide and dimethyl trisulfide at different concentrations. Dichloromethane (DCM) was used as the solvent. Control plants were not exposed to volatile compounds. Different letters indicate statistically significant differences (one-way ANOVA, P < 0.05).

FIG 6

Global visualization of GO terms assigned for differentially expressed genes (DEGs) of Arabidopsis exposed to volatiles from Microbacterium strain EC8. Functional groups of upregulated DEGs of shoots (A) and roots (B) are shown in red, whereas downregulated DEGs are shown in green. Functional groups with upregulated and downregulated DEGs are shown in gray. Single cluster analysis was performed using Cytoscape software with the ClueGO plugin. The fusion option was used to reduce redundancy of GO terms. Networks with terms functionally grouped with GO pathways are indicated as nodes (two-sided hypergeometric test corrected with the Benjamini-Hochberg procedure; P < 0.05) linked by their kappa score levels (≥0.4), with only the label of the most significant term per group shown. The node size represents the term enrichment significance; smaller nodes indicate larger P values, while larger nodes indicate smaller P values.

FIG 7

Differentially expressed genes (DEGs) involved in sulfur and nitrogen transport and metabolism of Arabidopsis seedlings exposed to volatiles from Microbacterium strain EC8. One-week-old seedlings were exposed to the bacterial volatiles for 1 week. Shoot DEGs are shown in blue and root DEGs are shown in green. Fold change (FC) was calculated using the log₂FC (volatile-exposed seedlings/control).