Endophytic colonization of Vitis vinifera L. by plant growth-promoting bacterium Burkholderia sp. strain PsJN

Abstract

Patterns of colonization of Vitis vinifera L. cv. Chardonnay plantlets by a plant growth-promoting bacterium, Burkholderia sp. strain PsJN, were studied under gnotobiotic conditions. Wild-type strain PsJN and genetically engineered derivatives of this strain tagged with gfp (PsJN::gfp2x) or gusA (PsJN::gusA11) genes were used to enumerate and visualize tissue colonization. The rhizospheres of 4- to 5-week-old plantlets with five developed leaves were inoculated with bacterial suspensions. Epiphytic and endophytic colonization patterns were then monitored by dilution plating assays and microscopic observation of organ sections. Bacteria were chronologically detected first on root surfaces, then in root internal tissues, and finally in the fifth internode and the tissues of the fifth leaf. Analysis of the PsJN colonization patterns showed that this strain colonizes grapevine root surfaces, as well as cell walls and the whole surface of some rhizodermal cells. Cells were also abundant at lateral root emergence sites and root tips. Furthermore, cell wall-degrading endoglucanase and endopolygalacturonase secreted by PsJN explained how the bacterium gains entry into root internal tissues. Host defense reactions were observed in the exodermis and in several cortical cell layers. Bacteria were not observed on stem and leaf surfaces but were found in xylem vessels of the fifth internode and the fifth leaf of plantlets. Moreover, bacteria were more abundant in the fifth leaf than in the fifth internode and were found in substomatal chambers. Thus, it seems that Burkholderia sp. strain PsJN induces a local host defense reaction and systemically spreads to aerial parts through the transpiration stream.

FIG. 1.

Comparison of the plant growth-promoting effects of the PsJN::gfp2x, PsJN::gusA11, and wild-type PsJN strains on V. vinifera L. cv. Chardonnay plantlets 15 days after rhizosphere inoculation. (A and B) Percentages of relative fresh weight of the root system (A) and aerial parts (B). (C and D) Percentages of relative lengths of the root system (C) and aerial parts (D). The bars indicate means, and the error bars indicate standard deviations.

FIG. 2.

Population dynamics of the PsJN::gfp2x strain for different parts of V. vinifera L. cv. Chardonnay plantlets after rhizosphere inoculation. (A) Rhizoplane; (B) root internal tissues; (C) fifth internode internal tissues; (D) fifth leaf internal tissues. The error bars indicate the standard deviations for mean log-transformed bacterial population sizes. The values are means and standard deviations. FW, fresh weight.

FIG. 3.

Microphotographs of rhizoplane and root internal tissues of V. vinifera L. cv. Chardonnay plantlets after rhizosphere inoculation with Burkholderia sp. strain PsJN. (A and B) Fluorescence stereomicroscope images of roots after inoculation of PsJN::gfp2x, showing strong fluorescence at the site of emergence of lateral roots of the primary root (A) and on a lateral root due to gfp-marked cells (A and B) (arrowheads). (C and D) Epifluorescence microscope images of rhizoplane, showing gfp-marked cells close to the cell walls (C) or around the whole outline of some rhizodermis cells (D) (arrowheads). (E to G) Light microscope images of a secondary root after PBS inoculation (E) and after inoculation with PsJN::gusA11 (F and G), showing a blue color due to gusA-marked cells at the root tips (arrowheads). (H and I) Epifluorescence microscope images of primary root internal tissues of V. vinifera L. cv. Chardonnay plantlets after treatment with PBS (H) or after inoculation of the rhizosphere with PsJN::gfp2x (I), showing yellow fluorescence in several cortical cell layers corresponding to a host defense reaction (arrow). (J to M) Light microscope images of resin-embedded primary roots after inoculation with PsJN, showing host defense reactions (arrows) in the exodermis and cell wall of a cortical cell (J), intercellular colonization of cortical cells (J and K) (arrowheads), a break in the endodermis (arrow) caused by PsJN (L) (arrowheads), and PsJN in xylem vessels (M) (arrows). (A and B) Bars = 500 μm; (C) bar = 25 μm; (D) bar = 10 μm; (E and F) bars = 250 μm; (G) bar = 125 μm; (H and I) bars = 100 μm; (J, K, and L) bars = 50 μm; (M) bar = 20 μm.

FIG. 4.

Photographs of the fifth internode and leaf internal tissues of V. vinifera L. cv. Chardonnay plantlets after rhizosphere inoculation with PsJN. (A and B) Light stereomicroscope images of the fifth internode cut longitudinally with a razor after treatment with PBS (A) or after inoculation with PsJN::gusA11 (B), showing the blue color (arrowheads). (C to E) Epifluorescence microscope images of hand-cut sections of the fifth internode after treatment with PBS (C) or after inoculation with PsJN::gfp2x (D and E), showing PsJN::gfp2x cells in xylem vessels (E) (arrowheads). (F) Light microscope image of resin-embedded stem after inoculation with wild-type strain PsJN, showing PsJN cells in xylem vessels (arrowheads). (G and H) Light micrographs of the fifth leaf after treatment with PBS (G) or after inoculation with PsJN::gusA11 (H), showing the blue color in veins due to gusA-marked cells (arrowheads). (I to K) Epifluorescence microscope images of hand-cut sections of the fifth leaf after treatment with PBS (I) or after inoculation with PsJN::gfp2x (J), showing PsJN::gfp2x cells in xylem vessels (K) (arrowhead). (L) Light microscope image of resin-embedded leaf after inoculation with wild-type strain PsJN, showing PsJN cells in xylem vessels (arrowheads). (M and N) Epifluorescence (M) and light (N) microscope images of the fifth leaf after inoculation of PsJN::gfp2x (M), showing bacteria exiting from stomata (arrowheads), or after inoculation of PsJN::gusA11 (N), showing blue cells (arrows) under stomata. (A and B) Bars = 500 μm; (C and D) bars = 150 μm; (E) bar = 30 μm; (F) bar = 30 μm; (G and H) bars = 500 μm; (I and J) bars = 150 μm; (K) bar = 20 μm; (L) bar = 50 μm; (M) bar = 10 μm; (N) bar = 5 μm.

FIG. 5.

Photographs of endoglucanase (A) and endopolygalacturonase (B) activities of PsJN. Bars = 3 mm.