Colonization process of olive tissues by Verticillium dahliae and its in planta interaction with the biocontrol root endophyte Pseudomonas fluorescens PICF7

Abstract

The colonization process of Olea europaea by the defoliating pathotype of Verticillium dahliae, and the in planta interaction with the endophytic, biocontrol strain Pseudomonas fluorescens PICF7 were determined. Differential fluorescent protein tagging was used for the simultaneous visualization of P. fluorescens PICF7 and V. dahliae in olive tissues. Olive plants were bacterized with PICF7 and then transferred to V. dahliae-infested soil. Monitoring olive colonization events by V. dahliae and its interaction with PICF7 was conducted using a non-gnotobiotic system, confocal laser scanner microscopy and tissue vibratoming sections. A yellow fluorescently tagged V. dahliae derivative (VDAT-36I) was obtained by Agrobacterium tumefaciens-mediated transformation. Isolate VDAT-36I quickly colonized olive root surface, successfully invaded root cortex and vascular tissues via macro- and micro-breakages, and progressed to the aerial parts of the plant through xylem vessel cells. Strain PICF7 used root hairs as preferred penetration site, and once established on/in root tissues, hindered pathogen colonization. For the first time using this approach, the entire colonization process of a woody plant by V. dahliae is reported. Early and localized root surface and root endophytic colonization by P. fluorescens PICF7 is needed to impair full progress of verticillium wilt epidemics in olive.

Figure 1

Microscopy analysis of Verticillium dahliae EYFP‐tagged mutant derivative VDAT‐36I.
A. Light microscopy image of the different fungal structures produced in a colony of Verticillium dahliae VDAT‐36I after 3 days growing on basal medium.
B. Single confocal optical section of the area in A, showing expression of the eyfp gene. c, conidia; h, hypha; m, microsclerotium.

Figure 2

Confocal laser scanning microscopy images showing the time‐course of colonization olive cv. Arbequina tissues by Verticillium dahliae VDAT‐36I. Confocal analysis was performed on whole representative roots to show surface colonization (A–E). In addition, vibratome tissue sections were made to demonstrate inner root (F–J), stem (K and L) and petiole (M) tissues colonization. Images are projections of 10 adjacent confocal optical sections in all the panels except in A and B where a single optical section is shown, F where the projection was made from 45 adjacent sections, and K–M where projections of 20 adjacent confocal sections are shown. The focal step size between confocal optical sections was 1 µm for all panels except for K–M where the focal step size was 0.5 µm.
A. Germinating conidium showing expression of the eyfp gene on the root surface (1 DAI).
B. View of the apical region of an olive root profusely colonized by hyphae (2 DAI). Fungal colonization was detected on the meristematic, elongation and differentiation root zones.
C and D. Surface colonization of root hairs on (C) the differentiation zone and (D) the elongation zone (3 DAI).
E. Developing microsclerotia on the elongation zone of a profusely colonized olive root (6 DAI).
F. Vibratome longitudinal section of a similar zone showed in E, demonstrating dense hyphal colonization and a developing microsclerotium on the epidermis (6 DAI).
G. Hyphae internally colonizing an epidermal root cell on the differentiation zone (7 DAI).
H. Vibratome longitudinal section of the elongation root region (9 DAI). Inter‐ and intracellular hyphal colonization of the cortex tissue (inset 1), and vascular vessel cells (inset 2).
I and J. Higher magnification of the areas inset 1 and 2 in H.
K and L. (K) Vibratome longitudinal and (L) transverse sections of an olive stem showing the secondary xylem vessel tissue colonized by the fungus.
M. Vibratome transverse section of the petiole from a fallen leave where the secondary xylem vessel cells are colonized by the fungus (arrow) 30 DAI. Asterisk in I marks the swelling of a hypha before penetrating the cell wall of an adjacent cortical cell. Arrows point to hypha branching within the cortical cell. Bars represent 10 µm in all panels except in K–M where it represents 20 µm. me, meristematic zone; e, elongation zone; d, differentiation zone; hy, hyphae; rh, root hair; m, microsclerotia; c, conidia; ep, epidermal cell; co, cortex; vt, vascular tissue; a, appressoria; xy, xylem vessel cell.

Figure 3

Confocal laser scanning microscopy showing simultaneous colonization of olive cv. Arbequina tissues by Pseudomonas fluorescens PICF7 and Verticillium dahliae VDAT‐36I.
A. Detection of EGFP‐tagged PICF7 cells inside a root hair cell, 3 DAI. Three non‐colonized root hairs are also shown. Expression of eyfp and egfp genes in VDAT‐36I and PICF7 strains, respectively, on the differentiation zone of the olive root surface at (B) 1, (C) 2, (D) 5 and (E) 8 DAI. Confocal analysis was performed on whole representative roots (B–E) or on vibratome olive tissue sections (F) to show the VDAT‐36I/ PICF7 interaction on the olive root surface and internally respectively. Images were single confocal optical sections (B, C), projections of 10 (D), 20 (F) and 30 adjacent confocal optical sections (A and E). The focal step size between confocal optical sections was 1 µm (A, D–F). VDAT‐36I hyphae colonized by strain PICF7 cells are indicated by arrows in B–D. Arrows in E show two non‐fluorescent hyphae.
F. Vibratome longitudinal section of a representative olive root from a plant sampled 10 DAI showing PICF7 inside the root hairs and VDAT‐36I hyphae localized intracellularly within the cortex region. Asterisks in E and F indicate root hairs colonized by PICF7. Bars, 20 µm in all the panels except in D where it represents 15 µm. rh, root hair; co, cortex; hy, hypha.

Figure 4

Confocal laser scanning microscopy analysis of transversal vibratome root sections of representative olive cv. Arbequina plants sampled 10 DAI. Images are projections of five adjacent confocal optical sections. The focal step size between adjacent optical sections was 1 µm. A central column of vascular tissue is visible in both panels.
A. Detection of Verticillium dahliae VDAT‐36I (arrows) within the cortex and the vascular tissue in a root of an ‘Arbequina’ plant which was not previously treated with Pseudomonas fluorescens PICF7.
B. Successful endophytic colonization of the intercellular spaces of the root cortex by strain PICF7 (arrow). In this plant root, fluorescent hyphae of isolate VDAT‐36I were not detected. Bars, 20 µm. co, cortical cells; ep, epidermal cells; rh, root hair; vt, vascular tissue.