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From the root to the stem: interaction between the biocontrol root endophyte Pseudomonas fluorescens PICF7 and the pathogen Pseudomonas savastanoi NCPPB 3335 in olive knots.

Maldonado-González MM, Prieto P, Ramos C, Mercado-Blanco J - Microb Biotechnol (2013)

Bottom Line: While PICF7 was not able to suppress disease development, its presence transiently decreased pathogen population size, produced less necrotic tumours, and sharply altered the localization of the pathogen in the hyperplasic tissue, which may pose epidemiological consequences.However, presence of the BCA seemed to confine P. savastanoi at inner regions of the tumours.This approach has also enabled to prove that the pathogen can moved systemically beyond the hypertrophied tissue.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Protección de Cultivos, Instituto de Agricultura Sostenible, Consejo Superior de Investigaciones Científicas-CSIC, Alameda del Obispo s/n, Apartado 4084, E-14080 Córdoba, Spain.

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Confocal laser scanning microscopy images of transversal vibratome tumour sections (40 μm thick) showing localization of Pseudomonas savastanoi (Psv-GFP, green) and Pseudomonas fluorescens (PICF7-RFP, red). Images were taken at 1 (A, B) and 4 (C, D) weeks after inoculation with Psv-GFP and PICF7-RFP. A. Vibratome transversal section of a representative stem 1 week after inoculation. Fluorescence located inside xylem vessels is due to the presence of intermixed Psv-GFP and PICF7-RFP cells (inset). B. Inset in (A) showing Psv-GFP and PICF7-RFP cells intermixed inside the vascular vessel cells. C. Tumour sampled 4 weeks after inoculation showing events of inner (arrowed) and surface (inset) localization of Psv-GFP and PICF7-RFP cells respectively. D. Inset in (C) showing PICF7-RFP cells at the surface of the knot and a small individual colony of Psv-GFP cells in a different focus plane and not mixed with PICF7-RFP cells (visible as weak green fluorescence at the bottom of the panel). Scale bar represents 100 μm in A, 20 μm in B, 150 μm in C and 25 μm in D. e, epidermis; s, sclereids; p, parenchyma; ph, phloem; x, xylem.
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fig04: Confocal laser scanning microscopy images of transversal vibratome tumour sections (40 μm thick) showing localization of Pseudomonas savastanoi (Psv-GFP, green) and Pseudomonas fluorescens (PICF7-RFP, red). Images were taken at 1 (A, B) and 4 (C, D) weeks after inoculation with Psv-GFP and PICF7-RFP. A. Vibratome transversal section of a representative stem 1 week after inoculation. Fluorescence located inside xylem vessels is due to the presence of intermixed Psv-GFP and PICF7-RFP cells (inset). B. Inset in (A) showing Psv-GFP and PICF7-RFP cells intermixed inside the vascular vessel cells. C. Tumour sampled 4 weeks after inoculation showing events of inner (arrowed) and surface (inset) localization of Psv-GFP and PICF7-RFP cells respectively. D. Inset in (C) showing PICF7-RFP cells at the surface of the knot and a small individual colony of Psv-GFP cells in a different focus plane and not mixed with PICF7-RFP cells (visible as weak green fluorescence at the bottom of the panel). Scale bar represents 100 μm in A, 20 μm in B, 150 μm in C and 25 μm in D. e, epidermis; s, sclereids; p, parenchyma; ph, phloem; x, xylem.

Mentions: To assess whether differences observed in the external, macroscopic anatomy between tumours developed in Psv-inoculated and Psv/PICF7 co-inoculated plants could correlate to changes in pathogen distribution mediated by PICF7 presence, fluorescent tagging of bacteria (Psv-GFP and PICF7-RFP), vibratome-sectioning of knot and stem tissues and CLSM were used. By combining these microscopy and biotechnological tools we aimed to explore the inner anatomy of tumours as well as the localization and distribution of the BCA and the pathogen on and within knots in vivo, without implementing further tissue manipulation, fixation and/or staining procedures. Overall, sectioning of knots from plants co-inoculated with Psv and PICF7 was more difficult, as they presented spongy consistency compared with tumours generated by single inoculation of Psv. CLSM images showed that, in co-inoculated plants, both bacteria could be found mixed within vascular vessels of the stem at early stages of the knot development (7 DAI, Fig. 4A and B). However, from 2 weeks after artificial inoculation of bacteria until the end of the experiments (6–9 weeks), each bacterial species tended to be allocated in different regions of the tumours in most of the observations (Fig. 4C). Indeed, while both fluorescently tagged Pseudomonas could be found mixed at any place within the knot, particularly at the beginning of the knot development (Fig. 4A and B), PICF7-RFP cells were predominantly visualized at the knot surface or in outer regions of the tumour (Fig. 4C and D). In contrast, Psv-GFP cells were mainly found at the inner regions of the hyperplasic tissue, particularly at later times of the experiment (Fig. 4C; Fig. 5B, D and F). Remarkably, localization of Psv colonies greatly differed depending on the presence of strain PICF7. Results showed that when strain NCPPB 3335 was inoculated alone the pathogen predominantly colonized the surface of the tumour (Fig. 5A, C and E).


From the root to the stem: interaction between the biocontrol root endophyte Pseudomonas fluorescens PICF7 and the pathogen Pseudomonas savastanoi NCPPB 3335 in olive knots.

Maldonado-González MM, Prieto P, Ramos C, Mercado-Blanco J - Microb Biotechnol (2013)

Confocal laser scanning microscopy images of transversal vibratome tumour sections (40 μm thick) showing localization of Pseudomonas savastanoi (Psv-GFP, green) and Pseudomonas fluorescens (PICF7-RFP, red). Images were taken at 1 (A, B) and 4 (C, D) weeks after inoculation with Psv-GFP and PICF7-RFP. A. Vibratome transversal section of a representative stem 1 week after inoculation. Fluorescence located inside xylem vessels is due to the presence of intermixed Psv-GFP and PICF7-RFP cells (inset). B. Inset in (A) showing Psv-GFP and PICF7-RFP cells intermixed inside the vascular vessel cells. C. Tumour sampled 4 weeks after inoculation showing events of inner (arrowed) and surface (inset) localization of Psv-GFP and PICF7-RFP cells respectively. D. Inset in (C) showing PICF7-RFP cells at the surface of the knot and a small individual colony of Psv-GFP cells in a different focus plane and not mixed with PICF7-RFP cells (visible as weak green fluorescence at the bottom of the panel). Scale bar represents 100 μm in A, 20 μm in B, 150 μm in C and 25 μm in D. e, epidermis; s, sclereids; p, parenchyma; ph, phloem; x, xylem.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3815922&req=5

fig04: Confocal laser scanning microscopy images of transversal vibratome tumour sections (40 μm thick) showing localization of Pseudomonas savastanoi (Psv-GFP, green) and Pseudomonas fluorescens (PICF7-RFP, red). Images were taken at 1 (A, B) and 4 (C, D) weeks after inoculation with Psv-GFP and PICF7-RFP. A. Vibratome transversal section of a representative stem 1 week after inoculation. Fluorescence located inside xylem vessels is due to the presence of intermixed Psv-GFP and PICF7-RFP cells (inset). B. Inset in (A) showing Psv-GFP and PICF7-RFP cells intermixed inside the vascular vessel cells. C. Tumour sampled 4 weeks after inoculation showing events of inner (arrowed) and surface (inset) localization of Psv-GFP and PICF7-RFP cells respectively. D. Inset in (C) showing PICF7-RFP cells at the surface of the knot and a small individual colony of Psv-GFP cells in a different focus plane and not mixed with PICF7-RFP cells (visible as weak green fluorescence at the bottom of the panel). Scale bar represents 100 μm in A, 20 μm in B, 150 μm in C and 25 μm in D. e, epidermis; s, sclereids; p, parenchyma; ph, phloem; x, xylem.
Mentions: To assess whether differences observed in the external, macroscopic anatomy between tumours developed in Psv-inoculated and Psv/PICF7 co-inoculated plants could correlate to changes in pathogen distribution mediated by PICF7 presence, fluorescent tagging of bacteria (Psv-GFP and PICF7-RFP), vibratome-sectioning of knot and stem tissues and CLSM were used. By combining these microscopy and biotechnological tools we aimed to explore the inner anatomy of tumours as well as the localization and distribution of the BCA and the pathogen on and within knots in vivo, without implementing further tissue manipulation, fixation and/or staining procedures. Overall, sectioning of knots from plants co-inoculated with Psv and PICF7 was more difficult, as they presented spongy consistency compared with tumours generated by single inoculation of Psv. CLSM images showed that, in co-inoculated plants, both bacteria could be found mixed within vascular vessels of the stem at early stages of the knot development (7 DAI, Fig. 4A and B). However, from 2 weeks after artificial inoculation of bacteria until the end of the experiments (6–9 weeks), each bacterial species tended to be allocated in different regions of the tumours in most of the observations (Fig. 4C). Indeed, while both fluorescently tagged Pseudomonas could be found mixed at any place within the knot, particularly at the beginning of the knot development (Fig. 4A and B), PICF7-RFP cells were predominantly visualized at the knot surface or in outer regions of the tumour (Fig. 4C and D). In contrast, Psv-GFP cells were mainly found at the inner regions of the hyperplasic tissue, particularly at later times of the experiment (Fig. 4C; Fig. 5B, D and F). Remarkably, localization of Psv colonies greatly differed depending on the presence of strain PICF7. Results showed that when strain NCPPB 3335 was inoculated alone the pathogen predominantly colonized the surface of the tumour (Fig. 5A, C and E).

Bottom Line: While PICF7 was not able to suppress disease development, its presence transiently decreased pathogen population size, produced less necrotic tumours, and sharply altered the localization of the pathogen in the hyperplasic tissue, which may pose epidemiological consequences.However, presence of the BCA seemed to confine P. savastanoi at inner regions of the tumours.This approach has also enabled to prove that the pathogen can moved systemically beyond the hypertrophied tissue.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Protección de Cultivos, Instituto de Agricultura Sostenible, Consejo Superior de Investigaciones Científicas-CSIC, Alameda del Obispo s/n, Apartado 4084, E-14080 Córdoba, Spain.

Show MeSH
Related in: MedlinePlus