Limits...
Endopathogenic lifestyle of Pseudomonas savastanoi pv. savastanoi in olive knots.

Rodríguez-Moreno L, Jiménez AJ, Ramos C - Microb Biotechnol (2009)

Bottom Line: The endophytic phase of Pseudomonas savastanoi pv. savastanoi in olive stems and the structural and ultrastructural histogenesis of olive knots have been studied.Hypertrophy of the stem tissue was concomitant with the formation of bacterial aggregates, microcolonies and multilayer biofilms, over the cell surfaces and the interior of plasmolysed cells facing the air-tissue interface of internal opened fissures, and was followed by invasion of the outer layers of the hypertrophied tissue.This is the first real-time monitoring of P. savastanoi disease development and the first illustrated description of the ultrastructure of P. savastanoi-induced knots.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biología Celular, Genética y Fisiología, Universidad de Málaga, Campus de Teatinos s/n, E-29071, Málaga, Spain.

Show MeSH

Related in: MedlinePlus

In situ visualization of P. savastanoi pv. savastanoi NCPPB 3335‐GFP cells by epifluorescence microscopy (A–I) and SCLM (J–L). (A) Cross‐section of a non‐infected olive stem. Autofluorescence emitted by plant tissues allowed differentiation of different histological structures; epidermis (e), schlereids (s), xylem (x), phloem (ph) and parenchyma (p). Epifluorescence images of cross‐sections of knots induced by NCPPB 3335‐GFP at 5 dpi (B and C), 9 dpi (D), 14 dpi (E and F), 21 dpi (G) and 120 dpi (H). (C) Detail of the area indicated in (B). (I) Detail of (H) showing the emission of green fluorescence within the lumen of xylem vessels localized within the hypertrophied tissue. (J–L) SCLM images of cross‐sections of olive knots. The green fluorescence emitted by NCPPB 3335‐GFP cells and the red autofluorescence emitted by the plant tissue was recorded. (J) GFP‐tagged P. savastanoi pv. savastanoi cells colonizing the intercellular spaces of the host tissue at 5 dpi. (K) Internal open fissure of a knot colonized by a biofilm of GFP‐tagged cells at 21 dpi. (L) SCLM visualization of GFP‐tagged cells forming a biofilm at 21 dpi in an internal knot cavity.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3815908&req=5

f5: In situ visualization of P. savastanoi pv. savastanoi NCPPB 3335‐GFP cells by epifluorescence microscopy (A–I) and SCLM (J–L). (A) Cross‐section of a non‐infected olive stem. Autofluorescence emitted by plant tissues allowed differentiation of different histological structures; epidermis (e), schlereids (s), xylem (x), phloem (ph) and parenchyma (p). Epifluorescence images of cross‐sections of knots induced by NCPPB 3335‐GFP at 5 dpi (B and C), 9 dpi (D), 14 dpi (E and F), 21 dpi (G) and 120 dpi (H). (C) Detail of the area indicated in (B). (I) Detail of (H) showing the emission of green fluorescence within the lumen of xylem vessels localized within the hypertrophied tissue. (J–L) SCLM images of cross‐sections of olive knots. The green fluorescence emitted by NCPPB 3335‐GFP cells and the red autofluorescence emitted by the plant tissue was recorded. (J) GFP‐tagged P. savastanoi pv. savastanoi cells colonizing the intercellular spaces of the host tissue at 5 dpi. (K) Internal open fissure of a knot colonized by a biofilm of GFP‐tagged cells at 21 dpi. (L) SCLM visualization of GFP‐tagged cells forming a biofilm at 21 dpi in an internal knot cavity.

Mentions: Autofluorescence emitted by plant tissues after excitation with blue fluorescence was used to differentiate different histological structures: parenchymatic cells (red), xylem and epidermis (yellow) and sclereids (dark‐green). The levels of background green autofluorescence emitted by the stem tissues of non‐inoculated plants (Fig. 5A), and by the knots induced by the wild‐type strain (data not shown), were clearly reduced in comparison to that of NCPPB 3335‐GFP cells inside olive knots. Sections of knots collected at 5 dpi showed only a few green fluorescent spots located near the inoculation point (Fig. 5B and C). At 9 dpi (Fig. 5D), the infection sites of NCPPB 3335‐GFP inside olive knots were clearly visible as numerous bright green spots, which increased over time in size and number. Figure 5E and F, Fig. 5G and Fig. 5H and I correspond to sections of knots collected at 14, 21 and 120 dpi respectively. As previously described for olive (Smith, 1920; Surico, 1977), oleander (Wilson and Magie, 1963), buckthorn (Temsah et al., 2007a) and myrtle (Temsah et al., 2007b), P. savastanoi cells were localized inside internal cavities of the hypertrophied knot tissue (Fig. 5E–H). Moreover, GFP tagging of the pathogen allowed its identification in other histological structures, such as the outer layers of the hypertrophied tissue (Fig. 5E and G). In spite of the low green fluorescence level emitted by whole tumours at 120 dpi (Fig. 3B), bright green fluorescent spots were clearly visible in transverse sections of these tumours, not only inside internal cavities or in outer layers of the knot, but also surrounding the stem vascular cylinder (Fig. 5H), and filling the internal lumen of newly formed xylem vessels (Fig. 5I).


Endopathogenic lifestyle of Pseudomonas savastanoi pv. savastanoi in olive knots.

Rodríguez-Moreno L, Jiménez AJ, Ramos C - Microb Biotechnol (2009)

In situ visualization of P. savastanoi pv. savastanoi NCPPB 3335‐GFP cells by epifluorescence microscopy (A–I) and SCLM (J–L). (A) Cross‐section of a non‐infected olive stem. Autofluorescence emitted by plant tissues allowed differentiation of different histological structures; epidermis (e), schlereids (s), xylem (x), phloem (ph) and parenchyma (p). Epifluorescence images of cross‐sections of knots induced by NCPPB 3335‐GFP at 5 dpi (B and C), 9 dpi (D), 14 dpi (E and F), 21 dpi (G) and 120 dpi (H). (C) Detail of the area indicated in (B). (I) Detail of (H) showing the emission of green fluorescence within the lumen of xylem vessels localized within the hypertrophied tissue. (J–L) SCLM images of cross‐sections of olive knots. The green fluorescence emitted by NCPPB 3335‐GFP cells and the red autofluorescence emitted by the plant tissue was recorded. (J) GFP‐tagged P. savastanoi pv. savastanoi cells colonizing the intercellular spaces of the host tissue at 5 dpi. (K) Internal open fissure of a knot colonized by a biofilm of GFP‐tagged cells at 21 dpi. (L) SCLM visualization of GFP‐tagged cells forming a biofilm at 21 dpi in an internal knot cavity.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815908&req=5

f5: In situ visualization of P. savastanoi pv. savastanoi NCPPB 3335‐GFP cells by epifluorescence microscopy (A–I) and SCLM (J–L). (A) Cross‐section of a non‐infected olive stem. Autofluorescence emitted by plant tissues allowed differentiation of different histological structures; epidermis (e), schlereids (s), xylem (x), phloem (ph) and parenchyma (p). Epifluorescence images of cross‐sections of knots induced by NCPPB 3335‐GFP at 5 dpi (B and C), 9 dpi (D), 14 dpi (E and F), 21 dpi (G) and 120 dpi (H). (C) Detail of the area indicated in (B). (I) Detail of (H) showing the emission of green fluorescence within the lumen of xylem vessels localized within the hypertrophied tissue. (J–L) SCLM images of cross‐sections of olive knots. The green fluorescence emitted by NCPPB 3335‐GFP cells and the red autofluorescence emitted by the plant tissue was recorded. (J) GFP‐tagged P. savastanoi pv. savastanoi cells colonizing the intercellular spaces of the host tissue at 5 dpi. (K) Internal open fissure of a knot colonized by a biofilm of GFP‐tagged cells at 21 dpi. (L) SCLM visualization of GFP‐tagged cells forming a biofilm at 21 dpi in an internal knot cavity.
Mentions: Autofluorescence emitted by plant tissues after excitation with blue fluorescence was used to differentiate different histological structures: parenchymatic cells (red), xylem and epidermis (yellow) and sclereids (dark‐green). The levels of background green autofluorescence emitted by the stem tissues of non‐inoculated plants (Fig. 5A), and by the knots induced by the wild‐type strain (data not shown), were clearly reduced in comparison to that of NCPPB 3335‐GFP cells inside olive knots. Sections of knots collected at 5 dpi showed only a few green fluorescent spots located near the inoculation point (Fig. 5B and C). At 9 dpi (Fig. 5D), the infection sites of NCPPB 3335‐GFP inside olive knots were clearly visible as numerous bright green spots, which increased over time in size and number. Figure 5E and F, Fig. 5G and Fig. 5H and I correspond to sections of knots collected at 14, 21 and 120 dpi respectively. As previously described for olive (Smith, 1920; Surico, 1977), oleander (Wilson and Magie, 1963), buckthorn (Temsah et al., 2007a) and myrtle (Temsah et al., 2007b), P. savastanoi cells were localized inside internal cavities of the hypertrophied knot tissue (Fig. 5E–H). Moreover, GFP tagging of the pathogen allowed its identification in other histological structures, such as the outer layers of the hypertrophied tissue (Fig. 5E and G). In spite of the low green fluorescence level emitted by whole tumours at 120 dpi (Fig. 3B), bright green fluorescent spots were clearly visible in transverse sections of these tumours, not only inside internal cavities or in outer layers of the knot, but also surrounding the stem vascular cylinder (Fig. 5H), and filling the internal lumen of newly formed xylem vessels (Fig. 5I).

Bottom Line: The endophytic phase of Pseudomonas savastanoi pv. savastanoi in olive stems and the structural and ultrastructural histogenesis of olive knots have been studied.Hypertrophy of the stem tissue was concomitant with the formation of bacterial aggregates, microcolonies and multilayer biofilms, over the cell surfaces and the interior of plasmolysed cells facing the air-tissue interface of internal opened fissures, and was followed by invasion of the outer layers of the hypertrophied tissue.This is the first real-time monitoring of P. savastanoi disease development and the first illustrated description of the ultrastructure of P. savastanoi-induced knots.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biología Celular, Genética y Fisiología, Universidad de Málaga, Campus de Teatinos s/n, E-29071, Málaga, Spain.

Show MeSH
Related in: MedlinePlus