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

Abstract

SummaryThe 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.

Verticillium wilt of olive: a case study to implement an integrated strategy to control a soil-borne pathogen

“…Indeed, CLSM imagery showed that entry of V. dahliae into root epidermal cells by an active mechanism, or at the points of emergence of lateral roots ( Fig. 5), could also take place although to a much lesser extent than entrance by available root breakages (Prieto et al 2009). An interesting finding was the detection of VDAT-36I MS on the olive root surface just a few days after artificial inoculation.…”

“…This provided an experimental design resembling conditions much closer to natural environments rather than those present in gnotobiotic systems frequently utilized in this type of study. Results have shown that inner colonization of olive root tissues predominantly takes place through a passive entry mechanism, facilitated by the presence of micro or macro root injuries (Prieto et al 2009). This scenario has been suggested as the most likely occurring in plant root systems growing under natural soil conditions (Pegg 1985;Pegg and Brady 2002).…”

“…About 1 week after inoculation, abundant VDAT-36I biomass on/in root tissues was visualized by CLSM. Then, VDAT-36I hyphae reached the root vascular tissue after root cortex cells were actively invaded by fungal biomass at both inter-and intracellularly levels (Prieto et al 2009). In fact, if the pathogen was able to penetrate the cortex tissue, little hindrance to final invasion of xylem vessels was observed in the susceptible cv.…”

“…The use of fluorescent tagging in conjunction with three-dimensional tissue sectioning and CLSM has been implemented to examine the infection and colonization processes of the susceptible olive cultivar Arbequina by V. dahliae (Prieto et al 2009). An EYFP (enhanced yellow fluorescent protein)-tagged derivative (VDAT-36I) of an olive-infecting D pathotype isolate was produced by ATMT.…”

Colonization process of Arabidopsis thaliana roots by a green fluorescent protein-tagged isolate of Verticillium dahliae

“…The colonization of cotton roots by V. dahliae in soil naturally leads to the colonization of vascular tissues in cotton (Garber and Houston, 1966;Gerik and Huisman, 1988). Recently, the development of confocal laser scanning microscopy (CLSM) has significantly increased our knowledge of the colonization process of V. dahliae on various plant roots (Eynck et al, 2007;Vallad and Subbarao, 2008;Prieto et al, 2009;Zhang et al, 2012). However, the comprehensive observation of the cotton living root colonization process by V. dahliae under CLSM is not likely to be practicable due to the large size of cotton roots.…”