Combining flow cytometry and gfp reporter gene for quantitative evaluation of Pectpbacterium carotovorum ssp. carotovorum in Ornithogalum dubium plantlets.

AIMS: Ornithogalum dubium is a natural host of the soft rot pathogen Pectobacterium carotovorum ssp. carotovorum (Pcc). The present study was aimed to develop a quantification system for Pcc expressing a gfp reporter gene, using fluorescent activated cell sorter (FACS) in planta. METHODS AND RESULTS: Several calibration steps were required to distinctly gate the GFP-labelled bacteria at FL1 mode and count the bacteria. To validate the bacterial counts obtained by FACS analysis, an internal standard of polystyrene green fluorescent microsphere beads was employed, resulting in high correlation with serial dilutions and plate counting. This allowed quantification of the bacteria, with no further need to culture, dilute or plate the cells. Micropropagation tools were developed to produce uniform plantlets of O. dubium, which were either inoculated with increasing concentrations of Pcc or elicited for resistance towards Pcc using methyl jasmonate. The rapid counting procedure allowed recovering, gating and counting the bacterial population in planta, separately from the plant cells background and from the microsphere beads. CONCLUSIONS: The FACS based quantification approach of Pcc was found accurate, reproducible and time saving, thus useful for counting bacteria in planta. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of time- and cost-saving approach for Pcc quantification with efficient screening tools during early stages of micropropagation may facilitate the preliminary process of selection for resistant cultivars.

Induction of defense responses in cucumber plants (Cucumis sativus L. ) By the biocontrol agent trichoderma harzianum

The potential of the biocontrol agent Trichoderma harzianum T-203 to trigger plant defense responses was investigated by inoculating roots of cucumber seedlings with Trichoderma in an aseptic, hydroponic system. Trichoderma-treated plants were more developed than nontreated plants throughout the experiment. Electron microscopy of ultrathin sections from Trichoderma-treated roots revealed penetration of Trichoderma into the roots, restricted mainly to the epidermis and outer cortex. Strengthening of the epidermal and cortical cell walls was observed, as was the deposition of newly formed barriers. These typical host reactions were found beyond the sites of potential fungal penetration. Wall appositions contained large amounts of callose and infiltrations of cellulose. The wall-bound chitin in Trichoderma hyphae was preserved, even when the hyphae had undergone substantial disorganization. Biochemical analyses revealed that inoculation with Trichoderma initiated increased peroxidase and chitinase activities within 48 and 72 h, respectively. These results were observed for both the roots and the leaves of treated seedlings, providing evidence that T. harzianum may induce systemic resistance mechanisms in cucumber plants.