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


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.


CONCLUSIONS
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.
Golan
A
A
Department of Ornamental Horticulture, ARO, The Volcani Center, Derech Hamacabim, Bet-Dagan, Israel.
Kerem
Z
Z
Tun
O M
OM
Luzzatto
T
T
Lipsky
A
A
Yedidia
I
I
D000085:Acetates / Q000494:pharmacology; D058491:Bacterial Load / Q000379:methods*; D003517:Cyclopentanes / Q000494:pharmacology; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000235:genetics*; D031442:Ornithogalum / Q000187:drug effects / Q000382:microbiology*; D054883:Oxylipins / Q000494:pharmacology; D016973:Pectobacterium carotovorum / Q000235:genetics* / Q000378:metabolism*; D010937:Plant Growth Regulators / Q000494:pharmacology
D000085:Acetates; D003517:Cyclopentanes; D054883:Oxylipins; D010937:Plant Growth Regulators; D049452:Green Fluorescent Proteins; C072239:methyl jasmonate