- Transfer 50μl of theXhpculture (OD 595 ¼0.25–0.3) and
slowly pipette the suspension on each disc allowing the suspen-
sion to be completely absorbed. For negative control, add 50μl
of sterile water to the discs. - Incubate the plates at 28C for 48 h.
- Examine the fluorescence emission in the area surrounding the
discs using a fluorescent binocular microscope. Detection of
green fluorescence indicates production of DSF that diffuses
from the tested strain to the reporter (Fig.3b).
3.2 Measurement
of DSF Production
- Collect with a plastic loop the cells of the reporter culture
surrounding the paper disc which contains the tested strain or
water. - Suspend each harvested culture in 1.5 ml tubes containing
500 μl of sterile water. - Transfer 100μl from each suspension and adjust OD 595 to 0.5
by diluting with sterile water. - Transfer each standardized culture to wells of a black microtiter
plate (seeNote 6). Load each sample by triplicate into wells. - Measure fluorescence with a fluorescence microplate reader
using an excitation of 485 nm and emission of 528 nm. - Fluorescence is recorded as Fluorescence Units (FU) of each
well normalized by subtracting the background fluorescence
value of the control wells containing only the reporter. Calcu-
late the average reading of 3 wells.
3.3 Pathogenicity
Test
- Puncture the plant stem three times with a needle, at approxi-
mately 2 cm above the soil. The punctures should be close. - Place at the wounds 10μl of the tested bacterial suspension
(10^8 cells/ml) or sterilized distilled water as control. For each
Fig. 3Bioassay for DSF production. (a) LB agar plate spread with the reporter strainXcc8523 containing paper
discs with the tested strains. Thesquareinblackdenotes the area to examine fluorescence with fluorescent
binocular microscope. (b) Fluorescent binocular images: ( 1 ) control water disc, no fluorescence; ( 2 ) and ( 3 )
discs with low and high DSF-producingXanthomonasstrains, respectively.Arrowspoint to the detection area
surrounding the discs
Quorum Sensing-Dependent Virulence of Phytopathogens 247