plant surface microbiology.pdf

2 Root Colonisation Following Seed Inoculation 19
ciated habitats, and phyllosphere. Commonly used marker genes include the
gusA, lacZ, phoA, xylE, luxAB, luc, and celB genes (Table 1).
The use of reporter genes such as b-galactosidase or b-glucuronidase as
reporter genes has greatly facilitated the localisation of bacteria on the root
surface. For b-galactosidase staining, roots or root sections can be directly
fixed in 1.25 % (v/v) glutaraldehyde in Z buffer (10 mM KCl, 1 mM MgSO 4,
50 mM KH 2PO 4, 50 mM K 2HPO 4, pH 7.0) for 30 min. Subsequently, the roots
are washed twice in Z buffer for 30 min and stained overnight at 28 °C in a
solution of X-Gal (0.8 mg/ml). The roots can be mounted for light microscopic
analysis after thorough rinsing in Z buffer. The use of cross-linking fixation
immobilises the bacteria on the root surface and the enzyme in the tissue.
Although plants are known to possess endogenous b-galactosidase
activity, this method gives no background of b-galactosidase activity from a
number of plant root systems including tomato and Arabidopsis, since
endogenous plant b-galactosidases are inactivated at high temperatures. By
making cross-sections of roots after staining, the method can also be used to
study bacterial-root associations in which bacteria penetrate deeper into the
root tissue. In a similar way bacteria carrying a b-glucuronidase gene can be
detected on the root system after staining with 5-bromo-4-chloro-3-indolylb-D-glucuronide.
The major advantage of the use of b-glucuronidase is that
plants do not possess endogenous b-glucuronidase activity.
The optimal reporter system should provide an easy and non-invasive way
to follow the fate of individual cells in the rhizosphere. In addition, it should
provide the possibility to quantify the activity of specific promoters in the rhizosphere.
Many of the reporters have several drawbacks and restrictions,
which limit their application. Some make use of specific substrates, have high
background signals, or require sophisticated and expensive equipment for
detection (Table 1). Compared to these reporters, autofluorescent proteins
possess several advantages and have been shown to be good tools for the
detection of cells (see Chap. 23, Visualisation of rhizosphere interactions of
Pseudomonas and Bacillus biocontrol strains), and are promising tools for the
measurement of gene activities in the rhizosphere. Nowadays, an argon laser
(488-nm wavelength) is often used to excite red-shifted gfp-variants. An epifluorescence
microscope equipped with a standard fluorescein isothiocyanate
filter is effective for the detection of gfp red-shifted mutants which have excitation
and emission maxima at 488 and 510 nm, respectively. A DAPI (4¢6diamidino-2-phenylindole)
filter set with excitation at 330–380 nm and barrier
filters at 435 nm can be used to detect wild-type Gfp. Autofluorescently
labelled colonies on agar plates can be detected under a hand-held UV-lamp
or a low-resolution binocular microscope equipped with a UV lamp. Other
methods such as flow cytometry can be used to quantify gfp-labelled bacteria.
Individual cells can be detected, quantified, and sorted with high speed and
accuracy. On media without added iron, fluorescent pseudomonads tend to
emit background fluorescence, which can obscure the GFP fluorescence. For