Agriculture Reference
In-Depth Information
differentiate the endophyte from a more highly resistant background fl ora. After dem-
onstrating that the derived antibiotic resistant strain is similar to the wild type strain
in terms of plant surface adhesion, biofi lm formation, growth rate, survivability, and
other important characteristics, the labeled strain is introduced into the plant or envi-
ronment and recovered using a selective medium that contains the appropriate
antibiotic(s) (Kluepfel 1993; Yan and others 2003). This simple, rapid, sensitive, and
inexpensive technique has been widely successful.
Alternatively, serological techniques based on antibodies raised against bacterial
cell wall protein components or whole bacterial cells have been used to detect endo-
phytes. After fl uorescently tagging the specifi c antibody with fl uorescein isothiocya-
nate (FITC) (Mahaffee and others 1997) or colloidal gold (Quadt-Hallmann and
others 1997), the endophyte can be viewed using fl uorescence or electron microscopy.
This method was further improved by combining immunofl uorescence with pour
plating in a technique termed
immunofl uorescent colony staining (IFC)
(Kluepfel
1993). With respect to foodborne pathogens, Itoh and others (1998) used a fl uores-
cein-conjugated goat antirabbit immunoglobulin to observe surface colonization and
internalization of
E. coli
O157:H7 in radish sprouts.
Currently, the most common method is to introduce a reporter gene into the organ-
ism of interest. The gene encoding the green fl uorescence protein (GFP, originally
isolated from the jellyfi sh
Aequoria victoria
) has become extremely useful with this
fl uorescent protein tag having been introduced into bacteria, yeast, slime molds,
plants, drosophila, zebra fi sh, and mammalian cells. When used in combination with
confocal laser scanning microscopy (CLSM), this GFP marker allows direct in vivo
observation of the fl uorescently tagged organism both on and in a wide range of plant
tissues, including apples, tomatoes, sprouts, lettuce, and spinach.
Glucuronidase (GUS)—another useful marker to visualize bacteria in plants, is
based on the discovery that gene fusions comprising the
- glucuronidase gene
can be effectively expressed in many organisms, leading to production of active
β
β
-glucuronidase. This enzyme, in turn, cleaves a chromagenic substrate that can be
directly observed as a green/blue precipitate in bacteria. Warriner and others (2003a,b)
used a GUS marker to confi rm internalization of
E. coli
in spinach and bean sprouts.
Other gene markers—such as
Lac
Z (
- galactosidase gene),
ina
Z (ice nucleation gene),
and
Lux
—have been used in plant pathology as described by Kluepfel (1993). However,
these methods generally have not been used to study foodborne bacteria in plants.
Autoradiography has also proven useful in assessing plant-microbe interactions
(Dumont and others 2006; Hallmann and others 1997b). After growing in a
broth medium amended with isotopes such as
14
C,
15
N, or
32
P, endophytic bacteria
can be introduced into plants and detected using autoradiography. Again, this isotopic
labeling method has not been used to study foodborne pathogens in plants.
β
Methods for Introducing Endophytic Bacteria into Plants
After proper labeling, various approaches can be used to introduce the target organism
into plants. One study by Musson and others (1995) compared 12 different methods
to introduce 21 bacterial endophytes into cotton seedlings. The endophytes were
delivered by vacuum infi ltration, soaking, methylcellulose coating, soil drenching,
root tip pruning, stem injection, stem stab, and foliar spray, with four methods also
