Agriculture Reference
In-Depth Information
maceration techniques, the pressure bomb method detected bacteria colonizing the
vascular system, while maceration mainly recovered endophytes residing in the root
cortex. Hence, a combination of methods is recommended to best understand the full
colonization pattern of endophytic bacteria. Due to the high pressures involved, the
pressure bomb technique favors hard plant tissue and is not suitable for fruits such as
tomatoes and cucumbers (Hallmann and others 1997b). It has not yet been tested on
root crops or leafy greens.
Centrifugation after surface decontamination—another alternative to maceration
(Hallmann and others 1997b)—is often used to collect intercellular fl uid from plant
tissue. Compared to vacuum and pressure extraction, the centrifugation method is
suitable for soft plant tissue.
Direct tissue printing is also useful in isolating endophytic bacteria. Radish sprout
hypocotyls, grown from seeds artifi cially inoculated with
E. coli
O157:H7, were
surface - sterilized by treating with 80% ethanol for 4 - 5 sec and 0.1% HgCl
2
for 4 or
10 min. The 1.5 cm sections were split and pressed onto agar plates for 10 min and
incubated thereafter. Presence of colonies on the agar surface indicated that
E. coli
O157:H7 infi ltrated radish sprouts (Itoh and others 1998). In another study, red bean
seedlings were stem-inoculated at the soil line with a fl uorescently labeled strain of
E. asburiae
JM22, a systemic endophyte isolated from cotton. After cutting the stems
at different distances from the root, the cut surfaces were squeezed and pressed onto
trypticase soy agar containing 100 ppm ampicillin to recover JM22 from stem tissue
10 cm above the soil line (Yan unpublished data).
Endophytic bacteria have been mainly studied by culturing. However, several
noncultural molecular approaches also have been used to examine the interactions
between bacterial endophytes and plants (Hallmann and others 1997b; Kluepfel 1993;
Rosenblueth and Martinez-Romero 2006; Ryan and others 2008). For example, Araújo
and others (2002) studied the interaction between endophytic bacterial communities
and
Xylella fastidiosa
(the causative agent of citrus variegated chlorosis) using culti-
vation-based plating techniques and a cultivation-independent method involving PCR-
generated 16S rRNA gene (rDNA) fragments and denaturing gradient gel electrophoresis
(DGGE). DGGE analysis of 16S rRNA gene fragments amplifi ed from total plant
DNA resulted in several bands that matched those from the bacterial isolates, indicat-
ing that DGGE profi les can be used to detect some endophytic bacteria in citrus plants.
However, some bands had no match with any isolate, suggesting the occurrence of
other nonculturable or undetected endophytic bacteria.
Labeling of Endophytic Bacteria
Endophytes can be detected and quantifi ed using the isolation procedures described
above. However, to understand the mechanisms of entry, localization, survival, and
interactions with other indigenous microorganisms, the specifi c endophytic organism of
interest must be introduced into its host plant or other plant species. Several labeling
methods, which are based on antibiotic resistance or fl uorescent tagging used to differ-
entiate the target endophyte from other background microorganisms, are described next.
The oldest and most common marker used in conjunction with conventional
plating is based on the selection of spontaneous antibiotic-resistant derivatives of
the wild-type bacterial strain. However, two or more antibiotics may be needed to
