Page 8 - Molecular Pathogens

Molecular Pathogens
MP2011, Vol.2, No.3 http://mp.sophiapublisher.com
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106 CFU/mL or 105 cell/well. Although, these antisera
showed cross-reactivity with X. axonopodis pv.
vesicatoria but this bacterium does not infect citrus
plant. They only infect tomato and Capsicum
pathogen (Mirik and Aysan, 2009). Therefore, the
cross-reaction detection should not be of alarm. The
antigenic molecules from the normal cell surface
components are often including in polyclonal
antibodies (or antiserum). This mixing of antibodies
has multiple specificities and cross-reactivities with
unrelated bacteria species. This study attempt to
diluted multiple specific antibodies and retain
dominant antibodies which showed acceptable
specificity for a given pathogen.
Many studies reported that, the ELISA sensitivity 105~
106 CFU/mL is sufficient for identification of bacteria
pathogens from symptomatic plants and colonies on
selective media (Jin et al., 2001; Alvarez, 2004; de
Leon et al., 2008; Kokoskova and Mraz, 2008).
However, boiling the bacteria samples could improve
the sensitivity of detection. Several previous reports
have mention that using extraction buffer containing
EDTA and lysozyme to removed cell surface
component could improve the sensitivity of ELISA
detection (Jones et al., 1997, Alvarez, 2004). However,
EDTA was not used in this study but the sensitivity of
detection was still high.
1.2.4 Detach leaf assay
Different concentrations of canker bacteria (107, 106
and 105 CFU/mL) were inoculated to Pan lime leaves
using detach leaf assay. The pathogen on the infected
leaves was detected by ELISA method every 2 days
post inoculation. The results showed that, infected
leaves on the first day of inoculation (day zero)
showed negative reactions in ELISA test whether the
bacterial cells were killed by heat treatment or not
(Table 4). The inoculation of this experiment were 20 µL
of bacterial suspensions dropped on each area. Thus,
bacteria populations on the leaves were 105, 104 and
103 cells on the inoculated leaves area. The sensitivity
of the ELISA (105 CFU/mL or 104 cells/well for dead
cells) should be sufficient for detection of dead cells.
However, protein or other polysaccharide molecules
from the leaf may interfered with the bacteria binding
to the plate therefore, low detection of target bacteria
were observed.
Infected leaves on day two showed positive reaction
when the leaves were inoculated with 105 cells.
Consequently, infected leaves 4 days post inoculation
showed positive reaction with all bacteria
concentration (105, 104 and 10 3 cells) whether the cells
were boiled or not. The boiled bacteria (dead cell)
from infected leaves showed higher reaction in ELISA
test than lived cells. These results were in accordance
with the results of antibody titer and sensitivity test
(Table 1 and Table 2). After day 4 post inoculation,
symptoms on the infected areas were observed as
slightly raised blister-like lesions. Thus, these results
indicated that, ELISA assay is a well-established
method for identification of bacterial pathogens from
symptomatic plants. However, enrichment techniques
can enhance ability of pathogen detection (Jin et al.,
2001) on natural samples but more time is needed for
the culturing period.
1.2.5 Molecular detection of Xac from canker lesions
Canker pathogen on symptom and non-symptom plant
materials (leaf and twig) in infected areas were
detected by PCR amplification using Xac specific
primers. The results showed that, the Xac specific
PCR products were detect in all samples of symptom
plant materials and some non-symptom plant
materials (Table 5).
These specific primers were also used to detect canker
pathogen on infected leaves from the detach leaf
experiment. The results showed that, canker pathogen
were detected by these specific primers on the first
day of inoculation while ELISA was unable (Table 4).
The amplification assay using specific primers have
been reported to detect at low pathogen population
(102~103 CFU/mL) (Cubero et al., 2001, Cubero and
Graham, 2002, Coletla-Filho 2006, Park et al., 2006,
Golmohammadi et al., 2007, de Leon et al., 2008).
The sensitivity of PCR amplification of this study
showed very high sensitivity at 103 CFU/mL or 1 cell
in the PCR reaction (Figure 3). Moreover, this
methodology showed no cross-reactivity with other
Xanthonomas species (Figure 2). However, PCR
method required laboratory equipment, special
reagents and skill to perform.