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Molecular Pathogens
MP2011, Vol.2, No.3
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included 10
5
, 10
6
and 10
7
CFU/mL infected parts and
one uninfected part. Each part of the leaf was
wounded by puncturing with a small needle, through
the lower surface (5 needle punctured wounds per
part). Each wounded leaf was placed on 1% water
agar in a Petri dish, with the back of the leaf up. 20 µL
of the different diluted bacterial suspension (10
5
~
10
7
CFU/mL
Xac
BP210) were dropped on each part
of the wounded leaf. The part of uninfected leaf, 20 µL
of sterile 0.85% NaCl was dropped to use as negative
control. The infected leaves were maintained in an
incubator at 28
℃
.
3.5.2 Serological detection of
Xac
in inoculated lime
tissues
The canker pathogen on each part of the infected leaf
was detected by ELISA method. The leaf was
aseptically cut and each part of the infected leaf was
ground in 150 µL coating buffer. 100 µL of leaf
extract was coated in one microtiter plate well and
incubated at 4
℃
overnight before detection with
1:2000 (v/v) diluted antiserum as mention above.
3.6 Molecular studies using specific primers
The sensitivity of PCR amplification was performed
with
Xac
isolate BP210 in tenfold dilution series
(10
8
~10
1
CFU/mL in 0.85% NaCl). 1 µL of each
dilution was used as template in the PCR reaction.
The detection of
Xac
in inoculated lime tissue was
also performed in
Xac
specific PCR amplification as
described above.
3.7 Canker detection from field samples
Symptom and non-symptom plant materials (leaf and
twig) from Pan, Nam Hom and M33 limes were taken
from the field and washed in 25 mL 0.85% NaCl and
shaked for 30 min. Then, the plant material was
removed, and the washing solution was boiled for 10 min.
1 µL of the boiled washing solution were used as
template for specific primers (XAC01 and XAC02)
amplification. The PCR condition was performed as
described above. The
Xac
BP210 isolate was used as
positive control. The detection results of inoculated
tissue were demonstrated as percentage (% detection),
calculated by; [number of sample that can be detect by
Xac
primers/total samples tested] × 100.
Author contributions
PS carried out all experiments as her M.Sc. Thesis work. She
also drafted the manuscript. MKC was the PI of the lab. She
conceived the study, participated in the experimental design,
coordinated, drafted and edited the manuscript. Both authors
have read and proved the final manuscript.
Acknowledgments
S. Loprasert (CRI) is thanks for the
Xanthomonas
reference
strains. PS was supported by SUT scholarship. This research
was funded by SUT Research and Development Fund.
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