Page 10 - Molecular Pathogens

Molecular Pathogens
MP2011, Vol.2, No.3 http://mp.sophiapublisher.com
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Figure 3 Xac specific PCR amplification from a tenfold dilution
series of X. axonopodis pv. citri cultured cells. M: 100 bp
marker (NEB); Lane 1: negative control; Lanes 2-7: 105 cells,
104 cells, 103 cells, 102 cells, 101 cells and 1 cell; lanes 8 -9: <1
cell respectively.
infected lime leaves tissue by commonly used
methods (OEPP/EPPO, 2005). Briefly, the infected
leaves were washed with sterile water and surface
sterilize by soaked in 1% sodium hypochlorite for 3 min.
Then, the lesions were rinsed in sterile water several
times and excised with scalpel. Xac isolates were
initially selected by streaked the water-soak tissue
from the lesion margins on sterile semi-selective
media (KCD medium, nutrient agar (NA) supplemented
with Kasugamycin (16 µg/mL), Cephalexin (16 µg/mL)
and Daconil (Chlorothalonil) (12 µg/mL) prior to
enrichment on NA media without antibiotics. The
bacteria were grown at 28℃ for 24~48 h. The
bacterial colonies were collected for Gram staining
and further used for pathogenicity tests.
Other Xanthomonas strains include X. axonopodis pv.
vesicatoria, X. campestris pv. campestris, X.
axonopodis pv. phaseoli, X. axonopodis pv. glycine , X.
oryzae pv. Oryzae obtained from Chulabhorn
Research Institute (CRI, Thailand) were used as
negative control in strain identification and cross-
reaction tests.
3.2 Specific primers identification and 16S rRNA
gene sequencing
Single colony of Xac isolates and other Xanthomonas
strains were resuspened in 1 mL DI-water and boiled
for 10 min. Then, the suspensions were used as
template for PCR reaction, with specific XAC01 and
XAC02 primers for Xac identification (Coletta-Filho,
2006). The PCR reactions were performed with 50 µL
reaction mixture, containing 1 µL of boiled cell
suspension, 1X reaction buffer (Promega GoTaq),
1.5 mmol/L MgCl2, 0.2 mmol/L dNTPs, 0.2 µmol/L
primers (XAC01: 5’C GC CAT CCC CAC CAC CAC
CAC GAC’3, XAC02: 5’AAC CGC TCA ATG CCA
TCC ACT TCA’3), 1.25U Taq DNA polymerase
(Promega). Unrelated bacterial DNA included
Escherichia coli, Sinorrhizobium and Agrobacterium
were also subjected to PCR reaction as negative
control. PCR conditions were as followed; An initial
cycle of 94℃ for 5 min followed by 35 cycles of
94℃ for 45 s, 60℃ for 45 s, and 72℃ for 45 s, with
a final step of 72 ℃ for 5 min. The PCR products
were observed under UV light after electrophoresis
through 1.0% (w/v) agarose gels and stained with
ethidium bromide.
The full length 16S rRNA gene of Xac isolates BP104
and BP210 were sequenced by Macrogen (Korea)
(http://dna.macrogen.com/eng/). The sequence results
were applied to BLAST program for gene comparison
with data in the GenBank (NCBI).
3.3 Pathogenicity test
3.3.1 Plant samples
M33 hybrid limes (highly resistant to canker) were
from Phichit horticulture research center, Samut
Sakorn and Prachinburi (Thailand), Nam Hom limes
(highly resistant to canker) were from Phichit
horticulture research center and Pan limes (susceptible
to canker) were from Samut Sakorn. These lime plants
were planted in the test field at Suranaree University
of Technology, Nakhon Ratchasima, Thailand.
3.3.2 Xac Inoculation on resistance and susceptible
limes
Half of the M33, Nam Hom and Pan limes leaves
were wounded with needles and celite, the other
halves were not wounded (healthy). 1 mL of the
bacterial isolate suspension (108 CFU/mL) was
sprayed on the leaves. The plants were then covered
with plastic bags overnight. In the morning, the bags
were removed and the plants were left in natural
condition for 2~3 weeks. The virulent canker disease
apparent on each leaf was evaluated.
3.4 Serological studies
3.4.1 Preparation of antigen
Antigen was prepared from the cells of Xac isolate
BP210. The bacterial cells were enriched on NB