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Molecular Pathogens
MP2011, Vol.2, No.3
http://mp.sophiapublisher.com
- 19 -
from lesion lime leaves. Total of nineteen bacterial
isolates were isolated from three different regions in
Thailand; Banpaew, Samut Sakhon, Nakhon Ratchasima
and Phichit. All bacterial isolates were initially
selected on KCD semi-selective medium. The Gram
stain indicated that, all isolates were Gram-negative
rod bacteria (data not shown). The bacterial isolates
were also confirmed the strain by
Xac
specific primers
(Coletla-Filho, 2006). The PCR results indicated that,
12 (included 10 isolated from Samut Sakhon (BP102,
BP104, BP105, BP107, BP109, BP201, BP202,
BP203, BP205 and BP210) and 2 isolates from Phichit
(PJ01 and PJ03) contained the 581 bp
Xac
regulation
of pathogenicity factors (
rpf
) gene fragments which
encoded pathogenicity effecter protein (Coletla-Filho,
2006). But this gene was not detected in samples from
Nakhon Ratchasima (K01, K02, SUT02 and SUT06)
and unrelated bacteria such as
E. coli
,
Sinorhizobium
and
Agrobacterium
that were used as negative control
(Figure 1). These primers were also not able to
produce specific PCR product from
X. axonopodis
pv.
vesicatoria
,
X. campestris
pv. campestris,
X.
axonopodis
pv.
phaseoli
,
X. axonopodis
pv.
glycine
,
X.
oryzae
pv.
oryzae
template (Figure 2). Non-target
DNA bands were detected in isolated from Nakhon
Ratchasima and some from Phichit (Figure 1).
Figure 1 Specific amplification of
Xac
target (581 bp fragment) using XAC01 and XAC02 specific primers
Note: Lanes 1 and 25: 100 bp DNA ladder (NEB); Lanes 2~11: Bacterial isolated from Samut Sakhon (BP); Lanes 12~15: Nakhon
Ratchasima (SUT02, 06 and K01, 02); Lanes 16~20: Phichit (PJ01
-
05); Lanes 21~23: Unrelated bacteria consist of
E. coli
,
Sinorhizobium
and
Agrobacterium
, respectively; Lane 24: Water control
Figure 2 Specific amplification of
Xac
target by PCR. Lane 1
and 9: 100 bp marker (NEB); Lance 2:
X. axonopodis
pv.
citri
(BP210); Lane 3:
X. axonopodis
pv.
vesicatoria
; Lane 4:
X.
axonopodis
pv.
phaseoli
; Lane 5:
X. campestris
pv.
campestris
;
Lane 6:
X. axonopodis
pv.
glycine
; Lane 7:
X. oryae
pv.
oryzae
,
respectively; Lane 8: negative control.
The bacterial isolates were inoculated to both
susceptible (Pan) and resistance limes (Nam Hom and
M33) for pathogenicity test. Ten bacterial isolates
from Samut Sakhon and 2 isolates from Phichit could
infect both susceptible and resistance lime tested.
These 12 pathogenic bacteria should be
Xanthomonas
species since the
Xac
specific genes product were
observed and they can infect limes. The lesion caused
by isolated BP104 and BP210 on both susceptible
(Pan) and resistance limes (Nam Hom and M33)
slowly turned brown after 3 week post-infection. The
BP104 and BP210 isolates seem to be virulence
pathogenic bacteria. These two virulence pathogenic
bacteria were identified by 16S rRNA gene
sequencing. The accession number HQ875739 and
HQ875740 were obtained. The 16S rRNA gene
sequences confirmed that both strains were
X.
axonopodis
pv.
citri
(
Xac
). In this experiment,
Xac
BP210 isolate was used as bacterial antigen for further
antibody production.
1.2 Serological studies
1.2.1 Determination of antiserum titer
Antisera were raised from dead cells of
Xac
BP210 in
two New Zealand white rabbits. The titer of
Xac