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CURRENT SCIENCE, VOL. 80, NO. 8, 25 APRIL 2001 1043

Assessment of genetic variability and
strain identification of Xanthomonas
oryzae pv. oryzae using RAPD-PCR
and I S1112-based PCR

V. S. Gupta
,
*, M. D. Rajebhosale

, M. Sodhi

,
Sukhwinder Singh

, S. S. Gnanamanickam
#
,
H. S. Dhaliwal

and P. K. Ranjekar

Plant Molecular Biology Unit, Division of Biochemical Sciences,

National Chemical Laboratory, Pune 411 008, India

Biotechnology Centre, Punjab Agricultural University,

Ludhiana 141 004, India
#
Centre for Advanced Studies in Botany, University of Madras,
Chennai 600 025, India

Sixteen isolates of Xanthomonas oryzae pv. oryzae
(X.o. pv. oryzae) representing different geographical
locations in India along with 2 isolates from the Phil-
ippines were analysed using polymorphic RAPDs.
The primers OPA-03, OPA-04, OPA-10, OPA-11,
OPK-7, OPK-12 and OPK-17 generated simple, spe-
cific and reproducible fingerprint patterns, indicat-
ing usefulness of RAPD markers in differentiating X.
o. pv. oryzae isolates. At a similarity index of 0.5 the
isolates were grouped into only 2 clusters, suggesting
that these primers are not very efficient in grouping
isolates. Primers PJEL1 and PJEL2, used in inser-
tion sequence I S1112-based PCR, also generated
specific and reproducible fingerprint patterns for
the same set of the X.o. pv. oryzae isolates. Based on
the RAPD-PCR (seven primers) and I S1112-PCR
(two primers) data, at a similarity of 0.57, sixteen
out of 18 isolates were grouped into 5 different clus-
ters and 2 isolates were loosely grouped with them.
The data using RAPD-PCR and I S1112-based PCR
approaches revealed their potential in rapid identifi-
cation of isolates, in assessment of genetic variation
in the Indian pathogen population and in generating
unique DNA fragments specific to 8 isolates of X.o.
pv. oryzae.

BACTERIAL blight of rice caused by Xanthomonas
oryzae pv. oryzae
1
(X.o. pv. oryzae) has become an in-
creasingly important problem affecting rice production
worldwide. Breeding rice varieties with genetic resis-
tance for bacterial blight is an effective and economical
strategy for the control of disease and is an important
component of rice improvement programme
2
. However,
it is a matter of concern that the widespread use of only
a few resistance genes might accelerate the evolution of
new pathogenic races leading to outbreak of this dis-
ease, causing serious losses in crop production
3
. In fact
virulent strains of X.o. pv. oryzae causing the bacterial
leaf blight have already been detected on the resistant
cultivars of rice
2
. Since host population influences the


*For correspondence. (e-mail: Vidya@ems.ncl.res.in)
genetic diversity and population structure of the patho-
gen, it is important to identify the causal pathogen
strains and isolate accurately in a plant pathogen sys-
tem
46
. Furthermore, X.o. pv. oryzae is seed-borne
7
, and
hence international tracking of the pathogen population
would be useful for quarantine programmes.
The development of the polymerase chain reaction
(PCR) technique
8
has provided a highly sensitive
method, where a few nanograms of template DNA are
enough to obtain simple and reproducible amplification
patterns from complex genomic DNA
9
. The amplifica-
tion reaction using random oligomeric primers produces
RAPD (randomly amplified polymorphic DNA) frag-
ments which are extensively used to detect genetic
variation in various eukaryotes and prokaryotes. In re-
cent years RAPD-PCR approach has been shown to be
useful in classifying a number of microbial strains and
species
10,11
, including various Xanthomonas spp. such
as Xanthomonas albilineas
12
, X. fragariae
13
, X. malto-
philia
14
and X. campestris pv. pelargonii
15
. RAPD-PCR
has also been used to identify races of Gremmeniella
albietina
16
, ZG groups of Rhizoctonia solani
17
, geo-
graphically distinct populations of Phytophthora
megasperma
18
and different populations and species of
arbuscular mycorrhizal fungi
19
.
The repeat probe pJEL101, isolated from X.o. pv.
oryzae, has already been shown to be a potential DNA
marker. It could differentiate X.o. pv. oryzae isolates
from other Xanthomonas pathovars
20
, and could also be
used to assess genetic diversity in Philippino
21
and
Asian isolates
22
. Further the repeat element pJEL 101 is
found to contain an insertion element, IS1112, with a
relatively high copy number (about 80 copies). Out-
wardly-directed primers complementary to IS1112, des-
ignated as pJEL1 and pJEL2, are shown to have the po-
tential in pathotyping of X.o. pv. oryzae
23
. Another
PCR-based approach, namely rep-PCR has also been
shown to be useful in measurement of haplotypic varia-
tion in X.o. pv. oryzae
24
.
In this laboratory, studies were initiated to examine
the Indian isolates of X.o. pv. oryzae at a molecular
level. Since very little information was available regard-
ing the molecular pathotyping of Indian isolates, this
study was considered to be important in assisting the
national rice breeding efforts in relation to bacterial
blight disease. In a previous report, potential of various
multilocus probes, like pV47, (TG)
10
and pBS101 and a
gene probe avrXa10 was shown in analysing the Indian
pathogen population
25
. All these probes needed South-
ern hybridization, which is time consuming and hence
there was a need for a rapid technique such as PCR-
based approach which could be more efficient and also
sensitive.
In this report, we demonstrate the utility of RAPD-
PCR and IS1112-based PCR in generating specific
genomic fingerprints and their potential in assess-
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CURRENT SCIENCE, VOL. 80, NO. 8, 25 APRIL 2001 1044
ing genetic variation in Indian isolates of X.o. pv.
oryzae.
The Indian isolates were obtained from various
sources in the form of pure single-cell isolates (Table
1). All the isolates were routinely subcultured on
MGYP (malt extract 0.3%, glucose 1%, yeast extract
0.3%, peptone 0.5%) agar slants and were stored as par-
affin oil stocks at 4C and as glycerol stocks at 70C.
The race grouping of X.o. pv. oryzae used in the present
study was based on virulence tests with differential rice
varieties IR8, IR20, IR1545339, Cas 209 and DV 85
which contain the bacterial blight resistance genes
Xa-11, Xa-4, Xa-5, Xa-10 and both Xa-5 and Xa-7,
respectively
2
. For all the PCR reactions, total DNA
from bacterial isolates was extracted by a procedure of
Rajebhosale et al.
25
. In the present study RAPD-PCR
was performed by using a set of two series of primers,
namely OPA and OPK (Operon Technologies Inc, USA)
each comprising twenty primers. Reaction mixtures for
PCR amplification were prepared in a final volume of
25 l, overlaid with 25 l of mineral oil. The reaction
mixture contained 10 mM Tris HCl (pH 8.3), 50 mM
KCl, 2.5 mM MgCl
2
, 0.001% gelatin, 100 M each of
dATP, dCTP, dGTP and dTTP, 5 pmol of primer, 20 ng
of genomic DNA and 0.3 U Taq polymerase. PCR am-
plification was performed in a Perkin Elmer Cetus
Thermocycler by using following cycles

:

initial stage at
95
o
C for 5 min, 45 cycles each of denaturation at 94
o
C
for 1 min, annealing at 36
o
C for 1 min and extension at
72
o
C for 2 min, followed by a final extension at 72
o
C
for 5 min and a final soak at 4
o
C. The amplified prod-
ucts were electrophoresed on a 1.4% agarose gel in 1X


Table 1. List of Xanthomonas oryzae pv. oryzae isolates used in
the present investigation
Strain/ Race/ Geographical Reference/
isolate group origin source

IXoIa Ia Hyderabad A. P. K. Reddy
IXo3863 Ia Chinsurah S. S. Gnanamanickam
IXo10 Ia S. S. Gnanamanickam
IXo2 Ia S. S. Gnanamanickam
IXoIb Ib Hyderabad A. P. K. Reddy
IXo3813 Ib Pantanagar S. S. Gnanamanickam
IXo3841 Ib Gurdaspur S. S. Gnanamanickam
IXo15 Ib S. S. Gnanamanickam
IXoII II Hyderabad A. P. K. Reddy
IXo3856 II Maruteru S. S. Gnanamanickam
IXo3858 II Hyderabad S. S. Gnanamanickam
IXo14 II S. S. Gnanamanickam
IXo9 ND* Cuttack R. N. Misra
IXoCI ND Ludhiana Sukhwinder Singh
IXoCII ND Ludhiana Sukhwinder Singh
IXoCIII ND Ludhiana Sukhwinder Singh
PXo35 I Philippines S. S. Gnanamanickam
PXo86 II Philippines S. S. Gnanamanickam
*ND, Race not determined.
TAE buffer, stained with ethidium bromide and photo-
graphed on a UV transilluminator. All the amplification
reactions were performed more than thrice for each iso-
late/primer combination and only those primers giving
reproducible patterns were used for scoring.
A total of 16 Indian isolates along with two Philip-
pino isolates of X.o. pv. oryzae were examined for their
RAPD-PCR patterns on agarose gel electrophoresis. In
all, forty random primers from OPA and OPK series
were used to amplify the bacterial DNA in PCR reac-
tions. Four primers (OPA-03, OPA-04, OPA-10 and
OPA-11) from OPA series and three primers (OPK-07,
OPK-12 and OPK-17) from OPK series generated
polymorphic and reproducible patterns in three different
replicates. All these polymorphic primers yielded mul-
tiple DNA amplification products, the sizes of which
ranged from approximately 0.1 to 3.0 kb. Both the
intense as well as faint bands were scored for calculat-
ing similarity index values, so as to maximize the num-
ber of scorable characters and minimize the statistical
errors
26
.
Figure 1 depicts a representative electrophoretic pat-
tern of RAPD-PCR amplified products from X.o. pv.
oryzae Indian isolates using primer OPK-12. As seen in
the figure, majority of amplification products are in the
form of strong and well-defined bands in the range of
~ 2.5 kb to ~ 0.3 kb. The electrophoretic profile with
this primer is highly distinct and polymorphic, generat-
ing isolate-specific DNA fingerprint pattern. A few iso-
lates such as IXoCI, PXo86 and IXo3813 (lanes 1, 4
and 18) show presence of many closely-spaced bands in
a ladder-like pattern with this primer. The other poly-
morphic RAPD primers also generated useful informa-
tion (Figures 27). For example, OPA-03, as in Figure
2, amplified at least 23 intense bands for each isolate,
in addition to faint bands. The isolates IXoII (lane 8) of



Figure 1. RAPD profile of X.o. pv. oryzae DNA using primer
OPK-12. Lanes 118 contain amplification products obtained using
DNA of isolates IXoCI (1), IXoCII (2), IXoCIII (3), PXo86 (4),
PXo35 (5), IXoIa (6), IXoIb (7), IXoII (8), IXo2 (9), IXo9 (10),
IXo10 (11), IXo14 (12), IXo3856 (13), IXo3858 (14), IXo3863 (15),
IXo3841 (16), IXo15 (17) and IXo 3813 (18). Lane L contains
/HindIII and 174/HaeIII marker.
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CURRENT SCIENCE, VOL. 80, NO. 8, 25 APRIL 2001 1045


Figure 2. RAPD profile of X.o. pv. oryzae DNA using primer
OPA-03. Lanes 118 contain amplification products obtained using
DNA of same isolates as indicated in Figure 1. Lane L contains
/HindIII.




Figure 3. RAPD profile of X.o. pv. oryzae DNA using primer
OPA-04. Lanes 118 contain amplification products obtained using
DNA of same isolates as indicated in Figure 1. Lane L contains 1 kb
ladder.


race II and IXo2 (lane 9) of race Ia indicated an identi-
cal profile with OPA-03, while isolate IXo3858 (lane
14) revealed presence of many intense bands. OPA-04
(Figure 3) showed presence of intense and unique bands
for a few isolates, namely IXo3858 (lane 14) and IXo2
(lane 9). OPA-10 (Figure 4) generated an intense band
of approximately 2 kb present in all the isolates, except
for IXo 3856 (lane 13), where it appeared to be slightly
faint compared to other isolates, accompanied by sev-
eral faint bands. Although the molecular weight of this
fragment of 2 kb is similar in all the isolates, the DNA
sequence homology still needs to be confirmed. Never-
theless such intense and common amplification product
might be pathovar-specific and cloning and sequencing
of such products might be useful in generating diagnos-
tic markers. With primer OPA-11 (Figure 5), a band of
~ 2.5 kb was present in most of the isolates, except
IXoII (lane 8) and IXo2 (lane 9). Some isolates had a
distinctly similar pattern, except for 1 or 2 band varia-
tions. OPK-07 (Figure 6) showed the presence of a
range of 4 to 11 distinct bands in all the 18 isolates.
OPK-17 (Figure 7) showed the presence of intense and
distinct bands for each isolate.
In another approach using PCR, variable length se-
quences between copies of the repetitive element




Figure 4. RAPD profile of X.o. pv. oryzae DNA using primer
OPA-10. Lanes 118 contain amplification products obtained using
DNA of same isolates as indicated in Figure 1. Lane L contains 1 kb
ladder.




Figure 5. RAPD profile of X.o. pv. oryzae DNA using primer
OPA-11. Lanes 118 contain amplification products obtained using
DNA of same isolates as indicated in Figure 1. Lane L contains
/HindIII.




Figure 6. RAPD profile of X.o. pv. oryzae DNA using primer
OPK-07. Lanes 118 contain amplification products obtained using
DNA of same isolates as indicated in Figure 1. Lane L contains
/HindIII and 174/HaeIII marker.
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CURRENT SCIENCE, VOL. 80, NO. 8, 25 APRIL 2001 1046


Figure 7. RAPD profile of X.o. pv. oryzae DNA using primer
OPA-17. Lanes 118 contain amplification products obtained using
DNA of same isolates as indicated in Figure 1. Lane L contains
/HindIII and 174/HaeIII marker.




Figure 8. IS1112-based PCR profile of X.o. pv. oryzae DNA using
primer pJEL1 and pJEL2. Lanes 119 contain amplification products
obtained using DNA of isolates IXo15 (1), PXo86 (2), IXoCII (3),
IXoCI (4), IXoIb (5), IXo14 (6), IXo10 (7), IXo9 (8), IXo3841 (9),
IXoCIII (10), IXoII (11), IXo2 (12), IXo3863 (13), IXo3813 (14),
IXoIa (15), PXo35 (16) IXo3844 (17) not included in present analy-
sis, IXo3858 (18) and IXo3856 (19). Lane L contains 1 kb ladder.


IS1112 were amplified using two outwardly-directed
primers (PJEL1 and PJEL2) specific to the ends of the
element as described by George et al.
23
. Reaction mix-
tures for PCR amplification were prepared in a final
volume of 24 l overlaid with 24 l of mineral oil. The
reaction mix contained 1X final concentration of Taq
polymerase buffer, 185 M each of dATP, dCTP, dGTP
and dTTP, 50 pmol each of primer PJEL1 and PJEL2,
10% of dimethylsulphoxide, 31 mM of Tris (pH 9.5),
10 ng of genomic DNA and 2 U Taq polymerase. PCR
amplification was performed with a Perkin Elmer Cetus
Thermocycler using the following cycles

:

1 initial stage
at 94
o
C for 1 min, 30 cycles of denaturation at 94
o
C for
10 s, annealing at 62
o
C for 1 min and extension at 65
o
C
for 10 min, followed by a final extension at 65
o
C for
15 min and a final soak at 4
o
C. The amplified products
were loaded on a 0.5% agarose/0.75% Synergel
TM

(equivalent of 2% agarose) in 0.5X TBE buffer, stained
with ethidium bromide and photographed on a UV tran-
silluminator. All the amplification reactions were
repeated more than thrice for each isolate and only
reproducible band patterns were used for scoring.
Figure 8 demonstrates the amplification profile of 16
Indian isolates along with two isolates from Philippines
of X.o. pv. oryzae using pJEL1 and pJEL2 primers. All
the isolates yield multiple DNA amplification products
in the range of approximately 10 to 0.2 kb. The number
of scorable bands obtained using pJEL1 and pJEL2
primers ranged from 6 to 18 bands per isolate. Many
intense bands of 3 kb and above were present in most of
the isolates, whereas bands of approximately 2 kb and
1.6 kb were present in 13 isolates, except for isolates
PXo86, IXoIb, IXo2, IXoIa and IXo3856 (lanes 2, 5,
12, 15 and 19). Similarly only few bands below 3 kb are
present uniformly in all isolates, except for isolate
IXo10 (lane 7), where a ladder-like pattern of low
intensity bands in addition to intense bands is observed.
A similarity index X
D
was calculated for all pairwise
comparisons expressing the probability that a fragment
in one genotype was also found in another
27
. Different
fingerprint parameters were studied for in-depth analy-
sis of each primer; these are represented in Table 2. The
average number of bands detected by the RAPD-PCR
approach was as high as 7.4 and the level of band shar-
ing between isolates was as low as 0.21, indicating high
level of polymorphism. The primer OPA-04 generated
highly polymorphic bands with the least probability of
identical match of 1.19 10
4
. Although OPK-7 had the
highest number of average bands per lane, many of
them were monomorphic thus increasing the average
similarity index value. The fingerprint parameters using
PJEL1 and PJEL2 primer indicate the average number
of bands as approximately 12 per isolate; however, most
of the bands are monomorphic. The number of poly-
morphic bands between pairs was the lowest (3.72) with
these two primers. The average similarity index value
and the value of probability of identical match were
considerably higher than those generally obtained with
RAPD markers.
The RAPD and IS1112 gel profiles were also ana-
lysed critically for presence of a unique fragment for
each isolate. The molecular weight of these fragments
was calculated using SEQUAID program
28
. Table 3
gives information on such unique fragments obtained in
8 specific isolates using specific primers. The isolate
IXo3858 is found to generate more unique fragments
compared to other isolates, indicating that it might con-
tain more diverse sequence compared to other isolates
in the present study. The identification of unique frag-
ments for each isolate will help in rapid identification of
the isolate and also can be further utilized to design a
diagnostic marker which is isolate-specific. Such mark-
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CURRENT SCIENCE, VOL. 80, NO. 8, 25 APRIL 2001 1047
Table 2. Analysis of RAPD-PCR and IS1112-based PCR DNA fingerprint patterns
Fingerprint parameter

Average Average Average Probability
number similarity number of of identical
of bands index polymorphic bands match by
Marker (N SD) (XD SD) between pairs chance (XD)
N


RAPD markers
OPA-03 6.31 2.00 0.44 0.26 7.06 5.62 10
3

OPA-04 5.79 2.65 0.21 0.26 9.14 1.19 10
4

OPA-10 6.84 2.58 0.43 0.23 7.79 3.11 10
3

OPA-11 5.68 2.03 0.58 0.23 4.77 4.53 10
2

OPK-07 7.47 2.52 0.59 0.49 6.12 2.03 10
2

OPK-12 6.26 2.92 0.38 0.49 7.76 2.64 10
3

OPK-17 4.73 2.07 0.28 0.45 6.81 2.38 10
3

IS1112-based primers
pJEL1 and pJEL2 11.95 3.72 0.73 0.15 3.72 2.32 10
2

Similarity index was calculated as
2NAB
XD = ,
(NA NB)
where NAB is number of bands present in both lanes; NA is the total number of bands in lane A; and NB is the
total number of bands in lane B.


Table 3. Unique fragments for specific isolates
Molecular weight of
Primer unique fragments* (bp) Isolate

OPA-03 2020 IXo3858
OPA-04 4400 IXo3858
OPA-04 1950 IXo2
OPA-10 1460 IXoIb
OPA-11 1460 IXo3858
OPK-07 530 IXoCIII
OPK-12 2630 IXoCII
OPK-17 420 IXo3858
pJEL1 and pJEL2 3700 IXo3858
pJEL1 and pJEL2 1830 IXo9
pJEL1 and pJEL2 1450 IXo10
pJEL1 and pJEL2 960 IXo3841
*Molecular weights calculated by using computer software
SEQAID
21
.

ers can be utilized for tracking the pathogenic isolates
and also to study their fitness in field.
Virulence typing of X.o. pv. oryzae is done by using
differential rice cultivars
2
, which gives an estimate of
pathogenicity of these isolates. However, this approach
is time consuming, laborious and shows changes in dis-
ease resistance with changes in rice cultivars, plant age
and environmental conditions. The racial classification
is based on the interaction of an avr gene in the patho-
gen and Xa gene in the host. In rice, more than twenty-
one Xa genes are identified which makes the assessment
of the overall genetic similarity among the Indian iso-
lates rather difficult. In previous studies, X.o. pv. oryzae
population in India has been shown to consist of three
races/groups, Ia, Ib and II, on the basis of pathogenicity
patterns on five indica varieties
29
. According to another
report, out of 86 X.o. pv. oryzae Indian isolates, 70 have
been classified into either Ia, Ib or II, while 5 belong to
new races 3 and 4, and the remaining 11 less-virulent
strains have not been assigned any race
30
. Biological
pathotyping can assess only a few genetic loci and
which in turn may be subject to selection, thus making
it difficult to infer the genetic diversity and evolution of
the pathogen.
Similarity indices calculated on the basis of PCR
band profiles from both the approaches, (RAPD-PCR
and IS1112-based PCR) were averaged to obtain a simi-
larity matrix. The purpose of averaging the similarity
matrices obtained by all the markers was to maximize
the number of loci. Moreover, if cluster analysis was
performed using each primer data, some of the isolates
would cluster similarly, while others would not. There-
fore to get an overall view about the genetic diversity, it
is essential to average the similarity matrices obtained
by maximum number of markers. The similarity matrix
was further utilized for cluster analysis using TAXAN
4.0 (ref. 31). The cluster analysis of X.o. pv. oryzae iso-
lates provides a new insight in their genetic relationship
(Figure 9). At a cutoff value of 0.57 similarity, 16 iso-
lates are grouped in five clusters and 2 isolates are
loosely grouped with them. This similarity level ob-
tained by PCR-based approaches appears to be similar
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CURRENT SCIENCE, VOL. 80, NO. 8, 25 APRIL 2001 1048
to our earlier observation
25
with hybridization-based
probes like (TG)
10
, pV47, pBS101 repeat element and
avr Xa-10. However, the clustering of isolates is differ-
ent in the present investigation. Our hybridization-based
approach
25
could group the two isolates from Philip-
pines together, whereas in the present investigation the
isolates from Philippines are grouped into separate clus-
ters. PXo86 groups with IXoIa and IXoIb in cluster III
and PXo35 clusters with IXo14 and IXo3856 in cluster
IV. The isolates which group together in hybridization
as well as PCR-based approach are IXoCI and IXoCII
in cluster I, IXoII and IXo2 in cluster II, IXo10 and
IXo14.
As depicted in Figure 9, cluster I includes IXoCI,
IXoCII, IXo15 and IXo3813, the latter two belong to
race Ib. IXoII and IXo3658 (race II) and IXo2 (race Ia)
group together in cluster II. Cluster III includes PXo86,
isolate from Philippines belonging to race II and IXoIa
and IXoIb, Indian isolates of race Ia and Ib, respec-
tively. IXoCIII and PXo35 group together in cluster IV.
Cluster V includes IXo10 and IXo3863 of race Ia,
IXo3841 of race Ib and IXo14 of race II. The isolates
IXo9 and IXo3856 (race II) are loosely grouped with
cluster I and V, respectively. These isolates can be rep-
resentatives of a new race and need to be characterized
by using more rice differentials. Earlier studies by Nel-
son et al.
32
have shown that the isolates belonging to a
different race, but clustering together on molecular
analysis when analysed using more R genes showed




Figure 9. Dendrogram of 18 isolates based on combined similarity
indices of the RAPD-PCR gel profile and IS1112-based PCR profile.
presence of new races. The loosely-grouped isolates can
also be good candidates for analysing new sources of
resistance.
In our molecular pathotyping work, we have used
selected isolates collected from different parts of the
country and most of them have been characterized ear-
lier using rice differentials. The analysis of such se-
lected isolates may serve as a platform study for refined
characterization of new isolates whose racial classifica-
tion has not been determined. The characterization of
isolates at a molecular level may further help in deline-
ating isolates belonging to the same race or group, in
studying the pathogenic diversity across the country and
monitoring isolates at field level. Handling of a large
number of isolates for biological pathotyping is practi-
cally difficult and hence alternatively a small group of
representative isolates clustering together on molecular
basis can be used for analysing the population structure
existing in the country, as well as looking for more re-
sistant lines in the host. With the use of arbitrary prim-
ers and IS1112-based primers, we have shown a new
range of molecular genetic markers which can be effi-
ciently used in strain or isolate identification and in as-
sessing genetic variability as well as population struc-
ture in X.o. pv. oryzae isolates.

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ACKNOWLEDGEMENTS. We thank Dr A. P. K. Reddy, and Dr
R. N. Misra for providing X.o. pv. oryzae isolates. M.D.R. is thank-
ful to the Council of Scientific and Industrial Research, New Delhi
for award of Senior Research Fellowship. This work was supported
in part by the Rockefeller Foundation, USA. M.S. acknowledges the
financial assistance and research facilities extended under Asian Rice
Biotechnology Network.


Received 16 May 2000; revised accepted 19 January 2001

Application of PCR fingerprinting
techniques for identification and
discrimination of Salmonella isolates

Anjali Tikoo

, A. K. Tripathi*, S. C. Verma*,
N. Agrawal* and Gopal Nath
,
**

Department of Microbiology, Institute of Medical Sciences and

*School of Biotechnology, Faculty of Science, Banaras Hindu
University, Varanasi 221 001, India

Different PCR-based DNA fingerprinting techniques
were evaluated for identification and discrimination
of bacterial strains. Fifteen strains of Salmonella
enterica subspecies enterica serotypes Typhi (10),
Paratyphi A (1) and Typhimurium (3) collected over
a period of 15 years from stool, blood, and urine
samples and river Ganga were tested. All the strains
were analysed by restriction analysis of the amplified
16s rDNA (ARDRA), random amplified polymorphic
DNA (RAPD) and BOX-PCR methods. In ARDRA,
strains belonging to the same species were identified
by identical fingerprints; RAPD on the other hand,
divided Salmonella into 9 different groups. Two
ciprofloxacin (CIP)-resistant strains showed similar
PCR fingerprints which were different from the rest
of the 12 strains. In BOX-PCR, all the strains of
Salmonella showed 6 different groups which showed
the presence of a common band. Serotype Typhi-
murium could be placed in the same group by BOX-
PCR. CIP-resistant strains also produced one char-
acteristic extra band by BOX-PCR. It was observed
that RAPD had higher discriminatory power than
BOX-PCR and was a simple and rapid technique for
use in epidemiological studies of isolates belonging to
S. enterica.

THE genus Salmonella consists of bacilli that are pri-
marily parasites of the intestine of a large number of
vertebrate species, including man. It causes enteric fe-
ver, gastroenteritis, septicaemia with or without focal
suppuration and also carrier state in human beings. Epi-
demiological studies are done for the prevention and
treatment of infections. The ability to distinguish iso-
lates of Salmonella may be very important to trace the
source of outbreak/s. The relationship, not only among
the isolates from patients and environments, but also
amongst the multiple isolations from the same patient
may be interesting. The methods that have been used for
deciphering the relatedness among the isolates are bio-
typing, antibiogram, plasmid typing, phage typing, sero-
typing, ribotyping and PCR fingerprinting. Some of the
above techniques have low discriminatory potential,
whereas others demand considerable amount of exper-
tise, time and equipment.


**For correspondence. e-mail: gnath@banaras.ernet.in