Indian Phytopath.

61 (4) : 449-455 (2008)

Genetic Differentiation of Bipolaris spp. based on Random

Amplified Polymorphic DNA markers

M. JAHANI1, RASHMI AGGARWAL*, K.D. SRIVASTAVA and RENU

Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi 110 012
1
Department of Agronomy, College of Agriculture, Birjand University, Iran

ABSTRACT: Seventeen Indian isolates of Bipolaris spp. which included 12 isolates of Bipolaris sorokiniana,
two isolates each of B. specifera and B. tetramera and one isolate of B. maydis were taken up for identification
of genetic markers and to provide a protocol for identification and characterization of isolates using Randomly
Amplified Polymorphic DNA (RAPD) technique. The genomic DNA was compared using 40 random decamer
primers (Operon Technologies Inc., U.S.A.) that generated 554 bands; out of which 549 were polymorphic
products. Genetic similarities of RAPD profiles were estimated on the basis of Jaccard’s coefficient. The
similarity index values ranged from 0.474 to 0.906 indicating the presence of wide range of genetic diversity.
These similarity coefficients were subjected to Unweighted Pair Group Method on Arithmetic Averages
(UPGMA) using paired matrix values. The dendrogram generated consisted of 2 major clusters, one having
all isolates of B. sorokiniana, and other cluster having isolates of B. specifera, B. tetramera and B. maydis
which were further distinguishable from each other by falling into different sub-groups. The banding pattern
of different isolates can also be used as reference for further comparisons.

Key words: Bipolaris sorokiniana, B. tetramera, B. specifera, B. maydis, RAPD markers, molecular characterization

Bipolaris species are important plant pathogens
of various cereal crops. The main symptoms with
which they are associated are dark spots on
leaves, and root rot of seedlings. In India spot
blotch of wheat caused by Bipolaris sorokiniana
(Sacc.) Shoem. [Drechslera sorokiniana (Sacc.)
Subram. & Jain] [ telomorph: Cochliobolus sativus]
is one of the most important diseases of wheat
causing up to 36 per cent loss under favourable
conditions (Anon., 1995). The classification and
identification of Bipolaris species is usually based
on morphological characteristics. But recently,
molecular biological techniques have been used to
explore genetic variability among plant pathogens.
Particularly, the polymerase chain reaction (PCR)
assay has provided a framework to understand the
taxonomy and population structure. In this context,
Randomly Amplified Polymorphic DNA (RAPD), the
procedure developed by Williams et al., (1990) is a
quick and easy technique for pathogen identification
and characterization. The RAPD method has been
*Corresponding author: rashmiiari@yahoo.com
successfully used to identify strains to characterize
races and to analyze virulence variability related to
genetic polymorphisms in phytopathogenic fungi
(Malvickan and Grau, 2001). It has also been used
in the study of inter- and intra-specific variability
among populations from different and from the
same geographic regions (Vakalounakis and
Fragkiadakis, 1999).
So far, not much work has been done on
molecular characterization of Indian isolates of
Bipolaris species. The present study was
undertaken to assess genetic variability among 12
isolates of B. sorokiniana, a pathogen of wheat
causing spot blotch, and compared with two isolates
of B. specifera, two isolates of B. tetramera and
one isolate of B. maydis by PCR based RAPD
technique. B. specifera and B. tetramera are other
two species, which occur as saprophytic fungi in
soil and on cereals. B. specifera attacks roots of
wheat and also is reported to be present on rice
and wheat grains. B. tetramera is reported to
attack sugarcane and paddy and also occurs
450 Indian Phytopathology [Vol. 61(4) : 2008]

saprophytically in soil. B. maydis is a pathogen of
maize causing leaf blight. B. specifera and B.
tetramera resemble each other and also with B.
sorokiniana in cultural characters, therefore, genetic
differentiation based on DNA polymorphism was
undertaken.
MATERIALS AND METHODS
Fungal isolates and Culture conditions: Bipolaris
sorokiniana (12 isolates), B. tetramera (2 isolates),
B. maydis (1 isolate) and B. specifera (2 isolates)
were obtained from the culture collection maintained
at the Wheat Pathology Laboratory and Indian
Type Culture Collection (ITCC), Division of Plant
Pathology, IARI, New Delhi (Table 1). Single spore
cultures of all the isolates were established and
cultivated on liquid culture media (Potato Dextrose
Broth, PDB) at 25±1 0C in shaker incubator at
100rpm. These cultures were harvested after 5
days of growth for extraction of DNA.
DNA extraction: DNA extraction was done as per
protocol of Murray and Thompson (1980) and Zolan
and Pukilla (1985) with slight modifications. Frozen
mycelial mats were weighed and about one gram
mycelium was ground in a sterile pestle and mortar
using liquid nitrogen to a fine frozen powder taking
care not to let the samples thaw. The fine mycelium
was transferred to a sterile centrifuge tube, and 10
ml of DNA extraction buffer [100 mM Tris-HCl (pH
8.0), 1.4 M NaCl, 150 mM EDTA (pH 8.0) and 2%
CTAB prewarmed at 65°C was added. The slurry
was incubated in water bath at 65°C with occasional
stirring for 1 hour. After incubation an equal volume
of phenol: chloroform: isoamyl alcohol (v/v 25: 24:
1) was added to each tube to denature proteins,
mixed gently and centrifuged at 10,000 rpm at
20°C for 20 min. The aqueous phase was transferred
to a new sterile tube and the DNA was precipitated
with 0.8 volume ice cold isopropanol and 0.1
volume of Sodium acetate (3M, pH 5.2). The DNA
was pelleted by centrifugation at 10,000 rpm for 10
min at 25°C. The supernatant was poured off and
the pellet was washed with cold 70% ethanol. Total
nucleic acid obtained was dissolved in 100µl sterile

Table 1. List of Indian isolates of Bipolaris spp used in the present investigations

Isolate Location Agroclimatic Cultivars/

Designation zone varieties

A. Bipolaris sorokiniana
BS-5 Jind-2 (Haryana) NWPZ HD 2329
BS-7 DWR-2,Karnal (Haryana) NWPZ PBW 502
BS-2 Jaipur 1 (Rajasthan) NWPZ PBW 396
BS-18 Jaipur 10 (Rajasthan) NWPZ LBSN
BS-25 Jaipur 17 (Rajasthan) NWPZ HD 2329
BS-27 Faizabad (U.P) NEPZ HD 2329
BS-47 Kalyani-3 (W.B.) NEPZ Local
BS-48 Shillongani-1(Assam) NEPZ WH 147
BS-53 Malan(Himanchal Pradesh) North Hill Zone HPW 89(Surabhi)
BS-54 Almora (Uttrakhand) North Hill Zone VL 832
BS-55 Almora (Uttrakhand) North Hill Zone HBW 184
BS-64 Powarkheda (MP) Central Zone GW 1220
B. Bipolaris specifera
BS 4922 ITCC (4922) IARI 2000
BS 5137 ITCC (5137) Wheat Seeds
C. Bipolaris tetramera
BT 3466 ITCC (3466) Soybean Seeds, Behrampur
BT 4805 ITCC (4805) Banana, New Delhi
D. Bipolaris maydis
BM-1 Maize Lab., IARI, New Delhi
[Vol. 61(4) : 2008]
distilled water and stored at -20°C. The concentration
of DNA was determined spectrophotometrically by
taking UV absorbance at 260 nm, and was
rechecked by running samples in 0.8% agarose gel
at 100 volt for an hour in TAE buffer along with
lambda DNA standard (Genei, India).
Optimization of Polymerase Chain Reaction
(PCR) and primer survey: In order to obtain
scorable and reproducible results, composition of
PCR reaction was optimized by varying the
concentration of template DNA (25 ng, 50 ng, 75
ng and 100 ng), Taq DNA polymerase (0.5, 1.0 and
1.5 units) and MgCl2 (1.5 mM, 2.5 mM and 3.5
mM). RAPD conditions for isolates of Bipolaris
spp. isolates in the present investigations were
standardized and the amplification assay contained:
template DNA -50 ng; Taq DNA polymerase (Genei,
India) - 1.5 unit; MgCl2- 3.5 mM; dNTP (Genei,
India)-200µM each of dATP, dGTP, dCTP, dTTP;
Primer (Operon Technology, USA) -1.0 µM; Assay
Buffer (Genei, India)-1X in a reaction volume of 25
µl. The polymerase chain reaction was performed
using Bio-Rad Gen CyclerTM with the following
temperature profile: the initial denaturation at 94°C
for 2 min, followed by 40 cycles of denaturation at
94°C for 1 min, annealing at 55°C for 30 sec and
extension at 72°C for 1 min with final elongation of
72°C for 10 min. Primer survey was carried out and
60 primers from OPM, OPN, OPA and OPB series
(Operon Technologies, Inc., USA) were screened
using DNA from all the 17 isolates. The primers
that gave reproducible and scorable amplification
products were used in the analysis of all the
isolates.
Agarose gel electrophoresis: Amplified products
obtained after the PCR amplification were loaded
into individual channels of 1.2 % agarose horizontal
gel in TAE (0.04 M Tris-Acetate, 0.001 M EDTA,;
pH 8.0) buffer. Electrophoresis was carried out at
60 volts for 3 hours. Gels were stained with
ethidium bromide (1 µg/ml) (Sambrook et al., 1989)
and observed in a trans-illuminator under UV light.
One Kb ladder (MBI, Fermentas) was used as a
marker.
Scoring of bands and data analysis: Data entry
was done in matrix form in which all observed
bands or characters were listed. The RAPD pattern
of each isolate was evaluated assigning ‘character
state’ ‘0’ or ‘1’ depending on presence or absence
Indian Phytopathology 451
of all bands that could be observed in gel with
certain primers. The estimates of genetic similarity
were calculated. Jaccard’s pair wise similarity
coefficients were subjected to unweighted pair-
group method on arithmetic averages (UPGMA)
and cluster analysis based on their overall similarities
was done to group the isolates. NTSYSPc version
2.02 software (Rohlf, 1990) was used for cluster
analysis and the dendrogram was prepared.
RESULTS AND DISCUSSION
The DNA content extracted from mycelium of
all the isolates ranged from 1.0 - 4.0 µg/ml. The
PCR protocol was first optimized in terms of
concentration of template DNA, Taq DNA
Polymerase and MgCl2 concentration. A titration of
different concentrations of Taq DNA Polymerase
and MgCl2 showed that 1.5 unit of Taq DNA
Polymerase and 3.5 mM MgCl2 gave optimum
results for PCR amplification. Out of 60 primers
screened for amplification of DNA of all the
mentioned isolates of Bipolaris sorokiniana, 2
isolates each of B. specifera and B. tetramera and
1 isolate of B. maydis, 20 resulted in either sub-
optimal or non-distinct amplification products.
Therefore, these were discarded and remaining 40
primers were used for PCR amplifications (Table 2).
Hence, the final numerical taxonomic analysis
included the results from only 40 primer
amplifications. Number of amplification products
obtained was specific to each primer and ranged
from 1 to 21 and also almost all primers showed
100 per cent polymorphism except OPA 8, OPA
20, OPB 17 and OPB 20 with fragment size varying
from 200 bp to 4 Kb. Out of 554 amplification
products, 549 amplicons (99.09 %) were polymorphic
in nature.
A high degree of polymorphism, in general,
was obtained with most of the primers. At intra
specific level a wide range of diversity existed (Fig.
1a,b). Jaccard’s similarity coefficients values of this
index ranged from 0.42 to 0.91, again indicating the
presence of wide range of genetic diversity among
the species and isolates used. The most diverse
pair showing 42% similarity comprised of B. maydis
an isolate from Indian Agricultural Research Institute,
Maize Pathology Laboratory and B. sorokiniana
isolate BS 53 collected from Malan, Himachal
Pradesh, India whereas maximum closeness (91%)
452 Indian Phytopathology [Vol. 61(4) : 2008]

Table 2. Primers used for determination of polymorphism in Bipolaris spp.

Primer Total band Polymorphic band Monomophic band Polymorphic percentage

OPA 1 16 16 - 100
OPA 5 11 11 - 100
OPA 7 21 21 - 100
OPA 8 10 8 2 80
OPA 9 14 14 - 100
OPA 10 19 19 - 100
OPA 11 10 10 - 100
OPA 12 19 19 - 100
OPA 13 14 14 - 100
OPA 18 10 10 - 100
OPA 19 15 15 - 100
OPA 20 17 16 1 94.11
OPB 08 14 14 - 100
OPB 09 17 17 - 100
OPB 10 14 14 - 100
OPB 11 15 15 - 100
OPB 14 15 15 - 100
OPB 16 15 15 - 100
OPB 17 15 14 1 93.33
OPB 18 14 14 - 100
OPB 19 13 13 - 100
OPB 20 13 12 1 92.30
OPM 01 9 9 - 100
OPM 02 16 16 - 100
OPM 04 13 13 - 100
OPM 08 1 1 - 100
OPM 10 17 17 - 100
OPM 11 3 3 - 100
OPM 12 20 20 - 100
OPM 13 14 14 - 100
OPM 14 12 12 - 100
OPM 15 17 17 - 100
OPM 17 15 15 - 100
OPN 04 16 16 - 100
OPN 05 16 16 - 100
OPN 09 13 13 - 100
OPN 10 9 9 - 100
OPN 12 15 15 - 100
OPN 13 12 12 - 100
OPN18 15 15 - 100

Total no. of bands observed: 554; Polymorphic bands: 549; Percentage polymorphism: 99.09

was observed between B. sorokiniana isolates BS The dendrogram obtained after cluster analysis
7 and BS 18 collected from Karnal and Jaipur also exhibited variable degree of relationships among
respectively (both from North western Plain Zone of the isolates as well as species (Fig. 2). There were
India. two major clusters; one including all isolates from
[Vol. 61(4) : 2008] Indian Phytopathology 453

Fig 1. Agarose gel electrophoresis of PCR amplification products obtained with primers OPN12 (a); and OPM12
(b) in Bipolaris spp . Lane 1 is 1Kb ladder marker (MBI, Fermentas) and last lane is 100 bp marker (Genei, India)

Fig 2. Dendrogram showing relationship between 12 isolates of B. sorokiniana, two isolates of B. Siecifera, two
isolates of B. tetramera and one isolate of B. maydis based on RAPD analysis
454 Indian Phytopathology
B. sorokiniana; and other having isolates of B.
specifera, B. tetramera and B. maydis. In the first
cluster isolate BS 53 which was from Himachal
Pradesh was totally different from other isolates
and was placed separately. In the second cluster,
an isolate of Bipolaris maydis (BM) was clearly
distinguishable from isolates of Bipolaris specifera
and Bipolaris tetramera, which were also
distinguishable from each other as they occupied
their respective sub- groups.
The random amplifed DNA (RAPD) is a PCR
based technique which reveals polymorphism within
completely unknown samples without the need of
probe hybridization or DNA sequencing. It can be
used also for characterization of fungal isolates by
constructing specific fingerprint or for the genetic
stability testing of an individual isolate. RAPDs
have also been used in race, pathotype and
population studies (Crowhurst et al., 1991; Manulis
et al., 1994). This feature combined with their easy
identification makes it a valid type of marker
potentially useful in many areas of genetic research
such as gene mapping and strain differentiation of
Trichoderma strains (Zimand et al., 1994), Mycogone
perniciosa, T. harzianum and Verticillium fugicola
var. fugicola isolates (Muthumeenakshi et al., 1995),
Pyricularia grisea (Sharma et al., 2000).
In our present studies, molecular
characterization of Bipolaris spp. undertaken by
PCR using 40 random primers has revealed genetic
variability. Among all isolates, genetic variation was
maximum with least similarity of 42 per cent,
observed between the isolates BS 53 from Malan
(Himachal Pradesh) and BM of B. maydis from
IARI. Isolate BM (Bipolaris maydis) showed 55%
similarity with other isolates of B. sorokiniana,
whereas, this isolate showed around 65% similarity
with isolates of B. specifera and B. tetramera. Our
findings have clearly shown that PCR-RAPD can
genetically differentiate the isolates at interspecific
level. According to the location where the isolates
have been collected, no clustering could be seen.
It could be noted that there are two major clusters
formed, one with 12 isolates of B. sorokiniana and
the other major cluster with two isolates of B.
specifera (from IARI and Karnal) and two isolates of
B. tetramera (from Behrampur and Delhi) and one
isolate of B. maydis (from IARI). The first major
cluster was further subdivided into two subclusters,
one with all the isolates of B. sorokiniana except
[Vol. 61(4) : 2008]
BS-53 fron Himachal Pradesh, which formed a
separate sub-group. The second major cluster had
two sub-groups, one with BS 4922 from IARI, BS
5137 from Karnal and isolate Bt 3466 from
Behrampur and Bt 4805 from New Delhi and other
sub-group having isolate BM from IARI which formed
a separate group in this subcluster.
Isolates of B. sorokiniana from Brazil have
been analysed by RAPD technique to determine
the amount of intraspecific genetic variability and to
study host pathogen interactions (Oliveira et al.,
2002; Mehta, 2001). RAPD analysis showed
distinctive patterns for some isolates of Drechslera.
No clear difference between the black spot and the
leaf blotch isolates was observed either by the
molecular or by the pathogenicity analysis (Mehta,
2001). Our results are in agreement with those of
Oliveira et al., (2002). According to them, RAPD
profiles did not reveal polymorphism that directly
correlated climatic factors with geographic source
of the isolates of B. sorokiniana, and B. oryzae. At
this level, three groups were characterized: the first
included all six B. sorokiniana isolated from wheat,
the second B. oryzae isolated from rice, and the
third B. maydis and Exserohilum turcicum isolated
from maize. RAPD profiles of B. sorokiniana strains
showed an average of 89.2% similarity while RAPD
profiles of B. maydis species had 76% similarity to
E. turcicum. Geographic distribution analysis of B.
oryzae samples showed that both the clusters
contained isolates from distinct regions, which
suggests that there was no correlation between
geographic origin and RAPD groupings. The RAPD
profiles showed a high level of genetic variability
among the B. sorokiniana isolates. Variability was
observed among isolates originated from the same
cultivar, as well as between different host cultivars
(Oliveira et al., 2002). According to Burdon and Silk
(1997), plant pathogenic fungi most commonly rely
on mutation and recombination as the main source
of genetically based variation. Within a species,
gene flow between population supplement these
processes as propagules spread from one
epidemiological area to another. Gene flow, along
with other evolutionary forces can result in the
spread of single gene (or DNA sequences), and
even in the establishment of whole populations in
different regions (McDermott and McDonald, 1993).
Similarly, in our present findings on PCR-RAPD
based molecular studies it is observed that all the
isolates of B. sorokiniana irrespective of their
[Vol. 61(4) : 2008]
geographic origin formed one cluster except isolate
BS 53 from Himachal Pradesh which differed from
others. This indicates that among the isolates of B.
sorokiniana, there exists much variability which
needs to be characterized further.
ACKNOWLEDGEMENTS
The authors are thankful to Department of
Biotechnology, Government of India for financial
support in the form of project funding (BT/PR4436/
AGR/02/216/2003) and Head, Division of Plant
Pathology, IARI, New Delhi-110012 for providing
facilities. The senior author is thankful to Iran
Government for giving fellowship to take up PhD
research work at IARI, New Delhi.
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Received for publication August 27, 2007