RESEARCH ARTICLE
Legume Research- An International Journal, Volume 46 Issue 9: 1247-1252 (September 2023)
New Report of Cercospora canescens Isolates from Coastal
Regions of Odisha, India causing Cercospora Leaf Spot (CLS)
Disease in Mung Bean (Vigna radiata L.)
Sushree Suparna Mahapatra1, Surjya Kanta Beura1,
Dhaneswar Swain2, Kundan R. Jadhao2, Gyana Ranjan Rout2
10.18805/LR-4805
ABSTRACT
Background: Cercospora leaf spot (CLS) is one of the major diseases causing a major loss in production of mung bean of up to 60%.
Although it is cultivated all round the world, its major contribution is attributed to the Asian continent especially the countries of Indian
subcontinents which accounts for about 90% of the world’s mungbean production. Due to its high nutritional content, mung bean is a
major contributor to the Nation’s economy. The present study was to isolate and characterize the new isolates of Cercospora
canescens collected from coastal belts of Odisha, India.
Methods: Surveys were conducted in the mung bean growing fields of Bhubaneswar (20.26N, 85.8E) and Berhampur (19.36N,
84.77E), coastal regions of Odisha during 2018-20. New Cercospora canescens isolates were collected and characterised through
morphological, biochemical and molecular basis.
Result: New Cercospora isolates were characterized on the basis of morphological, biochemical and molecular analysis to understand
the gravity of the disease. Pathogenicity tests were conducted by artificial inoculation to identify the resistant and susceptible mung
bean genotypes. On the basis of molecular analysis (internal transcribed spacer (ITS) region), there was a difference between the
two isolates and forming different clades. These isolates were sequenced having the NCBI Acc. No. MZ475049 and MZ475050. Both
isolates are different from each other because of transitional substitution of thymine and cytosine. The findings will be a complementary
contribution for conservation and better management strategies of CLS disease in mung bean.
Key words: Cercospora canescens, Fungal isolates, Legume, Mung bean, Phylogenetic analysis.
INTRODUCTION
Mung bean or green gram (Vigna radiata L.) is an important
pulse crop belonging to the family Fabaceae. Although it is
cultivated all round the world, its major contribution is
attributed to the Asian continent especially the countries of
Indian subcontinents which accounts for about 90% of the
world’s mung bean production. India, being the largest
producer of mung beans in the world, contributes around
54% of the global mung beanproduction (Singh and Singh,
2011) and has provided the Indian farmers a highly profitable
source of income (Pal et al., 2021). But infestation of the
crop with various diseases and pests poses serious threat
to its cultivation and production (Raje and Rao, 2002).
Cercospora leaf spot (CLS) caused by the fungus
Cercospora canescens Ellis andMartin is one of the
designated diseases causing a sizable loss in production
upto 60% (Bharti et al., 2017). CLS disease is marked by
typical characteristic symptoms initially on leaves producing
irregular to angular lesions with reddish brown margin and
yellow halo around the spots with greyish centre. The
disease progresses better at 20-26C and at a relative
humidity of 90-100%, which is required for the optimum
growth and sporulation of the pathogen (Kumar et al., 2011).
The fungus Cercospora canescens can be identified and
characterized based on the morphological structure,
molecular characterisation of the genome, nature and
Volume 46 Issue 9 (September 2023)
1
Department of Plant Pathology, College of Agriculture, Odisha
University of Agriculture and Technology, Bhubaneswar-751 003,
Odisha, India.
2
Department of Agricultural Biotechnology, Odisha University of
Agriculture and Technology, Bhubaneswar-751 003, Odisha, India.
Corresponding Author: Gyana Ranjan Rout, Department of
Agricultural Biotechnology, Odisha University of Agriculture and
Technology, Bhubaneswar-751 003, Odisha, India.
Email: grrout@rediffmail.com
How to cite this article: Mahapatra, S.S, Beura, S.K, Swain, D.,
Jadhao, K.R. and Rout, G.R. (2023). New Report of Cercospora
canescens Isolates from Coastal Regions of Odisha, India causing
Cercospora Leaf Spot (CLS) Disease in Mung Bean (Vigna radiata
L.). Legume Research. 46(9): 1247-1252. doi: 10.18805/LR-4805.
Submitted: 01-10-2021
Accepted: 01-02-2022
Online: 16-04-2022
intensity of the disease symptom caused on the host plant
(Chand et al., 2000). Numerous Cercospora species till date
have been identified based on the studies of the partial
sequences of the fungi employing the ITS region because
of its higher divergence and faster evolution than other
coding regions of the rDNA genes (Crous et al., 2013). The
analysis of phylogeny is majorly based on the partial
sequence of the ITS region of the nuclear ribosomal DNA
1247
New Report of Cercospora canescens Isolates from Coastal Regions of Odisha, India causing Cercospora Leaf Spot (CLS)......
which is the most commonly sequenced regions for
taxonomy and systematic affiliation of the fungi (Feliner and
Rossello, 2007). It is regarded as the barcode for
identification and characterization of fungi (Schoch et al.,
2012). The present study is to identify and characterize the
isolates of Cercospora canescens collected from the coastal
regions of Odisha, India for CLS disease in mung bean.
MATERIALS AND METHODS
Sample collection and isolation of fungal isolates
Surveys were conducted in the mung bean growing fields
of Bhubaneswar (20.26N, 85.81E) and Berhampur
(19.36N, 84.77E), coastal regions of Odisha to identify
plants infected with Cercospora canescens by visualising
designated symptoms of CLS during 2018-2020. Samples
collected from Bhubaneswar and Berhampur were denoted
as BBS1 and BAM1 respectively. The infected samples were
surface sterilized with 0.8% (v/v) sodium hypochlorite for 2 min.
Lesions with dense and ample spores were selected after
evaluation under microscope. The collected spore was
spread over petri platescontaining 2% water-agar and
incubated at 25±1C for 6 hrs. The germinated spores were
identified under a microscope (100). The pure culture of
the isolated fungus was maintained on PDA at 25±1C and
sub cultured as and when required. The cultures were
visualised for structure, colour, texture, dimensions of the
fungal colonies at different time intervals.
Pathogenicity test
The fungal mass of the pure culture was multiplied by mass
production on sterilised parboiled sorghum grain for spore
production and multiplication (Chand et al., 2013). The pure
cultured fungal culture was transferred to the sorghum
grains aseptically incubated at 25±1C. Ten mung bean
genotypes (5 highly resistant and 5 highly susceptible)
along with Kopergaon as the susceptible check for CLS
were artificially inoculated with the spore suspension (10-4
spore ml-1) prepared by soaking the colonised sorghum
grains in distilled water suspension was uniformly sprayed
at 50% flowering stage or 35 days old mung bean plants
during evening hours. Light irrig ation was given
immediately after inoculation to ensure proper humidity and
further additional irrigations were applied as and when
needed to maintain the moisture level.
DNA Isolation
The isolated fungal pure culture was sub-cultured in potato
dextrose broth (PDB) mediumand incubated for 10-12 days
at 25±1C after shaking.The mycelia mats were harvested
from the broth by filtration using Whatman No.1 filter paper
and the fungal mats were well dried using sterile blotting
paper. The freshly harvested mycelium (1 g) was ground to
fine powder using liquid nitrogen in pre-chilled mortar and
pestle. The fungal DNA was isolated using cetrimide
tetradecyl trimethyl ammonium bromide (CTAB) method of
DNA extraction and purification (Murray and Thompson, 1980).
1248
PCR amplification
ITS 1 and ITS 4 primers were used for the amplification of
the ITS region of the ribosomal nucleic region including the 5.8
rDNA of the fungus. The PCR was carried in 25 l reaction
volume using two primers, ITS1- (5-TCCGTAGGTGAACCTGCG
G-3) and ITS4 (5- TCCTCCGCTTATTGATATGC-3) 1ll each,
1ll dNTP, 2.5 llPCR buffer, 1ll of DNA, 0.5ll PFE
polymerase. The PCR parameters were programmed in a
Thermocycler (BIORAD, USA) as follows: initial denaturation
at 95C for 3min, primer annealing at 60C for 40 secs, chain
extension and final extension at 72C for 1 min and 10 min
respectively. The PCR amplification products were checked
and characterized by gel electrophoresis in a 1TAE agarose
gel at 70V for 1 hr and the amplicons were visualized under
UV with Ethidium bromide as staining agent. 100 bp ladder
was used as reference standard molecular weight marker.
Elution of DNA fragment and Sequencing
The PCR amplified DNA product was purified using
PROMEGA GEL kit and PCR purification kit. The desired
DNA fragments obtained through electrophoresis were
excised from the gel using a sterilized scalpel by visualising
under UV light. The agarose slice containing DNA was
transferred to microcentrifuge and incubated after adding
membrane binding solution at 70C to melt the gel. The DNA
was recovered from the gel slice and purified through
minicolumn assembly. Further, eluted fragment was kept in
-20C for sequencing. Sequencing was performed by a
commercial service provider using Sanger sequencing
method in 96 capillary array-based ABI 3730l DNA analyzer.
The sequence assembly and alignment were performed
using Clustal tool. The sequence of the fungal isolates
was deposited in NCBI gene bank.
Phylogenetic analysis
Phylogeny study was performed by establishing the
relationship between the isolated fungal strains with genus
Cercospora and Cercospora related species based on ITS
nucleotide sequences using neighbour joining method
(Saitou and Nei, 1987) in MEGA 7.0 (Kumar et al., 2016).
RESULTS AND DISCUSSION
Morphological characterization
The Cercospora isolates were grown in PDA medium in petri
plates for 30 days to evaluate the growth rate. The growth
of the fungal colonies was very slow but the radial growth
took place between 15 to 21 days of re-culture. The average
radial growth of the BBS1 isolate was recorded as 4.2-5.5 cm
diameter whereas, BAM1 about 2.8-4.3 cm diameter. The
BBS1 fungal colony was marked by white fluffy cottony
appearance with a radish brown outer border. Whereas, the
BAM1 had a less fluffy growth with pale cream colour and
was not marked with red or brown border but had a feathery
growth at the margin (Fig 1a,b). Both the isolates had a
very slow growth on the artificial medium but did not produce
spore on the PDA medium. But the isolates sporulated on
Legume Research- An International Journal
New Report of Cercospora canescens Isolates from Coastal Regions of Odisha, India causing Cercospora Leaf Spot (CLS)......
sorghum grains and the spores were characterized with
t h e c o n id ia a n d b ran c h e d c o n id io p h o re , pa le
olivaceous, straight to slightly bent and multi-septate.
Cercospora canescens with slight stroma often with
d en se fascic le b earing c o nid io p ho res w h ic h w ere
generally branched, straight, geniculate, bearing few
to many septa, pale to medium dark brownin colouration
(Videira et al., 2017).
Pathogenicity test
Leaf spots formation initiated after 10 days after inoculation.
The findings showed that the inoculated plants showed
Fig 1: Fifteen-days-old culture of Cercopora canescens BBSI isolate (a), BAMI isolate (b) (c) Gel profile of internal transcribes spacer
(ITS) region of BBSI and BAMI isolates of Cercospora canescens. M represents the reference molecular marker.
Fig 2A: Phylogenetic tree of germs Cercospora based on ITS nucleotide sequences constructed using neighbor joining method in
MEGA 7.0. The Cercospora canescens isolate of the present study marked in red color in the phylogenetic tree. Black bold font on
tree node indicates bootstrap values in per cent.
Volume 46 Issue 9 (September 2023)
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New Report of Cercospora canescens Isolates from Coastal Regions of Odisha, India causing Cercospora Leaf Spot (CLS)......
typical lesions on the leaves which were irregularly circular
with dark reddish-brown outer margin with palecentre. The
smaller spots further coalesced together to form irregular
lesions of yellow chlorotic halo around the brown margin
and on maturity formed a greyish centre with spores. The
susceptible plants showed higher incidence of the disease
with a greater number of spots with greyish centres on the
leaves which further led to the infection of the pods.
Molecular characterization
Molecular analysis showed that the amplicons separated
on agarose gel through electrophoresis showed a similar
The Cercospora canescens of the present study is marked in red color in the phylogenetic tree. Black bold
font on tree node indicates bootstrap values in pre cent.
Fig 2B: Neighbour-Joining tree representing the phylogenetic relationship of Cercospora germs and its closely related
genera derived from ITS region in MEGA 7.0.
A: Rrepresents group-I species, B: Represents group-II species, C: Represent group III species, D: Represents
group-IV species. Black arrow indicates the variation in helices structures of these species.
Fig 3: Secondary structure model of Cercospora genus derived from ITS region with variable sites that distinguish related species in
four different groups in pgylogenetic analysis.
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Legume Research- An International Journal
New Report of Cercospora canescens Isolates from Coastal Regions of Odisha, India causing Cercospora Leaf Spot (CLS)......
banding pattern under UV trans-illumination (Fig 1c). The
sequence of the fungal isolates BBS1 and BAM1 were
deposited in NCBI gene bank and assigned the accession
numbers viz. MZ475049 and MZ475050 respectively. The
evolutionary history was inferred using the phylogenetic
comparison was based on the ITS sequences of the two
isolates of Cercospora in the present study and the
sequences of other related species available in the NCBI
GenBank database to infer the genetic relationships among
the isolates and establish the level of genetic diversity (Fig 2A).
The phylogenetic analyses were performed based on
alignments and comparisons of the sequences using
ClustalX integrated into MEGA 7 software. The tree is drawn
to scale, with branch lengths in the same units as those of
the evolutionary distances used to infer the phylogenetic
tree (Videira et al., 2017). The evolutionary distances were
computed using the number of differences method (Nei and
Kumar, 2000). The percentage of replicate trees in which
the associated taxa clustered together in the bootstrap test
(1000 replicates) to assess the clade stability are shown
next to the branches (Felsenstein, 1985). The dendrogram
based on the neighbour joining method in MEGA 7.0
establishing the relationships between different species of
Cercospora depicts that the species can be delineated into
four broad groups namely Group-I, II, III and IV of related
species. Among the 4 groups, most of the Cercospora
canescens isolates are constituted under the group II and
IV.The isolate BBS1 (Acc. No. MZ475049) falls under group IV
and BAM1 (Acc.No. MZ475050) under group II (Fig 2B). Based
on the phylogenetic tree representing the relationships
among different Cercospora species and other closely
related genera, it is implied that genus Cercospora shares
common ancestors with other genera like Septoria,
Pseudocercospora, Exutisphaerella, Neodeightoniella,
Ramulariaetc (Fig 3). The genera Cercospora and Septoria
were found to share a and more immediate ancestor than
between Cercospora and Pseudocercospora (To-Anun et al.,
2011). These genera are clustered into 5 different groups
based on their relatedness and Cercospora species clearly
fall under group IV and V (Fig 3). It is evident from the
secondary structure model of Cercospora genus derived
from ITS region with variable sites and parsimony informative
Table 1: Comparison of the parsimony informative sites of genusCercospora species.Red colour indicate the new isolates i.e. BBS1
(MZ475049) and BAM1 (MZ475050).
Acc. no.
Species name
T
A
C
Acc. no.
Species name
T
A
C
KT193707
C. cf. zinniae
.
.
T
MK752899
C. nicotianae
C
.
.
KT193699
C. musigena
.
.
T
KU870468
C. apiicola
C
.
.
KT193690
C. cf. malloti
.
.
T
MG372319
C. citrullina
C
.
.
KT193667
C. cypericola
.
.
T
MZ475049
C. canescens
C
.
.
KM979942
C. guatemalensis
.
.
T
KJ696542
C. chrysanthemi
C
.
.
KC776165
C. capsicigena
.
.
T
MK336506
C. kikuchii
C
.
.
MH777047
C. kikuchii
.
.
T
MG966193
C. cf. flagellaris
C
.
.
KY351634
C. cf. sigesbeckiae
.
.
T
MK442573
C. gomphrenigena
C
.
.
MW077081
C. lunata
.
.
T
MK039698
C. dichondrae
C
.
.
MN706610
C. manihobae
.
.
T
MH424448
C. beticola
C
.
.
MH129519
C. malayensis
C
.
.
KY549098
C. asparagi
C
.
.
Group-I
MZ475050
C. canescens
T
MN795679
C. canescens
T
.
.
KX287277
C. dubia
C
.
.
NR 147292
C. cyperina
T
.
.
MW692173
C. sojina
C
.
.
KM979960
C. zebrina
T
.
.
MN706608
C. manihobae
C
.
.
JQ995781
C. guatemalensis
T
.
.
MN744316
C. cf. chenopodii
C
.
.
HM631725
C. kikuchii
T
.
.
MK027103
C. sesami
C
.
.
MK027098
C. apii
T
.
.
MK027097
C. apii
C
.
.
MH854904
C. kikuchii
T
.
.
MK027095
C. physalidis
C
.
.
KT193700
C. cf. nicotianae
C
.
.
KT193672
C. lactucae-sativae
C
.
.
KT193665
C. cf. citrulina
C
.
.
Group-II
MW412745
C. arctii-ambrosiae
C
.
T
Group-III
KT193770
C. armoraciae
C
.
.
KT193661
C. capsici
C
.
.
KM979958
C. piaropi
C
.
.
KT193651
C. cf. apii
C
.
.
KC776170
C. codiaei
C
.
.
NR 147293
C. glycinicola
C
.
.
KC776158
C. cocciniae
C
.
.
NR 111827
C. chrysanthemoides
C
.
.
KC776162
C. zinniicola
C
.
.
GU214657
C. zebrina
C
.
.
GQ884184
C. rodmanii
C
.
.
MW412756
C. guatemalensis
C
.
.
Group-IV
Volume 46 Issue 9 (September 2023)
Group-IV
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New Report of Cercospora canescens Isolates from Coastal Regions of Odisha, India causing Cercospora Leaf Spot (CLS)......
sites of the genus (Table 1) that the related species can be
distinguished into four different groups in phylogenetic
analysis, which implies that species differ from one another
based on transitional substitution of thiamine (T) and cytosine
(C) (Fig 3). New Cercospora isolates, BBS1(MZ475049) falls
under group IV and BAM1 (MZ475050) falls under group II.
CONCLUSION
The present phylogenetic study showed that clade Cercospora
appears as a sister group to Septoria establishing a closer
relationship. Cercospora and Septoria share similar
morphological characteristics in the pattern of conidiophores
and structure of conidia. But they differ from each other with
respect to production of pycnidial conidiomata by Septoria only
(To-Anun et al., 2011). Although the two isolates BBS1 and
BAM1 showed similar banding pattern but they differ from each
other with regard to the position of C and T which make them
fall into two different groups. This report can be brought in
accordance with the fact that single morphological species do
not necessarily reflect a single phylogenetic unit (Taylor et al.,
2000). Cercospora and related genera like Septoria,
Pseudocercospora, Exutisphaerella, Neodeightoniella,
Ramularia share a common teleomorph genus
Mycosphaerella. The Mycosphaerella is considered to be a
complex constituted of a large number of species of diverse
group of plant pathogenic pathogens. It was previously thought
to be monophyletic on the basis of the phylogenetic analysis
of the ITS region (Goodwin et al., 2002). Later on, with advances
in the molecular data, it concluded that the genus to be polyand paraphyletic and closeto the taxa-bearing genera
Ramularia anamorphs. This investigation will help to identify
the occurrence and distribution of new Cercospora canescens
isolates for CLS disease in mung bean.
ACKNOWLEDGEMENT
The authors wish to acknowledge to the Departmentof
Biotechnology, Govt. of India for providing the financial grant
under the R and D project (BT/IN/UK/PORI/04/GRR/2018-19).
Sushree Suparna Mahapatra is thankful to Department of
Science and Technology, Government of India for financial
support in form of INSPIRE fellowship (IF180456).
Conflict of interest: None.
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