Biocatalysis and Agricultural Biotechnology 2 (2013) 285–287

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Biocatalysis and Agricultural Biotechnology

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Identification and phylogenetic correlation among

Colletotrichum gloeosporioides pathogen of anthracnose for mango
Madhu Kamle a, Pradeep Kumar a,b, Vijai K. Gupta c,n, Ajay K. Tiwari d, Ashok K. Misra a,
Brijesh K. Pandey a
a
Central Institute for Subtropical Horticulture, Lucknow 227107, UP, India
b
Department of Biotechnology Engineering, Ben Gurion University of The Negev, Be’er Sheva 84105, Israel
c
Department of Biochemistry, National University of Ireland Galway, Ireland
d
UP Council of Sugarcane Research, Shahjhanpur 242001, UP, India

art ic l e i nf o a b s t r a c t

Article history: Anthracnose is recognized as the most important disease of field and post-harvest disease of mango
Received 26 March 2013 worldwide. It has affected the fruit production in all countries where mangoes are grown, particularly
Received in revised form where high humidity prevails during the cropping session. Severe lesions along with black spots on
6 April 2013
leaves, inflorescence and fruits on mango (Magnifera indica L.) were observed during routine survey at
Accepted 9 April 2013
Available online 29 April 2013
several sites with mango trees in Eastern Uttar Pradesh indicating the occurrence of anthracnose disease.
The pathogen was isolated from several infected samples and matched characteristics of Colletotrichum
Keywords: gloeosporioides Penz. and Sacc. by microscopic, cultural studies and on the basis of spore formation. Pure
Mangifera indica L. cultures of C. gloeosporioides were maintained and further identification of this pathogen was conducted
Anthracnose
using the internal transcribed spacer region of the known C. gloeosporioides rDNA region 5.8S. Sequence
C. gloeosporioides
similarity of the collected isolates was 100% with some C. gloeosporioides isolates and phylogenetic
rDNA Phylogenetic
analysis also showed the close relationship. Although there are many prior reports on anthracnose
disease on mango in India, confirmation of this disease on mango from isolates in India is confirmed by
sequence analysis for the first time.
& 2013 Elsevier Ltd. All rights reserved.

1. Introduction which enlarged and coalesced frequently to destroy leaf edges or

entire inflorescences (Jeffries et al., 1990). Lesions often coalesce to
Mango (Mangifera indica L.) has been considered as one of the form large necrotic areas frequently along the leaf margins severely
most popular fruit crops in several tropical and subtropical countries. affecting the growth of the leaves which often become dry and fall
It is considered as the “King of Fruits” and India is the leading country out giving the older leaves a ‘shot hole’ appearance. Under favorable
in mango production in the world and it shares around 56 per cent of conditions conidia are dispersed that invade young twigs causing
total global production (Kumar et al., 2011). Being a cash crop for twig dieback in some cases (Ploetz, 1999). Relative humidity above 95
several rural people, currently mango production has been affected per cent and temperature ranging from 20 to 30 1C for 12 h are
by different biotic and abiotic factors. Among the biotic factors, essential for infection and development of C. gloeosporioides on
diseases take a heavy toll on production due to fungal pathogens, of mango fruit. Infection progresses faster in wounded tissues and in
which, Colletotrichum gloeosporioides, a pathogen of mango anthrac- ripe fruits (Prakash, 1996; Pandey et al., 2012a). Association of C.
nose, is the most important biological constraint which restricts gloeosporioides to the infection has been proved through histopatho-
mango production in the Southeast Asia (Ploetz, 1999; Charles et al., gical analysis by using in vitro artificial inoculation of pathogen to the
2012). The ubiquitous fungus C. gloeosporioides Penz. and Sacc. is the leaves of seedling plants (Pandey et al., 2012b). The variation of C.
anamorph stage (asexual stage of the pathogenic fungus). C. gloeos- gloeosporioides was confirmed in several experiments through mole-
poriodes is responsible for many diseases in different crops and it is cular polymorphism generated by RAPD (Gupta et al., 2010). C.
also referred to as ‘anthracnose’ on tropical and subtropical fruits gloeosporioides was found to be highly pathogenic on the fruits of
including banana, avocado, papaya, coffee, passion fruit and others. mango and also affects mango production both in the pre- and post-
Pathogen causes the infections on stems, leaves and young inflor- harvest stages particularly when attempting to extend storage life
escences are manifested as sub-circular or angular black lesions resulting in huge economic losses of about 5–20% in the form of
damage on the stems, leaves, fruit decay and damage (Dodd et al.,
1997). However, the pathogen C. gloeosporioides causes the same
n
Corresponding author. Tel.: +353 86 200 1820. disease in other fruit crops like grape (Leu and Chang, 1985), avocado
E-mail address: vijaifzd@gmail.com (V.K. Gupta). (Coates et al., 1993), strawberry (Xiao et al., 2004) and olive

1878-8181/$ - see front matter & 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bcab.2013.04.001
286 M. Kamle et al. / Biocatalysis and Agricultural Biotechnology 2 (2013) 285–287

(Talhinhas et al., 2011). This study identified the fungus responsible reference id no.—HM102505 for C. gloeosporioides from mango
for fruit anthracnose in mango and its sequence homology with the anthracnose.
other countries' isolates of anthracnose based phylogenetic analysis In agarose gel 560 bp amplicons were observed in all the eleven
by using multiple sequence alignments tools. isolates of C. gloeosporioides cultures from the leaves samples
During a routine survey of mango growing areas of Eastern while no amplicon was visualized in control (Fig. 2). The amplicon
Uttar Pradesh symptoms of anthracnose disease were observed. (560 bp) obtained from the symptomatic samples, from one of the
The incidence of the disease within crops was significant (30–50%) isolate bands showing the highest pathogenicity, was gel purified
and symptoms consisted of lesion and black spots on leaf, and complete sequencing using ITS1 and ITS4 primers from both
inflorescence, apical tip and fruits (Fig. 1). The characteristic the direction for accuracy is performed.
symptoms of the diseased plants indicate the possibility of BLAST (Basic Local Alignment Search Tool) analysis was performed
anthracnose disease. Microscopic study was performed by the for HM102505, which shows 100% identity with the C. gloeosporioides
isolated pure pathogen culture and on the basis of the spore affecting other subtropical fruits. The sequence HM102505 is highly
identification it was confirmed that the isolated pathogen from identical to other C. gloeosporioides which were reported from USA,
mango leaf, fruit or stem is C. gloeosporioides (Mullar, 1940). Later, China and Thailand to cause anthracnose disease on different fruits as
genomic DNA from the isolated plant pathogen is isolated using hosts. During sequence similarity of HM102505 for C. gloeosporioides
the Fast DNA Extraction Kit (MP Bio, Inc.) following the manufac- isolates from mango in India through Genomatix dialing programme
turer's instructions. Genomic DNA is visualized under UV light on with the selected identical isolate from the GenBank, 100% similarity
0.8% (w/v) agarose gel stained with ethidium bromide and stored was recorded with AY266390 isolate of C. gloeosporioides reported
at –20 1C. The ITS rDNA region includes ITS1, 5.8S and ITS4 primers from Thailand on Herbaceous plants. Lower similarities of selected
which were used to amplify 11 isolates of C. gloeosporioides sequence were found with HM146134, DQ062671, HM53032,
selected from different places using the universal primers ITS1/ HM575231, HM575260, AY266379, AY266373, EF423519 and AY2
ITS4 (White et al., 1990). Each PCR reaction contained 1  PCR 66389 i.e. 89%. Phylogenetic analysis of nucleotide sequence of
buffer, 2.5 mM MgCl2, 200 mM of each dNTP, 0.2 mM of each primer, HM102505 for C. gloeosporioides isolate with selected isolates from
0.7U of Taq DNA polymerase (Biochem) and 1 ml (30 ng) of Genbank which were all at least 90% identical, was performed using
template DNA. The PCR reaction mix was adjusted to a final the molecular evolutionary genetics analysis (MEGA) version 4.0 soft-
volume of 25 ml with nuclease free water. PCR amplifications were ware (Tamura et al., 2007). The analysis revealed 100% homology to
performed on a Thermal Cycler (Eppendorf, India Limited). The the C. gloeosporioides mango isolate (AY266390) from Thailand and
program consisted of an initial step of 1 min at 94 1C for 1 min,
followed by 30 cycles of 60 s at 94 1C, 2 min at 58 1C, and 60 s at
72 1C; and a final extension step of 5 min at 72 1C. PCR products
were analyzed in 1.5% agarose gel, stained with ethidium bromide
and visualized under UV light. 100-bp DNA ladder was used as
a molecular weight marker (Biochem). The sequencing reactions
were performed in an ABI prism sequencer (ABI Advanced
Biotechnological Institute, Perkin-Elmer Corporation, Foster City,
USA). 602 bp consensus sequences without ambiguities were
obtained from the sequence of isolates of C. gloeosporioides from Fig. 2. Amplification of PCR products in maintained cultures from mango leaves.
mango which are deposited at NCBI GenBank under Accession (M—ladder, 01–11: C. gloeosporioides and lane N: Negative control).

Fig. 1. (a) Black spot on leaf of mango, (b) on apical tip of mango, (c) symptoms of anthracnose on mango fruits, (d) pure culture of C. gloeosporioides and (e) spores of
C. gloeosporioides.
 M. Kamle et al. / Biocatalysis and Agricultural Biotechnology 2 (2013) 285–287 287

64 HM537045_Fungal endophyt _China

EU326190_C.g._China
HM537025_Fungal endophyt _China
HM537032_Fungal endophyt _China
HM537077_Fungal endophyt _China
4
JF826502_C.g._China
HM575266_C.g._China
41 HM537042_Fungal endophyt _China
AY266378_C.g._Thailand
48 AY266373_C.g._Thailand
AY266379_C.g._Thailand
AY266391_C.g._Thailand
59 AY266392_C.g._Thailand
HM575260_C.g._China
46
HM575231_C.g._China
52
HM575222_C.g._China
FJ823432_C.g._China

29 60 DQ062671_Glomerella_USA
HM146134_C.g._China
25 AY266389_C.g._Thailand
75 EF423519_Glomerella_USA
HM102505 _Present study isolate
100 AY266390 _C. gloeosporioides

0.002

Fig. 3. Phylogenetic tree constructed by using Clustal W algorithm and MEGA 4.2 version with ITS region sequences retrieved randomly from the Genbank along with the
present study isolate.

high homology with the other reported isolates of C. gloeosporioides Colletotrichum gloeosporioides Penz. By random amplified polymorphic DNA
(Fig. 3). Thus, on the basis of the sequence similarity and phylogenetic analysis. Afr. J. Biotechnol. 9, 4009–4013.
Kumar, P., Misra, A.K., Modi, D.R., 2011. Current status of mango malformation in
analysis it was confirmed that mango anthracnose disease in India is India. Asian J. Plant Sci. 10, 01–23.
caused by C. gloeosporioides. From India, the existence of anthracnose Leu, L.S., Chang, C.W., 1985. Histological study of Colletotrichum gloeosporioides on
disease of mango has been confirmed by serological and RAPD grape fruit. Plant Prot. Bull. (Taiwan) 27, 11–18.
Mullar, H.R.A., 1940. Survey of the most important mango disease in Dutch East
analyses (Gupta et al., 2010) but to the best of our knowledge no Indies. Meded Alg. Proefstt. Linab. Bataivia 40, 9.
prior information is available at sequence level phylogenetic analysis Pandey, A., Yadava, L.P., Misra, R.K., Pandey, B.K., Muthukumar, M., Chauhan, U.K.,
of C. gloeosporioides from India. 2012a. Studies on the incident and pathogenesis of Colletotrichum
gloeosporioides penz. causes anthracnose of mango. Int. J. Sci. Nat. 3, 220–232.
Pandey, A., Pandey, B.K., Muthukumar, M., Yadava, L.P., Chauhan, U.K., 2012b.
Histopathological study of infection process of Colletotrichum gloeosporioides
Acknowledgments Penz and Sacc. on Mangifera indica L. Plant Pathol. J. 11, 18–24.
Ploetz, R., 1999. Anthracnose: the most important disease in much of the mango
producing world. News Lett. Plant Pathol. 3, 1–6.
The authors are thankful to Director, Central Institute for Prakash, O., 1996. Prescribed diseases of mango causes and control, Advances in
Subtropical Horticulture (ICAR), Rehmankhera, Lucknow, India, Diseases of Crops in India. Kalyani Publisher, Ludhiana191–256.
for providing necessary research facilities and fund during the Talhinhas, P., Mota-Capitao, C., Martins, S., Ramos, A.P., Neves-Martins, J., Guerra-
Guimara~es, L., Va´rzea, V., Silva, M.C., Sreenivasaprasad, S., Oliveira, H., 2011.
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Colletotrichum acutatum and C. gloeosporioides in Portugal. Plant Pathol. 60,
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