Indian Phytopathology

https://doi.org/10.1007/s42360-019-00178-x

RESEARCH ARTICLE

Characteristics of Colletotrichum musae PHBN0002

and the susceptibility of popular banana cultivars to postharvest
anthracnose
Mark Angelo Balendres1   · JayVee Mendoza1 · Fe Dela Cueva1

Received: 28 June 2019 / Revised: 19 October 2019 / Accepted: 23 November 2019

© Indian Phytopathological Society 2019

Abstract
Anthracnose is one of the leading causes of quality losses in banana fruit. It is caused by several species in the genus Colle-
totrichum. In the Philippines, Colletotrichum musae has long been known as the only anthracnose pathogen of banana in
the Philippines, but molecular characterization is yet to be done. Further, little is known of cultivar susceptibility to anthrac-
nose in popular banana cultivars. Here we provide evidence for C. musae as a postharvest anthracnose pathogen of banana
cv. Cavendish, presents the first molecular characteristics of C. musae PHBN0002, and demonstrates the susceptibility of
three popular banana cultivars to postharvest anthracnose. The pathogen was isolated from anthracnose-infected fruit of cv.
Cavendish with morphological characteristics resembling to C. musae. The ITS-rDNA, GAPDH and ACT sequences of the
fungus had high similarities to that of the authentic sequences of epitype specimen of C. musae. In controlled unwounded
detached-fruit assays, fruit of cv. Lakatan, Latundan and Saba developed anthracnose seven days post inoculation of C. musae.
The results strengthen the knowledge of C. musae as the anthracnose pathogen of banana in the Philippines and demonstrates
the ability of the fungus to cross-infect to popular banana cultivars. This study may lead to further characterization of the
genetic variability of C. musae from different banana growing regions in the country, which is one of the key elements in
developing effective and durable control measures.

Keywords  Internal transcribed spacer (ITS) · Actin gene (ACT) · Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) ·
Lakatan · Saba · Latundan

Introduction tons banana production from April to June 2018 (Philippines

Banana (Musa sp.) is one of the world’s most important
tropical fruits and the Philippines is among the top produc-
ing countries alongside Ecuador (FAOSTAT 2015). Caven-
dish is the most produced and exported cultivar. In 2015,
nearly 9.1 m tons of bananas were produced and about 50%
of this was Cavendish and the rest were cultivars for the local
market (Philippines Statistics Authority 2015). Currently,
Cavendish, Saba and Lakatan and other cultivars accounts
for 51.7%, 29% and 9%, respectively, of the 2.7 m metric
* Mark Angelo Balendres
mobalendres@up.edu.ph
1 et al. 2013) and French Antilles (Chillet et al. 2006). Most
Institute of Plant Breeding, College of Agriculture
and Food Science, University of the Philippines Los Baños,
4031 Laguna, Philippines
Statistics Authority 2018).
Anthracnose is among the most destructive diseases
of tropical fruit worldwide. Banana anthracnose is one of
the most important diseases that reduces the quality and
postharvest life of fruit (Ploetz 1998). Severe anthracnose
accelerates senescence and early fall of fruit (Ootani et al.
2017), thereby reducing the number and quality of market-
able fruit. In the export market, anthracnose can be a con-
straint due to latent infection. Banana anthracnose is caused
by several species of Colletotrichum. The most commonly
associated species is C. musae (Su et al. 2011; Sutton and
Waterston 1970) that has been reported in many banana
growing regions including Brazil (Vieira et al. 2017), India
(Thangavelu et al. 2004), Thailand (Udayanga et al. 2013),
Malaysia (Sakinah et al. 2014), Sri Lanka (Abayasekara
recently, a draft genome of C. musae has been described
(da Silva Junior et al. 2018). Other species associated with

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banana anthracnose are C. scovillei (Zhou et al. 2017), C.
siamense (Kumar et al. 2017), and C. gloeosporioides (Saki-
nah et al. 2013). In Brazil, in addition to C. musae and C.
siamense, multiple Colletotrichum spp. have been associ-
ated with the banana anthracnose. Molecular characteriza-
tion revealed these species as C. tropicale, C. theobromicola,
and C. chrysophilum (Vieira et al. 2017). In the Philippines,
C. musae has been associated with banana anthracnose
(Reinking 1918) and was identified based on morphological
characteristics. Nevertheless, during the last 100 years since
its first report, no information on the banana anthracnose
pathosystem in the country has become available (based on
Web of Science literature search). Because anthracnose is a
major banana production constraint, it is crucial to determine
the current etiology of banana anthracnose in the country.
Research on anthracnose etiology has increased in the last
10 years due, partly, to advances in molecular biology that
have now influenced fungal taxonomy studies. Using the
DNA sequences for molecular analysis, further discrimina-
tion of fungal isolates to different species, as in the case of
banana anthracnose in Brazil (Vieira et al. 2017) and other
related fungi (Cannon et al. 2012) and precise diagnosis of
the fungal pathogens were made possible. Molecular data,
in addition to traditional characterization methods, thus pro-
vided precise information on the current etiology of banana
anthracnose, which is important in proper disease control
deployment.
Several management measures have been proposed to
mitigate the effect of postharvest anthracnose on banana
fruit. These include, but not limited to, hot water and essen-
tial oil treatments (Bazie et al. 2014; Vilaplana et al. 2018)
and use of biological control agents (Peeran et al. 2014; Shu
et al. 2017). Durable control measures to avoid pathogen
infection from fruiting to pre-harvest are needed, which
could be achieved by developing banana with resistance to
anthracnose. However, to effectively develop resistant plants,
current knowledge of the etiology of banana anthracnose is
essential, so that plants are screened against the appropriate
pathogen.
Experiments were conducted to identify the Colletotri-
chum spp. associated with postharvest anthracnose of banana
cv. Cavendish, using combined morpho-cultural and molecu-
lar characterization data, and to investigate the susceptibility
of other popular banana cultivars to postharvest anthracnose
in controlled detached-fruit assays.
Materials and methods
Source of fungal isolate
Colletototrichum musae PHBN0002 was isolated from
banana cv. Cavendish fruit which was grown in one of the
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Indian Phytopathology
field sites of the University of the Philippines Los Baños,
Laguna, Philippines (14° 10′ 0.15″ N 121° 14′ 20.70″ E).
Ripe banana fruit was incubated in transparent plastic box
(Sunnyware, Philippines), laid with wet tissue paper to cre-
ate a humid condition, for 14 days. Anthracnose lesions on
fruit tissues were cut into 2 × 2 mm and sterilized in 10%
hypochlorite solution (v/v, Zonrox, GreenCross Philippines)
for 60 s, then washed three times with sterile distilled water
at 120 s each. Fruit tissue-cuttings were then blot-dried in
sterile tissue paper. Dried fruit tissue-cuttings were plated in
potato dextrose agar (PDA) medium (Himedia Laboratories
Pvt Ltd, India) and plates were incubated at 28 °C for 7 days.
The pure culture was obtained and maintained in the PDA
medium in room temperature until use.
Morpho‑cultural assay
In morpho-cultural characterization, fungal disc plug was
grown in PDA for 7 days at 28 °C with 14 h light (in 24 h
cycle). Colony characteristics (color and radial growth) were
assessed visually and spore characteristics (size, shape) were
assessed microscopically. Radial growth (mm) measurement
was performed on two trials, with each trial consisting of
5 replicate plates. For spore measurement, the length and
width (µm) of the conidia were measured from 3 replicate
plates, with 31 conidia measured per plate and thus, a total
of 93 technical replications.
Molecular assay and gene sequencing
In molecular characterization, fungal genomic DNA was
extracted following the method used by Balendres and Dela
Cueva (2018). The fungal genomic DNA was then used as
the template for the amplification of three important fungal
gene regions, namely; the internal transcribed spacer (ITS),
glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and
the partial actin (ACT) gene regions. These gene regions
were commonly used to discern species with the Colletotri-
chum gloeosporioides species complex and are particularly
useful to resolve species within the “musae” clade (Weir
et al. 2012). The fungal ITS, GAPDH and ACT gene regions
were amplified using the primer sets ITS4/ITS5 (White et al.
1990), GDF1/GDR1 (Guerber et al. 2003) and ACT-512F/
ACT-783R (Carbone and Kohn 1999), respectively. Each
primer set (0.2 mmM each) was added into a 15-µL PCR
reaction volume containing 1× buffer (Vivantis Technolo-
gies, Malaysia), 2.5 mM M ­ gCl2 (Vivantis Technologies,
Malaysia), 0.2 mM DNTPS (Invitrogen, USA) and 1 unit
of Taq polymerase (Invitrogen, USA) (Dela Cueva et al.
2018). PCR assay was performed in C1000 thermal cycler
(Bio-Rad Laboratories, USA) using the following cycling
conditions; 94 °C initial denaturation for 5 min, 30 cycles
of 94 °C denaturation for 45 s, 55 °C annealing for 45 s, 72
Indian Phytopathology
°C extension for 60 s, then a final extension of 72 °C for 7
min. The PCR products were then sent to Apical Scientific
Sdn. Bhd. (Malaysia) for DNA sequencing.
A consensus sequence of the forward and reverse
sequences of each gene region was assembled using the
MEGA7 software. The consensus sequence of each gene
region was then aligned with respective authentic sequences
(Cannon et al. 2012) of related Colletotrichum species within
the C. gloeosporioides species complex (Weir et al. 2012)
using CLUSTALW. Colletotrichum boninense was used as
an outgroup. The phylogenetic tree was inferred using the
Maximum Likelihood method based on the Tamura-Nei
model (Tamura and Nei 1993). The heuristic search was
obtained automatically by applying the Maximum Parsi-
mony method, and branch support of the trees was assessed
by bootstrapping with 1000 replicates (Felsenstein 1985).
Analyses were conducted in MEGA7 (Kumar et al. 2016).
Pathogenicity assay
In pathogenicity assay, to establish Koch’s postulate, rein-
fection of the isolated fungus and symptom expression of
the fruit were studied in banana cv. Cavendish fruit. Spore
suspension (10 µL) of 7-days old culture, containing 1­ 05
spores/mL water, was drop-inoculated on surface-sterilized
unwounded banana fruits and were incubated in the same
humid box as described above. Observation was made 7
days post inoculation (dpi). Re-isolation of the fungus from
infected fruit was also performed. Fruit inoculated with
distilled water served as negative controls. The assay was
replicated three times and was performed twice.
Cross‑infection assay
In species cross-infection assay, to determine the patho-
genicity of C. gloeosporioides, fungal cultures of C. musae
PHBN0002 and C. gloeosporioides (from pepper and
mango) were inoculated in banana cv. Cavendish fruit. The
same detached fruit method was used as described in the
pathogenicity assay, but fruits were pricked with 200-µL
capacity yellow pipette tip (to create a wound) prior to dis-
pensing the 10 µL inoculum suspension. The C. gloeospori-
oides PHPR0171 isolate used in this assay originated from
anthracnose-infected pepper, while the C. gloeosporioides
PHMg12 isolate originated from anthracnose-infected
mango cv. Carabao. Fungal isolates were obtained from the
Fungal Repository of the Plant Pathology Laboratory (Insti-
tute of Plant Breeding, College of Agriculture and Food Sci-
ence, University of the Philippines Los Baños), of which
their identity was previously determined using combined
morphological, pathogenicity and molecular approaches
(data not shown). Fruit inoculated with distilled water served
as negative controls. The species cross-infection assay was
performed once and replicated four times.
Susceptibility assays
Susceptibility of fruit of the banana cultivars Lagkitan,
Latundan and Saba to postharvest anthracnose caused by C.
musae was investigated in detached fruit assay (as described
above) that was replicated eight times (4 replicate fruit × 2
inoculation sites per fruit) and was performed twice. Cultivar
Cavendish, as the original source of C. musae PHBN0002,
was used as the susceptible check. Fruit inoculated with dis-
tilled water served as negative controls. Anthracnose lesion
measurement was taken at 7 dpi and 14 dpi for Trial 1. How-
ever, fruits used in Trial 2 were slightly relatively mature/
ripe compared to the fruits used in Trial 1, and anthracnose
developed more severely and rapidly on fruit used in Trial
2. Hence, lesion measurement was only taken at 7 dpi in
Trial 2.
Results
Morphology of C. musae
The same fungal isolate was isolated from all small-cut
anthracnose-infected fruit tissues placed on the PDA
medium after 7 days (Fig. 1). A representative isolate, des-
ignated as PHBN0002, was used for further characteriza-
tion. The fungus grew to 29.40 ± 1.24 mm (n = 10) 3 days
after incubation and had a 9.80 ± 0.41 mm growth per day
(n = 10). The mycelia were white, cottony, aerial, which
turns to orange with age. The conidia were cylindrical,
hyaline, with round ends and measures 15.61 ± 0.42 µm in
length and 4.34 ± 0.07 µm in width (n = 93). These morpho-
cultural characteristics resemble that of C. musae.
Molecular characteristics of C. musae
The ITS region of PHBN0002 had a 95% similarity to the
ITS of the epityped strain C. musae CBS116870 (Fig. 2).
The GAPDH and partial ACT gene regions of PHBN0002
had, respectively, 100% and 99% match with C. musae
CBS116870 (Figs. 3 and 4, respectively). Together, among
the species within the C. gloeosporioides species com-
plex, the DNA sequences of all sequenced gene regions of
PHBN0002 had the highest similarity to epityped C. musae
CBS116870. Additionally, the single PCR condition opti-
mized and used in this study was successful in amplifying
the three gene regions.
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 Indian Phytopathology

Fig. 1  Banana cv. Cavendish

fruit infected with anthracnose
(a), growth (b) and of character-
istics of condia (c, d) of Colle-
totrichum musae PHBN0002 in
potato dextrose agar medium 7
days post incubation.

Pathogenicity of C. musae and non‑cross infection Susceptibility of banana cultivars

of C. gloeosporioides
Anthracnose developed on the inoculated banana cv. Cav-
endish fruit 7 dpi in the detached-fruit assay. The lesions
were similar to the initial symptoms observed in cv. Cav-
endish fruit collected from the field. The re-isolated fun-
gus was the same fungus initially isolated and inoculated
and thus, established Koch’s postulate. Both C. gloe-
osporioides isolates of pepper and mango did not infect
the banana fruit. Anthracnose only developed in fruit
inoculated with C. musae. Anthracnose was not observed
in the negative controls.
Anthracnose lesions were observed in all test cultivars
when inoculated with C. musae PHBN0002. Anthracnose
lesion developed relatively slow in Trial 1 compared to
Trial 2 (Fig. 5). In Trial 1, the longest lesion was observed
in Lagkitan (mean of 14.13 ± 3.83 mm, n = 8) and the
shortest in Saba (mean 2.13 ± 1.04 mm, n = 8), but lesion
grew longer at 14 dpi in all cultivars. In Trial 2, anthrac-
nose was severe in Cavendish and Saba, but anthracnose
also developed in Lagkitan (mean of 19.50 ± 1.15 mm,
n = 8) and Latundan (mean of 17.88 ± 3.83 mm, n = 8).

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Indian Phytopathology

Fig. 2  Phylogenetic tree
generated from a maximum
likelihood analysis, based on
the Tamura-Nei model (Tamura
and Nei 1993), of C. musae
PHBN0002 (highlighted)
Philippine isolate’s internal
transcribed spacer (ITS) gene
region sequence. Colletotrichum
boninense was used as an out-
group. The isolates were com-
pared with authentic sequences
of Colletotrichum species
according to Cannon et al.
(2012). Percentage bootstrap
support was calculated from
1000 replicates (Felsenstein
1985). Analyses were conducted
in MEGA7 (Kumar et al. 2016)

Fig. 3  Phylogenetic tree
generated from a maximum
likelihood analysis, based on
the Tamura-Nei model (Tamura
and Nei 1993), of C. musae
PHBN0002 (highlighted)
Philippine isolate’s glyceral-
dehyde 3-phosphate dehydro-
genase (GAPDH) gene region
sequence. Colletotrichum bonin-
ense was used as an outgroup.
The isolates were compared
with authentic sequences of
Colletotrichum species accord-
ing to Cannon et al. (2012). Per-
centage bootstrap support was
calculated from 1000 replicates
(Felsenstein 1985). Analyses
were conducted in MEGA7
(Kumar et al. 2016).

Although variations in lesion size were observed between

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 Indian Phytopathology

Fig. 4  Phylogenetic tree
generated from a maximum
likelihood analysis, based on
the Tamura-Nei model (Tamura
and Nei 1993), of C. musae
PHBN0002 (highlighted)
Philippine isolate’s partial actin
(ACT) gene region sequence.
Colletotrichum boninense
was used as an outgroup. The
isolates were compared with
authentic sequences of Colle-
totrichum species according to
Cannon et al. (2012). Percent-
age bootstrap support was
calculated from 1000 replicates
(Felsenstein 1985). Analyses
were conducted in MEGA7
(Kumar et al. 2016).

Fig. 5  Reaction of popular
banana cultivars to anthrac-
nose caused by C. musae
PHBN0002. Vertical bars are
standard error of means (n = 8).

the two trials, the results indicate that all test cultivars
were susceptible to C. musae.
Discussion
Banana anthracnose was first reported in the Philippines by
Reinking (1918). The causal agent was C. musae and was
characterized by morphology. Recently, there have been
some changes in the taxonomy of fungal species associ-
ated with anthracnose of tropical fruit due to advances in
molecular biology. Some species have been renamed and
new species have been identified (Cannon et al. 2012; Weir
et al. 2012). Many species with overlapping morphological
features have now been resolved by molecular analyses
(Cannon et al. 2012). In this study, the combined mor-
phological, cultural, pathogenicity and molecular charac-
teristics of fungal isolate PHBN0002, causing postharvest
anthracnose of banana cv. Cavendish in the Philippines
is highly similar to C. musae CBS116870 (the epityped
specimen). Further, cross-infection of C. gloeosporioides
from pepper (PHPR0171) and mango (PHMg12) to banana
fruit did not induce the anthracnose symptom, indicating
host specificity of the C. gloeosporioides isolates to their
original hosts and hence, to date, C. musae is the only
anthracnose-associated species known to infect banana.

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Indian Phytopathology
This study also demonstrates for the first time that fruits
of banana cultivars Lakatan, Latundan, and Saba are sus-
ceptible to postharvest anthracnose caused by C. musae
PHBN0002.
The morphology of C. musae PHBN0002 (15.61 µm
× 4.34 µm) is within the acceptable range reported by Su
et al. (2011) on the epityped specimen CBS116870 of C.
musae (11.5–19.5 µm × 4–5 µm) and to that of the first
description of C. musae (11–17 µm x 3–6 µm) provided by
Sutton and Waterston (1970). The same colony morphol-
ogy was also observed in PDA. The identity of the fungus
was further supported by the molecular analyses of three
gene regions (ITS, GAPDH and ACT) which all had high
similarities to C. musae CBS116870 (Su et al. 2011; Weir
et al. 2012).
Colletotrichum musae PHBN0002 infected the banana
cultivars Cavendish, Latundan, Lakatan, and Saba within 7
days and severe infection follows within 7–14 days. Before
this study, there were anecdotal claims of anthracnose in
several banana cultivars, but there was no empirical evidence
of C. musae or any other Colletotrichum species infecting
the cultivars Cavendish, Latundan, Lakatan and Saba. The
current results, however, indicated that these widely known
and cultivated cultivars are likely to suffer losses when post-
harvest anthracnose becomes severe. Inoculation of two C.
gloeosporioides isolates did not result to infection, suggest-
ing that banana is likely to host only C. musae or C. gloe-
osporioides strains that are only specific to Musa species,
e.g. in Malaysia (Sakinah et al. 2013). Nevertheless, further
surveys in commercial banana farms will be needed to deter-
mine the genetic diversity of C. musae or to identify other
Colletotrichum species that may be associated with banana
anthracnose in the country.
By identifying the causal pathogen associated with the
severe anthracnose of banana, this study has taken an impor-
tant step and provided an essential information that will be
useful to commence selection for sources of resistance to
anthracnose in the Philippine Musa germplasm collection
and for breeding or development of disease resistant banana
plants.
Acknowledgements  We thank Fatima May Silva, Rizalina Tiongco
and Jose Ancheta for technical assistance.
Funding  This work was supported by the Institute of Plant Breeding,
College of Agriculture and Food Science, University of the Philippines
Los Baños.
Compliance with ethical standards  Sakinah MaI, Suzianti IV, Latiffah Z (2013) First report of Colle-
Conflict of interest  The authors declare that they have no conflict of
interests.
Ethical approval  This article does not contain any studies with human
participants or animals performed by any of the authors.
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