Plant Pathology (2008) 57, 562–572 Doi: 10.1111/j.1365-3059.2007.01782.

Characterization and pathogenicity of Colletotrichum

Blackwell Publishing Ltd

species associated with anthracnose on chilli

(Capsicum spp.) in Thailand

P. P. Thanab, R. Jeewonc*, K. D. Hydec**†, S. Pongsupasamitb, O. Mongkolpornde and

P. W. J. Taylorf
a
Mushroom Research Centre, 128 Moo3 Ban Ph Deng, T. Pa Pae, A. Mae Taeng, Chiang Mai 50150; bDepartment of Agronomy, Maejo
University, Sansai, Chiang Mai 50290; cCentre for Research in Fungal Diversity, School of Biological Sciences, University of Hong Kong,
Pokfulam Road, Hong Kong; dDepartment of Horticulture, Kasetsart University, Kamphaengsaen Campus, Nakhon Pathom 73140;
e
Center for Agricultural Biotechnology, Kasetsart University, Kamphaengsaen Campus, Nakhon Pathom 73140, Thailand; and fCentre
for Plant Health/BioMarka, School of Agriculture and Food Systems, University of Melbourne, Victoria 3010, Australia

Fungal isolates from chilli (Capsicum spp.) fruits in Thailand that showed typical anthracnose symptoms were identified
as Colletotrichum acutatum, C. capsici and C. gloeosporioides. Phylogenetic analyses from DNA sequence data of ITS
rDNA and β-tubulin (tub2) gene regions revealed three major clusters representing these three species. Among the
morphological characters examined, colony growth rate and conidium shape in culture were directly correlated with the
phylogenetic groupings. Comparison with isolates of C. gloeosporioides from mango and C. acutatum from strawberry
showed that host was not important for phylogenetic grouping. Pathogenicity tests validated that all three species isolated
from chilli were causal agents for chilli anthracnose when inoculated onto fruits of the susceptible Thai elite cultivar
Capsicum annuum cv. Bangchang. Cross-infection potential was shown by C. acutatum isolates originating from
strawberry, which produced anthracnose on Bangchang. Interestingly, only C. acutatum isolates from chilli were able
to infect and produce anthracnose on PBC 932, a resistant genotype of Capsicum chinense. This result has important
implications for Thai chilli breeding programmes in which PBC 932 is being hybridized with Bangchang to incorporate
anthracnose resistance into chilli cultivars.

Keywords: anthracnose, β-tubulin, chilli, Colletotrichum, ITS, morphology, pathogenicity, phylogeny

often wet. Chilli pepper fruits with blemishes have reduced

Introduction
Colletotrichum spp. are among the most important plant
pathogens worldwide, causing the economically important
disease anthracnose in a wide range of hosts, including
cereals, legumes, vegetables and tree fruits (Bailey & Jeger,
1992). Among these hosts, chilli (Capsicum annuum) con-
sidered the most important vegetable in Thailand (Poulos,
1992), is severely infected by anthracnose, with yield
losses of up to 50% (Pakdeevaraporn et al., 2005). Typical
anthracnose symptoms on chilli fruits include sunken
necrotic tissues, with concentric rings of acervuli that are
*E-mail: rajeshjeewon@yahoo.com
**E-mail: kdhyde@hkucc.hku.hk
†Present address: International Fungal Research &
Development Centre, Research Institute of Resource Insects,
Chinese Academy of Forestry, Kunming 650224, Yunnan, PR
China.
Accepted 20 August 2007
marketability (Manandhar et al., 1995).
Anthracnose of chilli has been shown to be caused
by at least four species of Colletotrichum: C. capsici and
C. gloeosporioides in India (Sharma et al., 2005), Indonesia
(Voorrips et al., 2004), Korea (Kim et al., 1999), Thailand
(Pakdeevaraporn et al., 2005); C. acutatum in Australia
(Simmonds, 1965) and Indonesia (Nirenberg et al.,
2002); and C. coccodes in New Zealand (Johnston &
Jones, 1997). Accurate taxonomic identification is neces-
sary for plant breeding purposes and disease management
(Freeman et al., 1998). Traditionally, identification and
characterization of Colletotrichum species was based on
morphological characters such as size and shape of
conidia and appressoria, existence of setae or presence of
a teleomorph, and cultural characters such as colony
colour, growth rate and texture (Smith & Black, 1990).
These criteria alone are not always adequate to differentiate
species because of variations in morphology and pheno-
type among species under different environmental con-
ditions. To overcome taxonomic problems associated with

© 2008 The Authors

562 Journal compilation © 2008 BSPP
 Chilli anthracnose
these traditional identification methods, DNA sequence
analyses were used to characterize and resolve the taxo-
nomic complexity of some fungal genera, e.g. Fusarium
(O’Donnell et al., 1998) and Pestalotiopsis (Jeewon
et al., 2004), as well as Colletotrichum (Sreenivasaprasad
et al., 1996; Photita et al., 2005). Cannon et al. (2000)
stated that data derived from nucleic acid analyses should
provide the most reliable framework to build a classifica-
tion of Colletotrichum as DNA characters were not directly
influenced by environmental factors. In particular, sequence
analysis of the ITS regions proved useful in studying
phylogenetic relationships of species of Colletotrichum
(Sreenivasaprasad et al., 1996; Photita et al., 2005). Apart
from rDNA, partial β-tubulin and translation elonga-
tion factor (TEF) sequence analyses were also applied to
resolve phylogenetic relationships among fungi, such
as in the Gibberella fujikuroi (O’Donnell et al., 1998;
Bogale et al., 2006), and C. acutatum species complexes
(Vinnere et al., 2001; Sreenivasaprasad & Tahinhas,
2005). Combined application of molecular diagnostic
tools, along with traditional methods, including mor-
phological characterization and pathogenicity testing, is
an appropriate and reliable approach for studying species
complexes of Colletotrichum (Cannon et al., 2000). The
objective of this study was to identify and characterize
Colletotrichum species causing chilli anthracnose in
Thailand.
Materials and methods
Isolation of Colletotrichum
Colletotrichum isolates were collected from anthracnose
lesions on chilli fruits (Capsicum annuum) in north
(Chiang Mai), northeast (Ubonratchathani) and west
(Kanchanaburi, Nakonpathon, Ratchaburi) districts of
Thailand. For a comparative study, isolates were collected
from infected fruits of mango (Mangifera indica) and
strawberry (Fragaria spp.) from a local market in Chiang
Mai (Table 1). Isolation was carried out by two methods,
depending on fungal sporulation. Isolates were obtained
from fruits without visible sporulation using the pro-
cedure described by Photita et al. (2005). Three 5 × 5-mm2
pieces of tissue were taken from the margins of infected
tissue, surface-sterilized by dipping in 1% sodium hypo-
chlorite for 3–5 min, and rinsed three times with sterile
water. They were then placed on the surface of water agar
(WA, Oxoid Ltd.) and incubated at room temperature
(28–30°C). The growing edges of any hyphal mycelium
developing from the disease tissue discs were then trans-
ferred aseptically to potato dextrose agar (PDA, Oxoid
Ltd.). The fungi were identified following sporulation and
single-spore isolation was carried out using the procedure
described by Choi et al. (1999), with modifications. Direct
examination and single-spore isolation from infected
fruits with sporulation was also carried out. Spore masses
were touched with a sterilized wire loop and streaked on
to the surface of WA plates which were then incubated
overnight. A single germinated spore was picked up
Plant Pathology (2008) 57, 562–572
563
with a sterilized needle and transferred onto PDA. Pure
cultures were stored at 4°C on PDA slants. Isolates were
deposited in the University of Hong Kong Culture Collec-
tion (HKUCC) (Table 1).
Morphological examination
Starter cultures were prepared by plating each isolate
onto PDA at room temperature (25°C). Three 4-mm
plugs were aseptically punched from actively sporulating
areas near the growing edge of a 5-day-old culture of
each isolate. Each plug was placed onto PDA plates
and incubated under the same conditions as starter
cultures. Three cultures of every isolate were investi-
gated. Cultures were incubated at room temperature
(25°C) for 7 days, after which the size and shape of 20
conidia harvested from every culture of each isolate
were recorded.
Colony diameter of every culture was recorded daily
for 7 days. Growth rate was calculated as the 7-day average
of mean daily growth (mm per day). After 7 days, colony
size and colour of the conidial masses and zonation were
recorded.
Appressoria were produced using a slide-culture
technique, where 10-mm squares of PDA were placed in
an empty Petri dish, with the edge of the agar inoculated
with spores taken from a sporulating culture, and a cover
slip placed over the inoculated agar (Johnston & Jones,
1997). After 5–7 days, appressoria formed across the
underside of the cover slip and their shape and size were
then recorded. Data were analysed using analysis of
variance (P < 0·05) with Duncan’s multiple range tests
(DMRT) and least significant difference (LSD) values used
with spss software version 13·0 (SPSS Inc.) (Kirkpatrick
& Feeney, 2006).
Molecular examination
DNA extraction
DNA was extracted from all isolates using a modification
of the protocol described by Promputtha et al. (2005).
Each culture derived from a single conidium from the
original isolate was subsequently cultured on PDA.
Cultures were incubated at room temperature for 10–14
days. Mycelium was scraped from the surface of the plate
and ground with 200 mg of sterilized quartz sand and
600 μL of 2 × CTAB extraction buffer (2% w/v CTAB,
100 mm Tris HCl, 1·4 m NaCl, 20 mm EDTA, pH 8) in a
1·5-mL Eppendorf tube. The whole contents were incu-
bated at 60°C in a water bath for 40 min with occasional
swirling. The solution was then extracted two or three
times with equal volumes of phenol and chloroform (1:1)
at 17 530 g for 30 min until no interface was visible. The
upper aqueous phase containing the DNA was precipi-
tated by addition of 2·5 volumes of absolute ethanol and
kept at –20°C overnight. The precipitated DNA was then
washed with 70% ethanol, dried under vacuum, suspended
in TE buffer (1 mm EDTA, 10 mm Tris-HCl, pH 8) and
treated with RNase (1 mg mL–1).
564 P. P. Than et al.

Table 1 Sources of Colletotrichum isolates used in this study and reference sequences from GenBank used in analysis

HKUCC
Acc. no.a ITS β-tubulin Isolate Colletotrichum species Location Host

10860 DQ454001 DQ454043 M1 C. gloeosporioides Chiang Mai, Thailand Mangifera indica

10863 DQ454004 DQ454041 M4 C. gloeosporioides Chiang Mai, Thailand Mangifera indica
10849 DQ454005 DQ454044 M5 C. gloeosporioides Chiang Mai, Thailand Mangifera indica
10891 DQ454018 DQ454066 S2 C. acutatum Chiang Mai, Thailand Fragaria sp.
10872 DQ454019 DQ454063 S3 C. acutatum Chiang Mai, Thailand Fragaria sp.
10873 DQ454020 DQ454062 S4 C. acutatum Chiang Mai, Thailand Fragaria sp.
10890 DQ454021 DQ454065 S5 C. acutatum Chiang Mai, Thailand Fragaria sp.
10814 DQ454022 DQ454064 S6 C. acutatum Chiang Mai, Thailand Fragaria sp.
10871 DQ454023 DQ454067 S7 C. acutatum Chiang Mai, Thailand Fragaria sp.
10882 DQ453991 DQ454035 Ku1 C. gloeosporioides Ratchaburi, Thailand Capsicum annuum
10883 DQ453992 DQ454036 Ku2 C. gloeosporioides Ratchaburi, Thailand Capsicum annuum
10892 DQ453993 DQ454040 Ku3 C. gloeosporioides Ratchaburi, Thailand Capsicum annuum
10884 DQ453994 DQ454029 Ku4 C. gloeosporioides Kanchanaburi, Thailand Capsicum annuum
10889 DQ453996 DQ454034 Ku6 C. gloeosporioides Kanchanaburi, Thailand Capsicum annuum
10887 DQ454000 DQ454032 Ku10 C. gloeosporioides Kanchanaburi, Thailand Capsicum annuum
10864 DQ453995 DQ454030 Ku5 C. gloeosporioides Kanchanaburi, Thailand Capsicum annuum
10881 DQ453998 DQ454031 Ku8 C. gloeosporioides Nakhonpathon, Thailand Capsicum annuum
10888 DQ453999 DQ454033 Ku9 C. gloeosporioides Nakhonpathon, Thailand Capsicum annuum
10848 DQ454006 DQ454058 Mj2 C. acutatum Chiang Mai, Thailand Capsicum annuum
10865 DQ454007 – Mj3 C. acutatum Chiang Mai, Thailand Capsicum annuum
10879 DQ454008 DQ454059 Mj4 C. acutatum Chiang Mai, Thailand Capsicum annuum
10893 DQ454010 DQ454061 Mj6 C.acutatum Chiang Mai, Thailand Capsicum annuum
10850 DQ454011 DQ454068 Mj9 C. acutatum Chiang Mai, Thailand Capsicum annuum
10894 DQ454012 DQ454069 Mj10 C. acutatum Chiang Mai, Thailand Capsicum annuum
10875 DQ453987 DQ454045 Ccmj2 C. capsici Chiang Mai, Thailand Capsicum annuum
10857 DQ453988 DQ454047 Ccmj3 C. capsici Chiang Mai, Thailand Capsicum annuum
10858 DQ453989 DQ454048 Ccmj7 C. capsici Chiang Mai, Thailand Capsicum annuum
10859 DQ453990 DQ454054 Ccmj10 C. capsici Chiang Mai, Thailand Capsicum annuum
10855 DQ454024 DQ454052 Skp4 C. capsici Chiang Mai, Thailand Capsicum annuum
10877 DQ454025 DQ454055 Skp16 C. capsici Chiang Mai, Thailand Capsicum annuum
10868 DQ454013 DQ454046 R4 C. capsici Chiang Mai, Thailand Capsicum annuum
10852 DQ454014 DQ454056 R5 C. capsici Chiang Mai, Thailand Capsicum annuum
10869 DQ454015 DQ454053 R7 C. capsici Chiang Mai, Thailand Capsicum annuum
10870 DQ454016 DQ454049 R11 C. capsici Chiang Mai, Thailand Capsicum annuum
10880 DQ454017 DQ454057 R12 C. capsici Chiang Mai, Thailand Capsicum annuum
10866 DQ454026 – U9 C. capsici Ubonratchathani, Thailand Capsicum annuum
10876 DQ454027 DQ454050 U10 C. capsici Ubonratchathani, Thailand Capsicum annuum
b
AY376525 AY376573 STEU-2289 C. boninense Zimbabwe Proteaceae
c
DQ195680 DQ195719 BRIP 26974 C. capsici QLD, Australia Capsicum frutescens
EF143971 EF143967 BRIP 4703a C. acutatum Townsville, QLD, Australia Fragaria × ananassa
EF143972 EF143968 BRIP 4704a C. acutatum Forest Glen, QLD, Australia Fragaria × ananassa
EF143974 EF143969 BRIP 11086a C. acutatum Nambour, QLD, Australia Fragaria × ananassa
EF143975 EF143970 BRIP 28519a C. acutatum Yandina, QLD, Australia Carica papaya

a
HKUCC, University of Hong Kong Culture Collection.
b
STEU, University of Stellenbosch Culture Collection.
c
BRIP, Queensland Department of Primary Industries Plant Pathology Herbarium.

PCR and sequencing with ethidium bromide on 1% agarose electrophoresis

DNA amplification and sequencing were performed by
PCR. Complete ITS/5·8S rDNA and partial β-tubulin
(tub2) sequences were amplified using fungal-specific
primers ITS 4 and ITS 5 (White et al., 1990) and Bt 2A
and Bt 2B (Glass & Donaldson, 1995), respectively. PCR
was carried out in a PTC-100 programmable thermal
cycler as follows: 95°C for 3 min; 30 cycles of denaturing
at 95°C for 1 min, annealing at 52°C for 50 s and elonga-
tion at 72°C for 1 min; and a final extension step of
72°C for 10 min. PCR products were verified by staining
gels. PCR products were then purified using the GFX
PCR Purification Kit (27-9602-01; Amersham Biosciences)
according to the manufacturer’s protocol. DNA sequenc-
ing using primers ITS 5 and Bt 2A was performed in the
Applied Biosystem 3730 DNA analyser at the Genome
Research Centre of the University of Hong Kong. For several
strains of the same species, however, sequencing was
performed with two primers (as above) in both directions to
ensure that there was no misreading. Sequences generated
from this study were deposited in GenBank (Table 1).

Plant Pathology (2008) 57, 562–572

 Chilli anthracnose
Phylogenetic analysis
Individual or combined datasets of ITS/5·8S rDNA
and β-tubulin (tub2) sequences were analysed. Sequences
from the collected isolates, along with sequences obtained
from isolates BRIP 26974, BRIP 4703a, BRIP 4704a,
BRIP 11086a and BRIP 28519a supplied by the Plant
Pathology Herbarium, Department of Primary Industries
and Fisheries, Queensland, Australia (Table 1), were
aligned in clustal x (Thomson et al., 1997) and optimized
manually. The partition homogeneity test (Farris et al.,
1995), as implemented in paup*, was used to examine
data for conflicting hierarchic signals and to evaluate
congruence of the combined dataset. Branch support of
the trees resulting from maximum parsimony analysis
was assessed by bootstrapping (Felsenstein, 1985). This
was performed with 1000 replications using the heuristic
search option to estimate the reliability of inferred mono-
phyletic groups. Descriptive tree statistics including tree
length (TL), consistency index (CI), retention index (RI) and
homoplasy index (HI) were calculated for all parsimony
trees. Colletotrichum boninense (STEU-2289) was the
designated outgroup in all analyses.
Pathogenicity testing
Three representative isolates of each species from each
host were used for pathogenicity testing. Isolates used
in this study were C. acutatum from chilli (Mj4, Mj5
and Mj10), C. capsici from chilli (R4, Ccmj10 and Skp4),
C. gloeosporioides from chilli (Ku4, Ku5 and Ku8),
C. acutatum from strawberry (S2, S4 and S5) and C.
gloeosporioides from mango (M1, M2 and M4). Isolates
were cultured on PDA at 27°C under continuous fluores-
cent light. Conidia from 7-day-old cultures were harvested
by adding 5–10 mL of sterilized distilled water onto the
culture, which was then gently swirled to dislodge the
conidia. The conidial suspension was filtered through two
layers of muslin cloth. Bangchang, a susceptible Thai elite
cultivar of C. annuum, and PBC 932, an anthracnose-
resistant accession of C. chinense, were supplied by the
Tropical Vegetable Research Center, Kasetsart University,
Thailand. Non-infected fruits were surface-sterilized with
1% sodium hypochlorite for 5 min and washed twice
with distilled water. The fruits were blotted dry with a
sterile paper tissue and inoculated using either the wound/
drop or non-wound/drop method (Lin et al., 2002;
Kanchana-udomkan et al., 2004). The wound/drop method
involved pin-pricking the chilli fruit wall to a 1-mm depth
and then placing 6 μL of conidial suspension (106 conidia
mL–1) over the wound. The non-wound/drop method
involved placing 6 μL of conidial suspension (2 × 106
conidia mL–1) onto the middle of each fruit. Preliminary
experiments showed that non-wound inoculation with
only 106 conidia mL–1 resulted in very little infection, hence
the higher concentration used for that method. The
inoculated fruits were incubated at 25°C, 98% RH in the
dark for 24 h in a 12-h light/dark cycle. Three fruits were
tested per isolate and the experiment was carried out twice.
Plant Pathology (2008) 57, 562–572
565
Disease reactions of the host were evaluated by measur-
ing the length, width and area of the typical anthracnose
lesion which developed on the fruits. Symptoms were
evaluated 9–15 days after inoculation (DAI). Fruit sizes
were also recorded. Disease reaction was scored on a 0–9
point scale that was modified from the disease scoring
scale described by Dasgupta (1981): 0 (highly resistant),
no infection; 1 (resistant), 1–2% of the fruit with a necrotic
lesion or a larger water soaked lesion surrounding the
infection site; 3 (moderately resistant), > 2 to 5% of the
fruit with a necrotic lesion, possibly acervuli may be
present, or a watery lesion covering up to 5% of the fruit
surface; 5 (susceptible), > 5 to 10% of the fruit showing a
necrotic lesion, possibly acervuli, or a water-soaked lesion
covering up to 25% of the fruit surface; 7 (very susceptible),
> 10 to 25% of the fruit covered with a necrotic lesion
with acervuli; and 9 (highly susceptible), > 25% of the
fruit showing necrosis, lesion often encircling the fruit,
abundant acervuli. The experiment was carried out twice.
Data of infected fruit areas were also analysed using
analysis of variance (P < 0·05), with DMRT and LSD values
used for multiple range tests with spss software version
13·0 (Kirkpatrick & Feeney, 2006).
Results
Collection and identification of isolates
Twenty-nine isolates of Colletotrichum spp. were
obtained from infected chilli fruits, three from infected
mango fruits and six from infected strawberry fruits, all
showing symptoms of anthracnose. Identification of the
isolates was based on the morphological descriptions
of Colletotrichum species outlined by Mordue (1971) and
Sutton (1992). From chilli, seven isolates fitted the descrip-
tion of C. acutatum, 13 fitted the description of C. capsici
and nine fitted the description of C. gloeosporioides. Three
isolates from infected mango fruits fitted the description
of C. gloeosporioides and six from infected strawberry
fruits fitted the description of C. acutatum (Table 2).
Morphological examination
Distinctness in spore morphology and colony characteris-
tics among the isolates resulted in morphological groups
being identified that correlated with the Colletotrichum
species regardless of the host species from which they
were obtained (Table 2).
Culture colony characteristics
Distinct morphological types on PDA were observed in each
morphological group after 7 days following subculturing
(Fig. 1). Isolates from group 1, mango C. gloeosporioides,
produced colonies with little aerial mycelium in alternat-
ing concentric zones of light orange at the centre turning
pale yellow towards the margin. Colonies produced by
isolates from group 2, chilli C. gloeosporioides, varied
from greyish-white to dark grey; some isolates (Ku1, Ku2,
Ku3, Ku4, Ku5 and Ku8) showed diurnal zonation of pale
566 P. P. Than et al.

grey to black aerial mycelium, whilst others (Ku6, Ku9

Growth rate
mm day–1
and Ku10) produced aerial mycelium in an even, felted

7·1 b
11·0 c
11·2 c

5·8 a
5·8 a

0·27
mat. Isolates from group 3, strawberry C. acutatum,
produced white to pale grey colonies showing diurnal
zonation of dense and sparse development of aerial
mycelia, sometimes with pinkish spore masses. Isolates

Width

6·0 b
6·5 c
6·3 c

5·5 a

6·5 c
(μm)

0·20
from group 4, chilli C. acutatum, produced pale orange
Appressoria
colonies with little aerial mycelium and a few orange
Length conidial masses around the centre. Isolates from group 5,

9·0 b
9·0 b

9·5 b
7·0 a
6·5 a
(μm) chilli C. capsici, produced colonies that were white to

0·75
grey; most of the isolates showed the diurnal zonation of
dense and sparse development of aerial mycelium, some-
Cylindrical
Cylindrical
times with beige-coloured spore masses.
Fusiform
Fusiform
Falcate
Shape

Growth rate
An important comparative character was the growth rate
of the colony in culture. There was no significant differ-
Width

4·5 d

3·5 b
3·5 b
4·0 c

3·0 a
(μm)

0·25

ence in growth rate among isolates of the same species, i.e.

among isolates of C. acutatum belonging to groups 3 and
For numerical characters, values followed by the same letter in a column did not differ significantly (0·01 level) in Duncan’s multiple range test.

4 (P = 0·168) or among isolates of C. gloeosporioides

Conidia

Length

belonging to groups 1 and 2 (P = 0·817). However, a

14·0 b
13·5 a
13·5 a

13·0 a

21·0 c
0·30
(μm)

statistical difference was observed in the growth rate of

the three different species. Isolates of C. gloeosporioides
from group 1 (11·0 mm day–1) and from group 2 (11·2 mm
White to grey colour with dark green centre and cottony mycelium
White to olive grey colour colony with very thick cottony mycelium

day–1) grew significantly faster than any other groups

(P = 0·001), followed by isolates of C. capsici from group
Light orange colony colour with delicate and thin mycelium
Pale grey to black zonated colonies with abundant orange

5 (7·1 mm day–1) and isolates of C. acutatum from group

3 (5·8 mm day−1) and group 4 (5·8 mm day–1) (Table 2).
Orange-coloured colony with slight mycelium

Conidial morphology
There were three types of conidia, viz. cylindrical, fusiform
and falcate, observed in the three species of Colletotrichum
(Table 2). Colletotrichum capsici isolates belonging to
conidial masses near the centre

group 5 produced falcate conidia and C. acutatum isolates

belonging to groups 3 and 4 produced predominantly
fusiform conidia (80% average occurrence). Colletotrichum
Table 2 Summary of morphological data for Colletotrichum species in groups 1–5

gloeosporioides isolates from groups 1 and 2 produced

Colony character

cylindrical conidia. However, there was little distinction

among the groups in size of conidia (Table 2).

Appressorial morphology
There were few differences in appressorial shape and size
between groups. Most of the appressoria formed in slide
C. gloeosporioides
C. gloeosporioides

cultures were irregularly shaped and only a few were ovoid.

Ovoid appressoria were commonly observed in slide cultures
C. acutatum
C. acutatum

from C. acutatum isolates from strawberry in groups 3 and 4.

C. capsici
Species

LSD (between group)

Phylogenetic analyses
PCR products obtained from the ITS regions (including 5.8 S)
Strawberry

ranged from 550 to 600 bp, whereas those from the β-tubulin
Mango

gene ranged from 450 to 500 bp. The final sequence

Chilli

Chilli
Chilli
Host

alignment of the concatenated ITS and β-tubulin dataset

comprising 43 taxa had 984 characters, of which 229 were
parsimony informative (23·27%), 575 were constant and
Morphological

180 were variable. Two trees were obtained when gaps were
treated as missing data in a weighted parsimony analysis.
group

The topologies of the two trees were not significantly

different and one of the trees (total length = 721 steps,
1
2

3
4
5

Plant Pathology (2008) 57, 562–572

 Chilli anthracnose 567

Figure 1 (a) Lower colony surface, (b) upper colony surface and (c) conidia of Colletotrichum species in groups 1–5. Bars = 15 μm.

consistency index = 0·861, retention index = 0·971, rescaled
consistency index = 0·836 and homoplasy index = 0·139)
is shown in Fig. 2.
In order to compare tree output with morphological
and cultural characters, the phylogeny generated from the
combined dataset was selected because most of the major
clusters and subclusters were more resolved and received
higher statistical support. As shown in Fig. 2, Colletotri-
chum isolates fell into three distinct lineages (clusters X, Y
and Z) supported by 100% bootstrap values. Cluster X
consisted of C. gloeosporioides isolates from chilli and
mango. This cluster only included isolates characterized
by cylindrical conidia and with a colony growth rate of
> 11 mm day–1. Cluster Y comprised C. acutatum isolates
from chilli and strawberry. All isolates from this cluster
had fusiform conidia and a growth rate of > 5 mm day–1.
Interestingly, C. acutatum isolates collected in Australia
from strawberry and papaya formed a subcluster distinct
from the isolates from Thailand, with high bootstrap
confidence. Cluster Z received high statistical support
and consisted only of C. capsici isolates from chilli.
All isolates in this cluster were characterized by falcate-
spored conidia and had an average growth rate of > 7 mm
day–1.

Plant Pathology (2008) 57, 562–572

568 P. P. Than et al.

Figure 2 Phylogenetic tree generated from a maximum parsimony analysis of a combined dataset of Colletotrichum ITS and β-tubulin (tub2) gene
sequences. The tree was rooted with C. boninense. Clusters X, Y and Z correspond to C. gloeosporioides, C. acutatum and C. capsici, respectively.
Values above branching nodes represent percentage bootstrap support calculated from 1000 replicates. Branch lengths are proportional to the
numbers of nucleotide substitutions and are measured by scale bars (bar = 10% sequence divergence).

C. acutatum from strawberry (group 3), C. capsici (group 5)

Pathogenicity testing
Pathogenicity of isolates on C. annuum cv. Bangchang
In both the wound/drop and non-wound/drop inoculation
methods, chilli fruits inoculated with the C. acutatum
isolates from strawberry (group 3); and the C. acutatum
(group 4), C. capsici (group 5) and C. gloeosporioides
isolates (group 2) from chilli showed symptoms of anthra-
cnose and lesions that were not significantly different in
size from one another. This was typical of a susceptible
host reaction, with a disease score in the range 7–9 (very
susceptible to highly susceptible) (Table 3). However, chilli
fruits inoculated with C. gloeosporioides from mango
(group 1) did not show any typical symptoms. With the
wound/drop inoculation method, lesions appeared 3 days
after inoculation, while with non-wound/drop inoculation,
lesions appeared 9 days after inoculation.
Pathogenicity of isolates on C. chinense PBC 932
In wound/drop inoculation at the inoculum concentration
of 106 conidia mL–1, only C. acutatum isolates from chilli
(group 4) produced typical anthracnose lesions on the
fruits of C. chinense cv. PBC 932, with a disease score of
7 (very susceptible) (Table 3). In contrast, other isolates of
from chilli and C. gloeosporioides from chilli and mango
(groups 2 and 1, respectively), did not produce any
symptoms on PBC 932 fruits. In non-wound/drop inocu-
lation, none of the isolates in any group produced
symptoms typical of anthracnose.
Discussion
A combined application of morphological characters,
molecular diagnostic tools and pathogenicity identified
three species of Colletotrichum, viz. C. acutatum, C. capsici
and C. gloeosporioides, as pathogens of commercial chilli
fruits in Thailand. The taxonomy of most taxa within
Colletotrichum was previously based primarily upon
variation in conidial size and shape, appressoria and
colony characters (Bailey & Jeger, 1992).
Morphological grouping (based on cultural morphology
and spore shape) was in agreement with phylogenies
derived from molecular data in this study, however, there
was an overlap in conidial size among the five morphological
groups studied. Sequence analyses from the ITS region
and β-tubulin genes also did not provide a clear indication
of possible phylogenetic relationships for isolates

Plant Pathology (2008) 57, 562–572

Plant Pathology (2008) 57, 562–572

Table 3 Host reactiona of Capsicum annuum cv. Bangchang and C. chinense PBC 932 fruits to isolates of Colletotrichum species 9 days after wound/drop inoculation and 15 days after non-wound/drop inoculationb

Bangchang PBC 932

Wound/drop Non-wound/drop Wound/drop Non-wound/drop

Infected Infected Infected Infected

Species Host Group Isolate fruit area (%) Host reaction fruit area (%) Host reaction fruit area (%) Host reaction fruit area (%) Host reaction

C. gloeosporioides Mango 1 M1 0 0 0 0
M2 0 0 0 0
M4 0 0 0 0
mean 0 0 0 0
C. gloeosporioides Chilli 2 Ku4 20·60 ac 17·5 a 0 0
Ku5 23·21 a 18·43 a 0 0

Chilli anthracnose
Ku8 24·99 a 19·39 a 0 0
mean 24·57 VS 17·27 VS 0 HR 0 HR
C. acutatum Strawberry 3 S2 21·24 a 14·47 a 0 0
S4 31·90 a 15·62 a 0 0
S5 20·48 a 15·26 a 0 0
mean 23·23 VS 15·12 VS 0 HR 0 HR
C. acutatum Chilli 4 Mj4 32·60 a 13·98 a 38·28 a 0
Mj5 34·00 a 24·99 a 20·47 a 0
Mj10 21·86 a 15·19 a 14·92 a 0
mean 28·97 HS 24·47 VS 24·56 VS 0 HR
C. capsici Chilli 5 R4 23·81 a 21·00 a 0 0
Skp4 26·34 a 18·60 a 0 0
Ccmj10 31·37 a 18·15 a 0 0
mean 27·17 HS 18·92 VS 0 HR 0 HR
LSD (between isolates) 17·46 21·33 47·43
LSD (between groups) 10·39 11·70

a
HS, highly susceptible; VS, very susceptible; HR, highly resistant.
b
Wound/drop inoculation used 106 conidia mL–1 and non-wound/drop inoculation used 2 × 106 conidia mL–1.
c
Values followed by the same letter in a column did not differ significantly (0·01 level) in Duncan’s multiple range test.

569
570 P. P. Than et al.
characterized by similar conidial size. These results
indicated that spore size was homoplasious. Similar con-
clusions were made by Hindorf (1973), who found a large
amount of morphometric overlap of conidial size within
Colletotrichum species. Differentiation of C. acutatum
from C. gloeosporioides and C. capsici was reliable based
on appressorial shape. However, species delineation
between C. capsici and C. gloeosporioides based on this
character was not possible. Sanders & Korsten (2003a)
considered that appressorial shape was unreliable for
species differentiation.
Cultural characteristics separated species from chilli,
mango and strawberry. Isolates from chilli were separated
into the three Colletotrichum species based on their
cultural characters, and this was robustly supported by
phylogenetic analysis.
Colony growth rate in vitro was one of the important
characteristics for distinguishing between the three species
of Colletotrichum. Phylogenies inferred from sequences
supported a close relationship of isolates with the same
growth rate. Isolates of C. acutatum had the slowest growth
rates. Simmonds (1965) and Sutton (1992) found that
C. acutatum could be differentiated from C. gloeosporioides
by its slower growth rate.
Molecular phylogenies did not show correlation between
DNA sequence data and host association within the
Colletotrichum species. Colletotrichum capsici from chilli
constituted a distinct monophyletic group, whereas
C. acutatum isolates from chilli were more related to other
C. acutatum isolates from strawberry. This showed that
spore morphology and cultural characters reflected phy-
logeny better than host association. Similar results were
obtained before for C. acutatum (Du et al., 2005) and
C. gloeosporioides (Guerber et al., 2003), although these
results were not in accordance with the importance
of host association in species such as C. graminicola (Du
et al., 2005). The subcluster of isolates of C. acutatum
from Australia may have reflected phylogenetic divergence
based on geographical isolation between Australia and
Thailand. Studies by Denoyes-Rothan et al. (2003) on
populations of C. acutatum on strawberry from a wide
geographic range revealed both a homogeneous group
and a highly variable group, with no direct correlation to
geographic areas. More isolates from Australia need to
be assessed to further understand geographic divergence
of C. acutatum.
Pathogenicity tests with the three Colletotrichum
species isolated from infected chilli fruits showed that all
the isolates were pathogenic on the susceptible Thai elite
cultivar Bangchang. This result proved that these three
species of Colletotrichum were casual agents of anthracnose
infection on chilli. Non-infection of the resistant genotype
C. chinense PBC 932 by C. capsici and C. gloeosporioides
reconfirmed the importance of the resistance in this
genotype to the interspecific breeding programme (Pak-
deevaraporn et al., 2005). The anthracnose symptoms
produced by all three isolates of C. acutatum in wound-
inoculated fruits of PBC 932 indicated that C. acutatum
was pathogenic and could overcome the resistance, but
infection could not occur in PBC 932 without wounding,
demonstrating the role of the cuticle in host resistance.
Wounding was noticed to greatly enhance the ability
of Colletotrichum to cause disease (Pring et al., 1995).
Oh et al. (1999) also showed the importance of cuticular
wax layers of green and red pepper fruits to infection
by C. gloeosporioides, where a negative correlation was
found between cuticle thickness and disease incidence.
Plant breeders need to be aware of the potential of
C. acutatum to be a major pathogen when developing
new chilli cultivars for resistance to anthracnose
disease.
The fact that C. acutatum from strawberry was a
pathogen of chilli confirmed numerous reports about the
cross-infection potential among different species of Colle-
totrichum on a multitude of hosts (Freeman et al., 1998).
In contrast to cross-inoculation studies by Sanders &
Korsten (2003b), who showed that isolates of C. gloe-
osporioides from mango could produce symptoms on
other hosts such as guava, chilli pepper and papaya,
isolates of C. gloeosporioides from mango did not show
any symptoms on inoculated chilli fruits in the present
study. Although mango isolates of C. gloeosporioides were
highly pathogenic when re-inoculated onto mango fruits
(data not shown), it is unclear why no symptoms were
produced on chilli fruits by the mango isolates. Further
microscopic work is needed to examine the host reaction
to initial infection by these pathogens. Despite the high
levels of infection potential on detached fruits, it is not
known whether isolates could pose a threat in the field,
since the inoculation studies were carried out under
optimal conditions to induce infection by the pathogen
(Sanders & Korsten, 2003b). Further studies with different
inoculation tests and different stages of ripeness are
needed to confirm these results.
Acknowledgements
We are grateful to the Mushroom Research Foundation,
Chiangmai, Thailand for funding. The University of Hong
Kong is thanked for providing funds for the molecular
work. Kasetsart University (Thailand) is acknowledged
for kindly supplying some isolates used in this study and
the Tropical Vegetable Research Center, Kasetsart Univer-
sity, is thanked for the supply of chilli fruits. Helen Leung
and Heidi Kong (University of Hong Kong) are thanked
for laboratory assistance and Chutchamas Kanchana-
udomkam (Kasesart University) is thanked for her assistance
in the pathogenicity tests.
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