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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.
these traditional identification methods, DNA sequence with a sterilized needle and transferred onto PDA. Pure
analyses were used to characterize and resolve the taxo- cultures were stored at 4°C on PDA slants. Isolates were
nomic complexity of some fungal genera, e.g. Fusarium deposited in the University of Hong Kong Culture Collec-
(O’Donnell et al., 1998) and Pestalotiopsis (Jeewon tion (HKUCC) (Table 1).
et al., 2004), as well as Colletotrichum (Sreenivasaprasad
et al., 1996; Photita et al., 2005). Cannon et al. (2000)
Morphological examination
stated that data derived from nucleic acid analyses should
provide the most reliable framework to build a classifica- Starter cultures were prepared by plating each isolate
tion of Colletotrichum as DNA characters were not directly onto PDA at room temperature (25°C). Three 4-mm
influenced by environmental factors. In particular, sequence plugs were aseptically punched from actively sporulating
analysis of the ITS regions proved useful in studying areas near the growing edge of a 5-day-old culture of
phylogenetic relationships of species of Colletotrichum each isolate. Each plug was placed onto PDA plates
(Sreenivasaprasad et al., 1996; Photita et al., 2005). Apart and incubated under the same conditions as starter
from rDNA, partial β-tubulin and translation elonga- cultures. Three cultures of every isolate were investi-
tion factor (TEF) sequence analyses were also applied to gated. Cultures were incubated at room temperature
resolve phylogenetic relationships among fungi, such (25°C) for 7 days, after which the size and shape of 20
as in the Gibberella fujikuroi (O’Donnell et al., 1998; conidia harvested from every culture of each isolate
Bogale et al., 2006), and C. acutatum species complexes were recorded.
(Vinnere et al., 2001; Sreenivasaprasad & Tahinhas, Colony diameter of every culture was recorded daily
2005). Combined application of molecular diagnostic for 7 days. Growth rate was calculated as the 7-day average
tools, along with traditional methods, including mor- of mean daily growth (mm per day). After 7 days, colony
phological characterization and pathogenicity testing, is size and colour of the conidial masses and zonation were
an appropriate and reliable approach for studying species recorded.
complexes of Colletotrichum (Cannon et al., 2000). The Appressoria were produced using a slide-culture
objective of this study was to identify and characterize technique, where 10-mm squares of PDA were placed in
Colletotrichum species causing chilli anthracnose in an empty Petri dish, with the edge of the agar inoculated
Thailand. 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
Materials and methods underside of the cover slip and their shape and size were
then recorded. Data were analysed using analysis of
Isolation of Colletotrichum
variance (P < 0·05) with Duncan’s multiple range tests
Colletotrichum isolates were collected from anthracnose (DMRT) and least significant difference (LSD) values used
lesions on chilli fruits (Capsicum annuum) in north with spss software version 13·0 (SPSS Inc.) (Kirkpatrick
(Chiang Mai), northeast (Ubonratchathani) and west & Feeney, 2006).
(Kanchanaburi, Nakonpathon, Ratchaburi) districts of
Thailand. For a comparative study, isolates were collected
Molecular examination
from infected fruits of mango (Mangifera indica) and
strawberry (Fragaria spp.) from a local market in Chiang DNA extraction
Mai (Table 1). Isolation was carried out by two methods, DNA was extracted from all isolates using a modification
depending on fungal sporulation. Isolates were obtained of the protocol described by Promputtha et al. (2005).
from fruits without visible sporulation using the pro- Each culture derived from a single conidium from the
cedure described by Photita et al. (2005). Three 5 × 5-mm2 original isolate was subsequently cultured on PDA.
pieces of tissue were taken from the margins of infected Cultures were incubated at room temperature for 10–14
tissue, surface-sterilized by dipping in 1% sodium hypo- days. Mycelium was scraped from the surface of the plate
chlorite for 3–5 min, and rinsed three times with sterile and ground with 200 mg of sterilized quartz sand and
water. They were then placed on the surface of water agar 600 μL of 2 × CTAB extraction buffer (2% w/v CTAB,
(WA, Oxoid Ltd.) and incubated at room temperature 100 mm Tris HCl, 1·4 m NaCl, 20 mm EDTA, pH 8) in a
(28–30°C). The growing edges of any hyphal mycelium 1·5-mL Eppendorf tube. The whole contents were incu-
developing from the disease tissue discs were then trans- bated at 60°C in a water bath for 40 min with occasional
ferred aseptically to potato dextrose agar (PDA, Oxoid swirling. The solution was then extracted two or three
Ltd.). The fungi were identified following sporulation and times with equal volumes of phenol and chloroform (1:1)
single-spore isolation was carried out using the procedure at 17 530 g for 30 min until no interface was visible. The
described by Choi et al. (1999), with modifications. Direct upper aqueous phase containing the DNA was precipi-
examination and single-spore isolation from infected tated by addition of 2·5 volumes of absolute ethanol and
fruits with sporulation was also carried out. Spore masses kept at –20°C overnight. The precipitated DNA was then
were touched with a sterilized wire loop and streaked on washed with 70% ethanol, dried under vacuum, suspended
to the surface of WA plates which were then incubated in TE buffer (1 mm EDTA, 10 mm Tris-HCl, pH 8) and
overnight. A single germinated spore was picked up treated with RNase (1 mg mL–1).
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
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.
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
Length
13·0 a
21·0 c
0·30
(μm)
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
Appressorial morphology
There were few differences in appressorial shape and size
between groups. Most of the appressoria formed in slide
C. gloeosporioides
C. gloeosporioides
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
Chilli
Chilli
Host
180 were variable. Two trees were obtained when gaps were
treated as missing data in a weighted parsimony analysis.
group
3
4
5
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 by cylindrical conidia and with a colony growth rate of
consistency index = 0·836 and homoplasy index = 0·139) > 11 mm day–1. Cluster Y comprised C. acutatum isolates
is shown in Fig. 2. from chilli and strawberry. All isolates from this cluster
In order to compare tree output with morphological had fusiform conidia and a growth rate of > 5 mm day–1.
and cultural characters, the phylogeny generated from the Interestingly, C. acutatum isolates collected in Australia
combined dataset was selected because most of the major from strawberry and papaya formed a subcluster distinct
clusters and subclusters were more resolved and received from the isolates from Thailand, with high bootstrap
higher statistical support. As shown in Fig. 2, Colletotri- confidence. Cluster Z received high statistical support
chum isolates fell into three distinct lineages (clusters X, Y and consisted only of C. capsici isolates from chilli.
and Z) supported by 100% bootstrap values. Cluster X All isolates in this cluster were characterized by falcate-
consisted of C. gloeosporioides isolates from chilli and spored conidia and had an average growth rate of > 7 mm
mango. This cluster only included isolates characterized day–1.
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).
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
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 infection could not occur in PBC 932 without wounding,
indicated that spore size was homoplasious. Similar con- demonstrating the role of the cuticle in host resistance.
clusions were made by Hindorf (1973), who found a large Wounding was noticed to greatly enhance the ability
amount of morphometric overlap of conidial size within of Colletotrichum to cause disease (Pring et al., 1995).
Colletotrichum species. Differentiation of C. acutatum Oh et al. (1999) also showed the importance of cuticular
from C. gloeosporioides and C. capsici was reliable based wax layers of green and red pepper fruits to infection
on appressorial shape. However, species delineation by C. gloeosporioides, where a negative correlation was
between C. capsici and C. gloeosporioides based on this found between cuticle thickness and disease incidence.
character was not possible. Sanders & Korsten (2003a) Plant breeders need to be aware of the potential of
considered that appressorial shape was unreliable for C. acutatum to be a major pathogen when developing
species differentiation. new chilli cultivars for resistance to anthracnose
Cultural characteristics separated species from chilli, disease.
mango and strawberry. Isolates from chilli were separated The fact that C. acutatum from strawberry was a
into the three Colletotrichum species based on their pathogen of chilli confirmed numerous reports about the
cultural characters, and this was robustly supported by cross-infection potential among different species of Colle-
phylogenetic analysis. totrichum on a multitude of hosts (Freeman et al., 1998).
Colony growth rate in vitro was one of the important In contrast to cross-inoculation studies by Sanders &
characteristics for distinguishing between the three species Korsten (2003b), who showed that isolates of C. gloe-
of Colletotrichum. Phylogenies inferred from sequences osporioides from mango could produce symptoms on
supported a close relationship of isolates with the same other hosts such as guava, chilli pepper and papaya,
growth rate. Isolates of C. acutatum had the slowest growth isolates of C. gloeosporioides from mango did not show
rates. Simmonds (1965) and Sutton (1992) found that any symptoms on inoculated chilli fruits in the present
C. acutatum could be differentiated from C. gloeosporioides study. Although mango isolates of C. gloeosporioides were
by its slower growth rate. highly pathogenic when re-inoculated onto mango fruits
Molecular phylogenies did not show correlation between (data not shown), it is unclear why no symptoms were
DNA sequence data and host association within the produced on chilli fruits by the mango isolates. Further
Colletotrichum species. Colletotrichum capsici from chilli microscopic work is needed to examine the host reaction
constituted a distinct monophyletic group, whereas to initial infection by these pathogens. Despite the high
C. acutatum isolates from chilli were more related to other levels of infection potential on detached fruits, it is not
C. acutatum isolates from strawberry. This showed that known whether isolates could pose a threat in the field,
spore morphology and cultural characters reflected phy- since the inoculation studies were carried out under
logeny better than host association. Similar results were optimal conditions to induce infection by the pathogen
obtained before for C. acutatum (Du et al., 2005) and (Sanders & Korsten, 2003b). Further studies with different
C. gloeosporioides (Guerber et al., 2003), although these inoculation tests and different stages of ripeness are
results were not in accordance with the importance needed to confirm these results.
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
Acknowledgements
based on geographical isolation between Australia and We are grateful to the Mushroom Research Foundation,
Thailand. Studies by Denoyes-Rothan et al. (2003) on Chiangmai, Thailand for funding. The University of Hong
populations of C. acutatum on strawberry from a wide Kong is thanked for providing funds for the molecular
geographic range revealed both a homogeneous group work. Kasetsart University (Thailand) is acknowledged
and a highly variable group, with no direct correlation to for kindly supplying some isolates used in this study and
geographic areas. More isolates from Australia need to the Tropical Vegetable Research Center, Kasetsart Univer-
be assessed to further understand geographic divergence sity, is thanked for the supply of chilli fruits. Helen Leung
of C. acutatum. and Heidi Kong (University of Hong Kong) are thanked
Pathogenicity tests with the three Colletotrichum for laboratory assistance and Chutchamas Kanchana-
species isolated from infected chilli fruits showed that all udomkam (Kasesart University) is thanked for her assistance
the isolates were pathogenic on the susceptible Thai elite in the pathogenicity tests.
cultivar Bangchang. This result proved that these three
species of Colletotrichum were casual agents of anthracnose
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