Characterization and pathogenicity of Colletotrichum species associated with anthracnose on chilli (Capsicum spp.) in Thailand

Authors

  • P. P. Than,

    1. Mushroom Research Centre, 128 Moo3 Ban Ph Deng, T. Pa Pae, A. Mae Taeng, Chiang Mai 50150;
    2. Department of Agronomy, Maejo University, Sansai, Chiang Mai 50290;
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  • R. Jeewon,

    Corresponding author
    1. Centre for Research in Fungal Diversity, School of Biological Sciences, University of Hong Kong, Pokfulam Road, Hong Kong;
      *E-mail: rajeshjeewon@yahoo.com
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  • K. D. Hyde,

    Corresponding author
    1. Centre for Research in Fungal Diversity, School of Biological Sciences, University of Hong Kong, Pokfulam Road, Hong Kong;
      **E-mail: kdhyde@hkucc.hku.hk
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    • Present address: International Fungal Research & Development Centre, Research Institute of Resource Insects, Chinese Academy of Forestry, Kunming 650224, Yunnan, PR China.

  • S. Pongsupasamit,

    1. Department of Agronomy, Maejo University, Sansai, Chiang Mai 50290;
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  • O. Mongkolporn,

    1. Department of Horticulture, Kasetsart University, Kamphaengsaen Campus, Nakhon Pathom 73140;
    2. Center for Agricultural Biotechnology, Kasetsart University, Kamphaengsaen Campus, Nakhon Pathom 73140, Thailand; and
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  • P. W. J. Taylor

    1. Centre for Plant Health/BioMarka, School of Agriculture and Food Systems, University of Melbourne, Victoria 3010, Australia
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*E-mail: rajeshjeewon@yahoo.com

**E-mail: kdhyde@hkucc.hku.hk

Abstract

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.

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) considered 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 often wet. Chilli pepper fruits with blemishes have reduced marketability (Manandhar et al., 1995).

Anthracnose of chilli has been shown to be caused by at least four species of Colletotrichum: Ccapsici 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 necessary 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 phenotype among species under different environmental conditions. To overcome taxonomic problems associated with these traditional identification methods, DNA sequence analyses were used to characterize and resolve the taxonomic 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 classification 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 elongation 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 morphological 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 procedure 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 hypochlorite 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 transferred 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 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 Collection (HKUCC) (Table 1).

Table 1.  Sources of Colletotrichum isolates used in this study and reference sequences from GenBank used in analysis
HKUCC Acc. no.aITSβ-tubulinIsolateColletotrichum speciesLocationHost
  • 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.

10860DQ454001DQ454043M1C. gloeosporioidesChiang Mai, ThailandMangifera indica
10863DQ454004DQ454041M4C. gloeosporioidesChiang Mai, ThailandMangifera indica
10849DQ454005DQ454044M5C. gloeosporioidesChiang Mai, ThailandMangifera indica
10891DQ454018DQ454066S2C. acutatumChiang Mai, ThailandFragaria sp.
10872DQ454019DQ454063S3C. acutatumChiang Mai, ThailandFragaria sp.
10873DQ454020DQ454062S4C. acutatumChiang Mai, ThailandFragaria sp.
10890DQ454021DQ454065S5C. acutatumChiang Mai, ThailandFragaria sp.
10814DQ454022DQ454064S6C. acutatumChiang Mai, ThailandFragaria sp.
10871DQ454023DQ454067S7C. acutatumChiang Mai, ThailandFragaria sp.
10882DQ453991DQ454035Ku1C. gloeosporioidesRatchaburi, ThailandCapsicum annuum
10883DQ453992DQ454036Ku2C. gloeosporioidesRatchaburi, ThailandCapsicum annuum
10892DQ453993DQ454040Ku3C. gloeosporioidesRatchaburi, ThailandCapsicum annuum
10884DQ453994DQ454029Ku4C. gloeosporioidesKanchanaburi, ThailandCapsicum annuum
10889DQ453996DQ454034Ku6C. gloeosporioidesKanchanaburi, ThailandCapsicum annuum
10887DQ454000DQ454032Ku10C. gloeosporioidesKanchanaburi, ThailandCapsicum annuum
10864DQ453995DQ454030Ku5C. gloeosporioidesKanchanaburi, ThailandCapsicum annuum
10881DQ453998DQ454031Ku8C. gloeosporioidesNakhonpathon, ThailandCapsicum annuum
10888DQ453999DQ454033Ku9C. gloeosporioidesNakhonpathon, ThailandCapsicum annuum
10848DQ454006DQ454058Mj2C. acutatumChiang Mai, ThailandCapsicum annuum
10865DQ454007Mj3C. acutatumChiang Mai, ThailandCapsicum annuum
10879DQ454008DQ454059Mj4C. acutatumChiang Mai, ThailandCapsicum annuum
10893DQ454010DQ454061Mj6C.acutatumChiang Mai, ThailandCapsicum annuum
10850DQ454011DQ454068Mj9C. acutatumChiang Mai, ThailandCapsicum annuum
10894DQ454012DQ454069Mj10C. acutatumChiang Mai, ThailandCapsicum annuum
10875DQ453987DQ454045Ccmj2C. capsiciChiang Mai, ThailandCapsicum annuum
10857DQ453988DQ454047Ccmj3C. capsiciChiang Mai, ThailandCapsicum annuum
10858DQ453989DQ454048Ccmj7C. capsiciChiang Mai, ThailandCapsicum annuum
10859DQ453990DQ454054Ccmj10C. capsiciChiang Mai, ThailandCapsicum annuum
10855DQ454024DQ454052Skp4C. capsiciChiang Mai, ThailandCapsicum annuum
10877DQ454025DQ454055Skp16C. capsiciChiang Mai, ThailandCapsicum annuum
10868DQ454013DQ454046R4C. capsiciChiang Mai, ThailandCapsicum annuum
10852DQ454014DQ454056R5C. capsiciChiang Mai, ThailandCapsicum annuum
10869DQ454015DQ454053R7C. capsiciChiang Mai, ThailandCapsicum annuum
10870DQ454016DQ454049R11C. capsiciChiang Mai, ThailandCapsicum annuum
10880DQ454017DQ454057R12C. capsiciChiang Mai, ThailandCapsicum annuum
10866DQ454026U9C. capsiciUbonratchathani, ThailandCapsicum annuum
10876DQ454027DQ454050U10C. capsiciUbonratchathani, ThailandCapsicum annuum
 AY376525AY376573bSTEU-2289C. boninenseZimbabweProteaceae
 DQ195680DQ195719cBRIP 26974C. capsiciQLD, AustraliaCapsicum frutescens
 EF143971EF143967BRIP 4703aC. acutatumTownsville, QLD, AustraliaFragaria×ananassa
 EF143972EF143968BRIP 4704aC. acutatumForest Glen, QLD, AustraliaFragaria×ananassa
 EF143974EF143969BRIP 11086aC. acutatumNambour, QLD, AustraliaFragaria×ananassa
 EF143975EF143970BRIP 28519aC. acutatumYandina, QLD, AustraliaCarica papaya

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 investigated. 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 incubated 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 precipitated 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).

PCR and sequencing

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 elongation at 72°C for 1 min; and a final extension step of 72°C for 10 min. PCR products were verified by staining with ethidium bromide on 1% agarose electrophoresis gels. PCR products were then purified using the GFX PCR Purification Kit (27-9602-01; Amersham Biosciences) according to the manufacturer's protocol. DNA sequencing 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).

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 monophyletic 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), Cacutatum 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 fluorescent 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.

Disease reactions of the host were evaluated by measuring 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 description 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).

Table 2.  Summary of morphological data for Colletotrichum species in groups 1–5
Morphological groupHostSpeciesColony characterConidiaAppressoriaGrowth rate mm day−1
Length (µm)Width (µm)ShapeLength (µm)Width (µm)
  1. 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.

1MangoC. gloeosporioidesLight orange colony colour with delicate and thin mycelium13·5 a4·0 cCylindrical9·0 b6·5 c11·0 c
2ChilliC. gloeosporioidesPale grey to black zonated colonies with abundant orange conidial masses near the centre13·5 a4·5 dCylindrical9·0 b6·3 c11·2 c
3StrawberryC. acutatumWhite to olive grey colour colony with very thick cottony mycelium13·0 a3·5 bFusiform7·0 a5·5 a 5·8 a
4ChilliC. acutatumOrange-coloured colony with slight mycelium14·0 b3·5 bFusiform6·5 a 6·0 b 5·8 a
5ChilliC. capsiciWhite to grey colour with dark green centre and cottony mycelium21·0 c3·0 aFalcate9·5 b6·5 c 7·1 b
 LSD (between group) 0·300·25 0·750·20 0·27

Morphological examination

Distinctness in spore morphology and colony characteristics 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 alternating 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 grey to black aerial mycelium, whilst others (Ku6, Ku9 and Ku10) produced aerial mycelium in an even, felted 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 from group 4, chilli C. acutatum, produced pale orange colonies with little aerial mycelium and a few orange conidial masses around the centre. Isolates from group 5, chilli C. capsici, produced colonies that were white to grey; most of the isolates showed the diurnal zonation of dense and sparse development of aerial mycelium, sometimes with beige-coloured spore masses.

Figure 1.

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

Growth rate

An important comparative character was the growth rate of the colony in culture. There was no significant difference in growth rate among isolates of the same species, i.e. among isolates of C. acutatum belonging to groups 3 and 4 (P = 0·168) or among isolates of C. gloeosporioides belonging to groups 1 and 2 (P = 0·817). However, a 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 day−1) grew significantly faster than any other groups (P = 0·001), followed by isolates of C. capsici from group 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).

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 group 5 produced falcate conidia and C. acutatum isolates belonging to groups 3 and 4 produced predominantly fusiform conidia (80% average occurrence). Colletotrichum gloeosporioides isolates from groups 1 and 2 produced 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 cultures were irregularly shaped and only a few were ovoid. Ovoid appressoria were commonly observed in slide cultures from C. acutatum isolates from strawberry in groups 3 and 4.

Phylogenetic analyses

PCR products obtained from the ITS regions (including 5.8 S) ranged from 550 to 600 bp, whereas those from the β-tubulin gene ranged from 450 to 500 bp. The final sequence 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 180 were variable. Two trees were obtained when gaps were treated as missing data in a weighted parsimony analysis. The topologies of the two trees were not significantly different and one of the trees (total length = 721 steps, consistency index = 0·861, retention index = 0·971, rescaled consistency index = 0·836 and homoplasy index = 0·139) is shown in Fig. 2.

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).

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, Colletotrichum 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.

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 Cacutatum 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 anthracnose 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.

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
SpeciesHostGroupIsolateBangchangPBC 932
Wound/dropNon-wound/dropWound/dropNon-wound/drop
Infected fruit area (%)Host reactionInfected fruit area (%)Host reactionInfected fruit area (%)Host reactionInfected fruit area (%)Host reaction
  • 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.

C. gloeosporioidesMango1M1 0  0  0 0 
  M2 0  0  0 0 
  M4 0  0  0 0 
  mean 0  0  0 0 
C. gloeosporioidesChilli2Ku420·60 ac 17·5 a  0 0 
  Ku523·21 a 18·43 a  0 0 
  Ku824·99 a 19·39 a  0 0 
  mean24·57VS17·27VS 0HR0HR
C. acutatumStrawberry3S221·24 a 14·47 a  0 0 
  S431·90 a 15·62 a  0 0 
  S520·48 a 15·26 a  0 0 
  mean23·23VS15·12VS 0HR0HR
C. acutatumChilli4Mj432·60 a 13·98 a 38·28 a 0 
  Mj534·00 a 24·99 a 20·47 a 0 
  Mj1021·86 a 15·19 a 14·92 a 0 
  mean28·97HS24·47VS24·56VS0HR
C. capsiciChilli5R423·81 a 21·00 a  0 0 
  Skp426·34 a 18·60 a  0 0 
  Ccmj1031·37 a 18·15 a  0 0 
  mean27·17HS18·92VS 0HR0HR
LSD (between isolates)17·46 21·33  47·43  
LSD (between groups)10·39 11·70     

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 Cacutatum from strawberry (group 3), C. capsici (group 5) 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 inoculation, 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 characterized by similar conidial size. These results indicated that spore size was homoplasious. Similar conclusions 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 phylogeny 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 (Pakdeevaraporn 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 Colletotrichum 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. gloeosporioides 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 University, 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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