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Keywords:

  • anthracnose;
  • epidermal cell;
  • hot pepper;
  • infection process;
  • pathogenicity

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This study showed that Colletotrichum acutatum penetrates the cuticle layer of Capsicum spp. fruits by forming a previously uncharacterized structure from appressoria. This unusual structure was localized in the cuticle layer. The structure, formed within 24 h post-inoculation (hpi), was a highly branched, well-differentiated hypha which penetrated the epidermal cell at 72 hpi. The novel structure, with abnormally thick walls (about 250 nm), often formed multiple branches in the affected chilli pepper. This dendroid structure, probably required for penetration, was formed exclusively in the cuticle layer of chilli pepper fruits and was not found when C. acutatum was inoculated onto pepper petals, mango leaves, or fruits of tomato and aubergine. Colletotrichum acutatum produced similar dendroid structures within resistant chilli pepper fruits, but eventually these structures turned dark brown and no further infection in the epidermal cells occurred, implicating the presence of inhibitors of the formation and development of the dendroid penetration structure in the resistant line.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The genus Colletotrichum includes many highly pathogenic species that affect numerous economically important crops, especially in subtropical and tropical areas. The establishment of infection in host plants by Colletotrichum species has been well studied (Bailey et al., 1992). Spores attach to the plant surface by secreting extracellular matrix. After germination, the germ tube produces a swollen and heavily melanized appressorium for penetration. The deposition of melanin is thought to decrease appressorial permeability, which allows this highly differentiated structure to accumulate turgor pressure when mature. The turgor pressure generated through the accumulation of glycerol forces a penetration peg to penetrate the host cuticle layer (O’Connell et al., 2000). As demonstrated on avocado and mango fruits, the penetration pegs can penetrate very thick cuticles (Prusky & Plumbley, 1992).

Colletotrichum acutatum is one of the major pathogens of chilli peppers in Taiwan. This fungal pathogen also infects many other economically important plants worldwide such as peach, almond, citrus, blueberry and strawberry (Wharton & Diéguez-Uribeondo, 2004; Peres et al., 2005). Colletotrichum acutatum displays a penetration process similar to other species in the genus Colletotrichum, but does not always rely on appressoria for penetration. It can penetrate through stomata on almond leaves (Wharton & Diéguez-Uribeondo, 2004) and directly penetrates citrus flowers without formation of appressoria, even though appressoria might be required to penetrate citrus leaves (Agostini et al., 1992; Zulfiqar et al., 1996).

Colletotrichum acutatum also displays different types of infection after breaching plant cuticles. On citrus, the C. acutatum pathotype causing postbloom fruit drop resides on citrus leaves as a quiescent infection, displaying biotrophic growth on epidermal cells without infecting the neighbouring cells (Zulfiqar et al., 1996). On apple, peach and blueberry fruits, C. acutatum also displays a biotrophic lifestyle by producing primary hyphae in the epidermal cells (Wharton & Diéguez-Uribeondo, 2004). Colletotrichum acutatum does not kill the affected host cells during biotrophic growth. The fungus produces slender secondary hyphae to initiate the necrotrophic stage, in which the infected host cells are killed by the fungus. On strawberry petioles and stolons or almond leaves and petals, C. acutatum hyphae exhibit intramural biotrophic growth after penetrating the host cuticles (Curry et al., 2002; Arroyo et al., 2005; Diéguez-Uribeondo et al., 2005).

Chilli pepper (Capsicum spp.) anthracnose has been reported to be caused by five species of Colletotrichum, viz. C. gloeosporioides, C. capsici, C. acutatum, C. coccodes and C. boninense (Hadden & Black, 1988; Than et al., 2008; Tozze et al., 2010). Among these, C. acutatum is the most widely distributed and aggressive species in Taiwan (Lin et al., 2007). Colletotrichum gloeosporioides has been reported to form appressoria on pepper fruit surfaces and penetrate the cuticle layer with penetration pegs to reach epidermal cells (Oh et al., 1998, 2003; Kim et al., 1999, 2004). The infection process of C. acutatum on other host plants is also well documented. However, the penetration strategy and infection process of C. acutatum on chilli pepper fruits have not yet been elucidated.

During the course of the present study, which aimed to elucidate the strategy used by C. acutatum to penetrate pepper fruits, an abnormal penetration structure was observed, formed by the fungus in the inoculated areas. The study sought to determine the nature of this previously uncharacterized structure using green fluorescent protein (GFP)-tagged transformants and microscopy.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Fungal isolates and cultivation

The C. acutatum isolates Coll-153, Coll-365, Coll-522, Coll-524 and Coll-573 were single-spore cultured from peppers with anthracnose symptoms and kindly provided by AVRDC-The World Vegetable Center (Tainan, Taiwan). Isolate CL-11 was obtained from the Bureau of Animal and Plant Health Inspection and Quarantine (Taipei, Taiwan). For sporulation, fungal isolates were cultured by spreading spore suspension (appropriately 1 × 106 spores mL−1) on PSA agar (200 g potato extract, 20 g sucrose and 18 g agar L−1) plates and incubating at 24°C for 5 days. Conidia were harvested, suspended in sterile water, filtered through a layer of Miracloth (Calbiochem) to remove mycelia, and used for in vitro and in planta inoculation as well as Agrobacterium-mediated DNA transformations. Agrobacterium-mediated T-DNA integration was performed as described previously (Lee & Bostock, 2006). The binary vector pPTGFPH (Marek et al., 2002), carrying a stable green fluorescent protein (sGFP) from pCT74 (Lorang et al., 2001), was kindly provided by Dr Bostock (University of California-Davis, USA).

Plant inoculation

Pepper plants used in this study include chilli peppers Capsicum chinense (AVRDC PBC-932), Ca. baccatum (AVRDC PBC-81), Ca. annuum cv. Gishin (ornamental chilli pepper), Ca. annuum cvs Groupzest and Chivalry and sweet pepper (Ca. annuum V-065). Seeds were provided by AVRDC or purchased from a local seed company (Known-You Seed). All plants were maintained in a greenhouse located at the National Chung-Hsing University (NCHU) campus (Taichung, Taiwan). Mature sweet pepper, tomato and aubergine fruits were purchased from local markets, washed with tap water, rinsed with reverse osmosis (RO) water and dried. Fruits, petals or leaves were detached and inoculated by placing 5 μL conidial suspension (1 × 105 spores mL−1) on surfaces, then incubated in a mist chamber at room temperature (approximately 27/22°C, day/night) for symptom development. For tomato and pepper petal inoculation, three drops of conidial suspension were placed on the surface of each petal or fruit. For inoculation on chilli pepper fruits (green and red) and leaves, as well as aubergine fruits, five to seven drops were placed on the surface from the end to the head of the leaf or fruit. Green and red fruits of all tested chilli pepper cultivars were used. Half of the inoculated materials were used for the examination of the formation of the infection structure and the other half were incubated further for symptom development. Colletotrichum acutatum isolates Coll-153, Coll-365 and Coll-524 were used to inoculate all the plant materials used in this study, while Coll-522, Coll-573 and CL-11 were used to inoculate the green fruits of chilli pepper cv. Groupzest. Sterile distilled water was used for mock-inoculated tissues.

Isolation of cuticle layer and inoculation

Cuticle layers were purified from chilli pepper fruits as described (Bostock et al., 1999). Fruit peels were sliced, soaked in water, and autoclaved twice. Peels were scraped gently with a teaspoon, extracted with chloroform/methanol (2:1, v:v) for 24 h, and dried in a fume hood to remove the organic solvents. Samples were treated with a cell-wall-degrading enzyme solution containing 1 mg cellulase (Onozuka RS), 1 mg macerozyme (Yakult Honsha) and 44 μL pectinase (Sigma, P-4716) dissolved in 20 mL 0·05 m sodium acetate buffer (pH 4·0) at 30°C for at least 14 h. After three rinses with Milli-Q water, cuticle strips were dried in air and extracted with chloroform/methanol (2:1, v:v) again. The extracted cuticle strips were stored at −20°C until needed. Cuticle strips were carefully placed on the surface of 1·5% water agar. Conidial suspension (10 μL; 1 × 105 spores mL−1) was placed onto the purified cuticle strips and the agar plate was incubated at room temperature.

Light and transmission electron microscopy

Conidial germination, appressorial formation, penetration, and fungal infection were examined using a Zeiss microscope equipped with a Spot CCD image system (Spot RT-2, Diagnostic Instruments). The affected fruit surface was carefully sliced into a thin layer with a double-edged blade and examined microscopically. Alternatively, a 3-mm2 fruit peel inoculated with C. acutatum was frozen and embedded with a cryogenic agent (Shandon cryomatrix, Thermo Scientific). The frozen tissues were transversely sectioned with a hand-adjusted microtome (PR-50F 130A, Yamato, Kohki Industrial) and examined microscopically. All experiments were performed at least three times with two or three fruits per cultivar.

To prepare ultrathin sections, samples were fixed with 2% glutaraldehyde (GA) in 0·2 m phosphate buffer (pH 7·2) at 4°C for 48 h. Samples were rinsed three times with 0·2 m phosphate buffer and incubated in 1% OsO4 (in 0·2 m phosphate buffer, pH 7·2) at 4°C for 16 h. Samples were washed with phosphate buffer, dehydrated with an ethanol series and embedded in a 100% ethanol/LR White resin (1:1, v:v) solution at 4°C for 24 h. The samples were carefully placed into a capsule filled with 100% LR White resin and heated at 60°C for 24 h. The solidified capsule was transversely sliced into small pieces (1·5 × 1·5 mm) using an ultramicrotome (Leica Ultracut R) equipped with a glass knife. Samples were stained with 1% toluidine blue and examined microscopically.

For transmission electron microscopy (TEM), samples were transversely sliced to 80 nm thick with a diamond knife, stained with 2% uranyl acetate and a lead citrate solution, and examined under an electron microscope (JEOL JEM-1400) at 1200 kV.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Formation of a novel penetration structure by C. acutatum in pepper fruits

Inoculation of the C. acutatum isolates onto the green fruits of susceptible chilli pepper cvs Chivalry and Groupzest resulted in characteristic anthracnose lesions 5 days post-inoculation (dpi). Microscopic analysis revealed that C. acutatum formed an irregular dendroid structure beneath the fruit surface (Fig. 1). The pigmented structure was not previously characterized in any Colletotrichum–plant interactions. This novel structure was often formed from the penetration pore of an immature or mature appressorium at 24 h post-inoculation (hpi) (Fig. 2). The structure was located primarily within the cuticle layer (Fig. 3), from where it apparently extended to epidermal cells of susceptible cultivars at 72 hpi.

image

Figure 1.  Formation of a previously unknown dendroid structure (ds) in chilli pepper fruit infected with Colletotrichum acutatum conidia (cn) 48 h post-inoculation. Scale bar = 10 μm.

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image

Figure 2.  Light microscopy images showing a novel dendroid structure (ds) produced from an appressorium (a) by Colletotrichum acutatum isolate Coll-524 on the green fruit of susceptible chilli pepper cv. Chivalry at 48 h post-inoculation (hpi) or cv. Groupzest at 72 hpi. Photos were taken serially focusing on the fruit surface, the bottom of the appressorium, or the cuticle layer. Fungal conidia (cn) germinated to produce germ tubes (gt), which subsequently formed mature appressoria (a) with distinct penetration pores (pp). Scale bar = 10 μm.

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image

Figure 3.  Localization of the novel dendroid structure (ds) in the cuticle layer (cu) of chilli pepper cv. Groupzest inoculated with Colletotrichum acutatum isolate Coll-153, 72 h post-inoculation. The dendroid structure facilitated fungal penetration into epidermal cells (ec). Fruit tissues were imbedded in freezing medium and sections were made with a hand-adjusted cryosectioning machine. Scale bar = 10 μm.

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The sequential development of this penetration structure and penetration process was revealed through a time-course analysis (Fig. 4). Colletotrichum acutatum produced an appressorium between 4 and 24 hpi and formed a highly branched structure in the cuticle layer between 24 and 48 hpi. The dendroid structure subsequently generated a swollen vesicle after penetration into epidermal cells at 72 hpi and primary hyphae proliferated within the cells. The dendroid structure is probably required for penetration and its formation might be the result of the unique cuticle structure in pepper fruits.

image

Figure 4.  The infection process of Colletotrichum acutatum in chilli pepper cv. Groupzest green fruit inoculated with field-collected isolate Coll-524. (a) Formation of small unmelanized appressoria (a) with conidia (cn) on the surface of the inoculated fruit at 12 h post-inoculation (hpi). (b) The novel dendroid structure (ds) with a pointed margin formed in the cuticle layer at 24–48 hpi. (c) Formation of a swollen infection vesicle (iv) underneath the dendroid structure at 48–72 hpi. (d) Formation of primary hyphae (ph) inside an epidermal cell from the swollen structure at 72 hpi. (e) Schematic diagram illustrating the infection process in chilli pepper inoculated with C. acutatum. Scale bar = 10 μm.

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Conidia of C. acutatum germinated and produced the dendroid structure in different ways on chilli pepper. Conidia could germinate to produce germ tubes and subsequently appressoria from one or multiple ends. The lengths of germ tubes varied considerably. The dendroid structure could be formed from both mature and immature appressoria. Most of the dendroid structures were highly branched hyphae with pointed or blunted tips, ranging from 0·5–2·2 μm in width. The primary hypha, formed in the primary infected epidermal cell, was measured as about 5 μm in width. Although C. acutatum was also able to infect detached petals of chilli pepper (cv. Chivalry), causing anthracnose lesions, it did not produce the dendroid structure within the pepper petals (data not shown). It was unable to cause necrotic lesions on detached pepper leaves (AVRDC PBC-81and cv. Groupzest) and did not produce any penetration structure.

When inoculated onto different pepper cultivars, including Ca. chinense (AVRDC PBC-932), Ca. baccatum (AVRDC PBC-81) and Ca. annuum (V-085 and cvs Gishin and Chivalry), all test fungal isolates, including Coll-153, Coll-365 and Coll-524, produced this structure. In addition to these latter three isolates, isolates Coll-522, Coll-573 and CL-11 also produced similar dendroid structures when inoculated onto green fruits of the Ca. annuum cv. Groupzest.

Confirmation of the dendroid structure formed by a GFP-tagged C. acutatum

To characterize this dendroid fungal structure further, GFP-tagged transformants from three C. acutatum isolates were generated by Agrobacterium-mediated transformation. After three rounds of single-spore isolations, fungal isolates Coll-365 GFP-1, Coll-153 GFP-197 and Coll-524 GFP-100 were selected for further analysis. After inoculation onto pepper fruits, green fluorescence was clearly observed in the dendroid structure (Fig. 5). The GFP fluorescence was shown to extend into some of the apices of the dendroid structures, indicating that the apices were filled with fungal hyphae. However, not all of the apices were filled with GFP fluorescence, suggesting that dissolved cuticle materials may participate in the formation of the dendroid structures.

image

Figure 5.  Microscopic examination of dendroid structures of the GFP-tagged transformant Coll-365 GFP-1 of Colletotrichum acutatum on green fruit of cv. Groupzest at 48 or 72 h post-inoculation (hpi). Distinct septa are indicated with arrows. Scale bar = 10 μm.

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Histological analysis

As stated above, the highly branched penetration structure was formed in the cuticle layers. Histological analysis revealed severe tissue degradation of host plants inoculated with C. acutatum (Fig. 6). Plant cuticles and intracellular spaces were stained a green-blue colour and cell walls stained red with toluidine blue. Fungal cells and some plant cellular contents, probably chloroplasts, appeared as a dark red colour. Light microscopy revealed that the cuticle layers and epidermal cells from the mock-inoculated control fruits were apparently intact and healthy (Fig. 6a). In contrast, inoculation of C. acutatum on chilli peppers resulted in apparent disruption of the epidermal cells at 48 hpi. The cells were completely collapsed at 96 hpi and cellular organelles were deformed. Germination of conidia (two-celled), formation of appressoria and penetration structures, and fungal hyphae, as well as extracellular matrix surrounding conidia, appressoria and penetration structures, were clearly present on fruit surfaces or within the degraded tissues (Fig. 6a). TEM further revealed that the structures were often surrounded by materials of low electron density – probably extracellular matrices or areas of cuticle dissolution (Fig. 6b). The structure had thick walls (approximately 0·25 μm) and pointed edges, as indicated in Figure 6b.

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Figure 6.  (a) Light microscopy of green fruits of chilli pepper cv. Groupzest 48 or 96 h post-inoculation (hpi) with sterile water (mock) or Colletotrichum acutatum isolate Coll-524. Samples were sectioned, stained with toluidine blue and examined microscopically, showing deformation of epidermal cells (ec). Appressoria (a), extracellular matrix (ecm), penetration structures (ps) and primary hyphae (ph) were clearly observed. Plant cuticles (cu) are indicated. Fruit tissues were fixed, dehydrated, imbedded, cut to ultrathin sections (500 nm thick) and stained with toluidine blue. Scale bar = 10 μm. (b) Transmission electron microscopy of the cuticle layer of chilli pepper fruit inoculated with C. acutatum, showing hyphal branching point (hbp), fungal cell (fc), fungal cell wall (fcw) and the penetration structure (psp) with a pointed edge. The transverse sections (80 nm) were prepared with a diamond knife.

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Formation of the structure in vitro

To determine the components essential for the formation of this penetration structure, cuticle layers of pepper fruits were carefully isolated and inoculated with C. acutatum. As observed at 24 hpi, the fungal pathogen formed the dendroid structures immediately after inoculation from immature appressoria on the isolated cuticle strip (Fig. 7). The dendroid structures formed at 24 hpi were much larger than those formed in the cuticles of intact fruits at 24 hpi (data not shown).

image

Figure 7.  Light microscopy images of cuticle strips from chilli pepper cv. Groupzest green fruits 24 h post-inoculation with Colletotrichum acutatum isolate Coll-524. Scale bar = 10 μm. (a) A small unmelanized appressorium (a) produced from a conidium (cn) on the surface of the inoculated cuticle strip. (b) Formation of dendroid structure (ds) from the appressorium in the cuticle strip.

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Absence of the dendroid structure in other members of the Solanaceae

To determine if C. acutatum would form the novel dendroid penetration structure on other plants of the Solanaceae, the C. acutatum isolates were inoculated onto fruits of tomato and aubergine. The results revealed no such dendroid structures in either of these species. On tomato fruits, fungal primary hyphae were found to grow inside the epidermal cells and form anthracnose lesions without forming any dendroid structure (data not shown). On aubergine, spores formed appressoria on the fruit surface with no penetration structure or anthracnose lesions.

The green fruit of AVRDC accession PBC-81 is highly resistant to C. acutatum (C-Y Liao, M-Y Chen, Y-K Chen, P-F Chang, M-H Lee, National Chung-Hsing University, Taiwan; T-C Wang, Z-M Sheu, AVRDC Taiwan; K-C Kuo, Bureau of Animal and Plant Health Inspection and Quarantine, Taiwan; K-R Chung, Citrus Research and Education Center, IFAS, University of Florida, USA, our unpublished data). Inoculation of isolates Coll-153, Coll-365 or Coll-524 onto green fruits of PBC-81 often resulted in no or small lesions, even after a prolonged incubation (Fig. 8a and data not shown). All fungal isolates germinated, produced appressoria on fruit surfaces and formed the dendroid penetration structures at 24 hpi (Fig. 8b), similar to those formed on the susceptible fruits. As observed at 13 dpi, the penetration structures and the host epidermal cells within the resistant cultivar became dark brown, implicating cell death (Fig. 8c). Fungal hyphae were not observed inside the epidermal cells of the resistant line. Fluorescence microscopy confirmed the presence of yellow-orange fluorescence inside the browned epidermal cells, suggestive of cell death (Fig. 8d). As tested in vitro, C. acutatum was also able to form dendroid structures on the cuticle strips purified from PBC-81 green fruits (resistant line) and continuously grew into epidermal cells.

image

Figure 8.  (a) Pinpoint lesions of Colletotrichum acutatum isolate Coll-524 on a resistant chilli pepper green fruit (AVRDC accession PBC-81) at 13 days post-inoculation (dpi). (b,c) Light microscopy showing dark pigmented dendroid structure (ds) (b) and necrotic epidermal cells (indicated by arrows in c). (d) Fluorescence microscopy showing formation of yellow-orange fluorescence (indicated with arrows) around dendroid structures. Scale bar = 10 μm.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This study demonstrated the formation of a novel penetration structure, showing a dendroid appearance, by C. acutatum to grow through the cuticle layer of pepper fruits. This penetration structure was produced exclusively in the cuticle layers of Capsicum spp. fruits, but not in petals, and was capable of expanding and penetrating into the epidermal cells.

The penetration structure is highly branched and has not been previously reported in any Colletotrichum–plant associations, even though the infection process of Colletotrichum species has been well documented (Politis & Wheeler, 1973; O’Connell et al., 1985; Mould et al., 1991; Shen et al., 2001; Curry et al., 2002; Kim et al., 2004; Arroyo et al., 2005; Diéguez-Uribeondo et al., 2005; Wharton & Schilder, 2008). Most studies have shown that Colletotrichum spp. penetrate the host cuticle layer and infect epidermal cells by the formation of penetration pegs, but in the present study the penetration structure was primarily produced from the penetration pore of an appressorium and was able to penetrate the cuticle of chilli pepper. This dendroid structure is composed of multiple, thick-walled hyphal branches with swollen or sharp ends. As evidenced using GFP-tagged transformants in chilli pepper, the radial branches contain multiple septa. The mechanism by which the fungus generates the highly branched structure remains unknown. Because this penetration structure was solely formed in chilli pepper fruits, it is tempting to speculate that the chilli pepper fruits may contain unique yet unidentified compounds in the cell walls that trigger C. acutatum to develop this unusual structure. This assumption was further supported by the fact that the cuticles purified from chilli pepper were able to induce the formation of dendroid penetration structures in vitro. All C. acutatum isolates from pepper were capable of causing anthracnose lesions on the tomato fruits and mango leaves (C-Y Liao, M-Y Chen, Y-K Chen, P-F Chang, M-H Lee, National Chung-Hsing University, Taiwan; T-C Wang, Z-M Sheu, AVRDC Taiwan; K-C Kuo, Bureau of Animal and Plant Health Inspection and Quarantine, Taiwan; K-R Chung, Citrus Research and Education Center, IFAS, University of Florida, USA, our unpublished data). However, no similar penetration structure was formed in these host plants, indicating further the uniqueness of chilli pepper cuticles in the formation of this dendroid penetration structure. Interestingly, although C. acutatum could cause necrosis lesions on detached flower petals of chilli pepper under high humidity, it did not produce the dendroid structure in this case. These results strongly indicate that C. acutatum produces the highly branched penetration structure specifically in response to the components of chilli pepper fruits in order to penetrate more effectively.

Colletotrichum acutatum utilizes various infection strategies to invade its host plant. Many C. acutatum pathotypes, such as those that infect almond leaves, strawberry petioles or blueberry fruits, can proliferate within subcuticular layers (Curry et al., 2002; Arroyo et al., 2005; Diéguez-Uribeondo et al., 2005; Wharton & Schilder, 2008). However, none of these subcuticular hyphae of C. acutatum are able to penetrate cuticle layers or differentiate to form a dendroid penetration structure as observed here in chilli pepper.

Colletotrichum acutatum could form the dendroid penetration structure in pepper fruit cuticles of both resistant (e.g. PBC-81) and susceptible cultivars (e.g. cv. Groupzest green fruit). However, the dendroid structure formed on the resistant fruit cuticle tended to turn dark brown, indicating death of the cells. The results implicate the presence of inhibitors of the formation and development of the dendroid penetration structure in the resistant line.

Fungi have been shown to vary their hyphal sizes during growth in different conditions. For example, C. lindemuthianum produced penetration pegs 100–200 nm in diameter to pass through the cuticle of bean hypocotyls (O’Connell et al., 1985). Furthermore, hyphae of Magnaporthe grisea were capable of enlarging plant plasmodesmata (approximately 50 nm) up to 10-fold during penetration (Kankanala et al., 2007). The hyphae of the novel dendroid structure produced by C. acutatum appear to be smaller, approximately 0·5–3 μm in width, compared to the primary hyphae (approximately 3–5 μm), which indicates that the fungal pathogen is under stress inside the chilli pepper. Thus, the formation of the dendroid structure may allow the fungal pathogen to invade chilli pepper effectively. The branched hyphae were eventually able to penetrate the pepper cuticle layer and grow inside the epidermal cells.

The novel dendroid penetration structure has thickened cell walls and pointed edges, which indicates that the pathogen encounters extreme conditions inside the cuticle of chilli pepper and requires this highly differentiated structure to penetrate host plants successfully. In fungi and oomycetes, only resting structures such as oospores or chlamydospores, or the supporting structures such as binding hyphae of basidiomycetes, have thick walls. The tramal binding hyphae of basidiomycetes are highly branched and are often dead cells serving as skeleton structures in basidiocarps (Sotome et al., 2007). Hyphal branching probably increases surface area for colonization. Alternatively, hyphal branching might facilitate hyphal fusion, thereby promoting exchange of genetic materials between different hyphae of the same or different fungi (Harris, 2008). Highly branched fungal structures, especially vegetative hyphae, are not commonly formed by fungi (Harris, 2008). However, the Aspergillus nidulans hbr mutant with unknown genotype and the Neurospora crassa cot1 mutant, with a defective protein kinase, have been shown to produce highly branched hyphae (Yarden et al., 1992; Gatherar et al., 2004). A Cot1 homologue (TB3) cloned from Colletotrichum trifolii was able to restore normal elongation of hyphae defective in the N. crassa cot1 mutant (Buhr et al., 1996). Genetic determination of a TB3 homologue for the formation of the dendroid penetration structure by the chilli pepper pathotype of C. acutatum is currently underway.

Because the novel dendroid penetration structure was consistantly formed in chilli pepper inoculated with C. acutatum, it could be an important pathogenicity factor. More research will be needed to identify the cellular and environmental factors regulating its formation and to determine if this penetration structure is absolutely required for pathogenicity in chilli pepper.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The authors thank Drs W.-H. Ko, C.-Y. Chen, P.-F. Chang (NCHU), P. J. Ann (Agricultural Research Institute) and Dr J.-F Wang and Mr Z.-M. Sheu (AVRDC) for their helpful comments and suggestions. Research was supported in part by grants from the Department of Plant Protection, Bureau of Animal and Plant Health Inspection and Quarantine, Council of Agriculture, Executive Yuan, Taipei, Taiwan, under project numbers 97AS-14.2.3-BQ-B5 (7) and 98AS-9.2.4-BQ-B6 (2) to MHL.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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