Light quality influences the virulence and physiological responses of Colletotrichum acutatum causing anthracnose in pepper plants

Authors


  • The first two authors contributed equally to this study.

Correspondence

Yong Hoon Lee, Division of Biotechnology, Chonbuk National University, 194-5 Ma-Dong, Iksan, Jeonbuk 570-752, Republic of Korea. E-mail: yonghoonlee@jbnu.ac.kr

Abstract

Aims

To explore the effects of light quality on the physiology and pathogenicity of Colletotrichum acutatum, we analysed the morphological traits, melanin production and virulence of the pathogen under different light wavelengths.

Methods and Results

The influence of light wavelength on the mycelial growth and conidial germination of C. acutatum was investigated using red, green, blue and white light sources. Red and green light reduced the mycelial growth in comparison with blue and white light, and dark conditions. The least percentage of conidial germination was observed under blue light, while the germination rate among white, red and green light, as well as in the dark, was insignificant. In comparison with its influence on mycelial growth and conidial germination, light wavelength significantly affected the pathogen's virulence towards hot pepper fruits. The highest disease severity was observed under blue light, which was at least a twofold increase compared with the disease severity under other light conditions. To elucidate the effect of light on the disparity in virulence, scytalone was assayed by HPLC, and scd1 gene expression was examined with real-time PCR. The highest and lowest scytalone production was observed in the cultures incubated under blue (10·9 mAU) and green light (1·5 mAU), respectively. Higher scd1 gene expression (~ 40-fold increase) was observed in cultures incubated under blue and white light in comparison with those incubated in the dark.

Conclusions

This study revealed that light affects the growth, colonial morphology and virulence of C. acutatum. The pathogen needs light for its active melanin production and also to attain higher virulence.

Significance and Impact of the Study

This is the first report on the effect of light quality on the virulence of C. acutatum. The findings of this study will broaden our knowledge of the influence of light on physiological responses of fungal pathogens.

Introduction

Light is an important environmental factor for most living organisms, including fungi, which use light as a signal in many metabolic pathways. More than 100 fungal species, representing all phyla, have been found to be reactive to light (Tisch and Schmoll 2010). It regulates many metabolic activities, including circadian rhythms, asexual conidiation, pigmentation, secondary metabolism and sexual development (Purschwitz et al. 2006). Although the interaction between light and fungi has been studied and reviewed by many researchers (Idnurm and Heitman 2005; Purschwitz et al. 2006; Chen et al. 2009; Idnurm et al. 2010), reports on the effect of light qualities on fungal pathogenicity or virulence are limited.

The Colletotrichum genus includes many important and devastating plant pathogens, which cause a significant yield loss to many crop varieties and agricultural products. Almost every plant grown in the world is susceptible to one or more species of Colletotrichum; hence, it was ranked first among the top 10 significant fungi in a recent survey (Dean et al. 2012). Among the Colletotrichum species, Colletotrichum acutatum is one of the principle plant pathogens, which can infect a range of host plants including strawberry, tomato, apple, mango, peach, almond, citrus and blueberry (Peres et al. 2005; Liao et al. 2012a,b). In particular, this pathogen is the major causal agent of pepper anthracnose in many regions of the world and can lead to significant economic losses (Liao et al. 2012a,b).

Colletotrichum infection starts with the attachment of conidia to the surface of the host plant. The attached conidium germinates and forms densely melanized appressorium and penetrates the cuticle layer of the plant. After entering the host plant, this pathogen establishes itself by producing highly differentiated hyphae, such as haustoria. In this infection process, melanin plays an important role, helping to create turgor pressure within the appressorium to penetrate the host plant cuticle layer. Chen et al. (2004) reported that unmelanized appressoria of Colletotrichum kahawae produced one quarter of the pressure of the melanized appressoria, which led to a much lower percentage of infection in green berries. Several other studies also indicated that melanin is essential for fungal pathogenesis (Langfelder et al. 2003; Tsuji et al. 2003). Some other fungi, such as Verticillium dahlia and Alternaria alternata, need melanin not only for pathogenesis but for longevity and survival of fungal propagules (Tanabe et al. 1995), which protect the cells from enzymatic activities and abiotic stresses like heat (Rosas and Casadevall 1997).

In Colletotrichum and many other fungi, melanin is produced by the 1,8-dihydroxynaphthalene (1,8-DHN) pathway (Tsuji et al. 2003), which starts with malonyl-CoA molecules and forms the final product with the following intermediates: 1,3,6,8-tetrahydroxynaphthalene (1,3,6,8-THN), scytalone, 1,3,8-trihydroxynaphthalene (1,3,8-THN), vermalone and 1,8-dihydroxynaphthalene (1,8-DHN); the 1,8-DHN polymerizes to form melanin. Among the intermediates, scytalone is an essential intermediate for melanin production, the loss of which leads to a loss of pathogenicity (Kubo et al. 1996). Scytalone is converted into 1,3,8-THN by scytalone dehydratase (scd1), a 23-kDa polypeptide, which was characterized by Kubo et al. (1996).

Recent studies reported that light plays an important role in plant–pathogen interactions (Rahman et al. 2003; Wang et al. 2010; Yu and Lee 2013) and controls several metabolic activities of pathogenic fungi. This raises questions regarding how different wavelengths of light affect fungal virulence and what factor is responsible for the disparity in virulence. Hence, in the present study, the effect of light wavelength on the pathogenicity of C. acutatum was examined, with regard to various morphological characteristics, such as mycelial growth rate, conidia germination and pigmentation. In addition, scytalone, which is a melanin intermediate product, was quantified by chromatography, and the expression pattern of the scd1 gene was also analysed. Results of these experiments indicated that light wavelength affected the select physiological traits evaluated as well as melanin biosynthesis, which is an essential component of pathogenicity for C. acutatum. Different wavelengths of light led to various quantities of melanin production and, consequently, various degrees of virulence. To the best of our knowledge, this is the first report on the influence of light wavelengths on the virulence of C. acutatum.

Materials and Methods

Fungal culture and light sources

A pathogenic isolate of Colletotrichum acutatum (KACC40804) was obtained from the Korean Agricultural Culture Collection (KACC). The pure culture was maintained on potato dextrose agar (PDA) and revived periodically by subculturing on PDA. A conidial suspension was prepared by pouring sterile distilled water (DW) with 0·01% of Silwet L-77 (OSi. Specialties, Inc., CT, USA) over the surface of 7- to 10- day-old cultures grown on PDA and rubbing with a sterile glass rod. The spores were diluted to the required concentration using a haemocytometer. LED light sources were custom designed and constructed by ODTech Co. Ltd. (Jeonju, South Korea). Light sources of white-, red- (645 nm), green- (524 nm) and blue- emitting (458 nm) diodes were connected to the circuit box, which enabled the intensity (photosynthetically active photon flux densities; PPFD) of the light to be controlled (Yu and Lee 2013). In all light-based experiments, light sources were adjusted to receive 120 PPFD, unless otherwise specified.

Mycelial growth assay under different light wavelengths

To analyse the effect of light wavelength on the mycelial growth of C. acutatum, an agar plug (8 mm diameter) from the margin of 7–10-day-old fungal cultures was placed at the centre of each PDA plate. Four different light wavelengths (red, green, blue and white) were illuminated from the top of the lids of individual Petri plates. The light source and Petri plate were covered with aluminium foil to avoid interference from environmental light. Control plates also were covered with aluminium foil without any light source. The plates were incubated at 25°C, and the temperature difference among the incubated plates with and without light source was negligible (±1°C). The diameter of the colony was measured with two perpendicular cross sections 7 days after inoculation (DAI). The experiment was repeated three times with three replications.

Effect of light wavelengths on conidial germination

Five millilitres of conidial suspension (1 × 105 spores ml−1) in 10% potato dextrose broth (PDB) was poured into each empty Petri plate. Four different light sources were placed on the lids of individual Petri plates and covered with aluminium foil. Control plates were covered with aluminium foil without light source. These plates were incubated at 25°C for 6 h. After incubation, germination was stopped with lactophenol cotton blue, and specimens were observed under a light microscope. Germinated and nongerminated conidia were counted, and germ tube lengths of conidia were also measured under a light microscope. Approximately 200 conidia were analysed, at random, per sample. The conidia were considered to be germinated when the total length of conidia was double the size of the actual spore length. The experiment was repeated twice with three replicates.

In vivo disease severity assay on hot pepper fruits

Fresh hot pepper fruits were surface sterilized with 70% alcohol and washed twice with sterile water. The sterilized fruits were wounded with a toothpick (~1 mm in depth), and 10 μl of conidial suspension (1 × 105 spores ml−1) was placed in each wound. Inoculated fruits were kept in a plastic box and incubated under red, green, blue and white light wavelengths or in the dark at 25°C. High humidity (95% RH) was maintained by keeping water-saturated filter paper inside the plastic boxes. The diameter of infected area was measured with two perpendicular cross sections at 5 DAI. Five fruits with five wounds per fruit were used under each light treatment. The experiment repeated five times.

Assay on scytalone production

Five millilitres of conidial suspension (1 × 105 spores ml−1) in 10% PDB was poured into each empty Petri plate. Light sources were placed on the lids of individual Petri plates and covered with aluminium foil. Control plates were covered with aluminium foil without a light source, and the plates were incubated at 25°C for 48 h. After incubation, the pigmentation of the cultures was visually observed. For the quantification of scytalone, the culture solution was extracted with ethyl acetate and evaporated to dryness (Kubo et al. 1986). Initially, the scytalone (intermediate of melanin) fractions were analysed with thin-layer chromatography on silica plates (G60 F254, 8 × 8 cm, Merck, Darmstadt, Germany). The fractions having scytalone were separated and purified by reverse-phase high-performance liquid chromatography (RP-HPLC) using Zorbax C18 column (4·6 × 150 mm, Agilent, Palo Alto, CA, USA). Intermediates were eluted with a linear gradient of 10–100% (v/v) acetonitrile/water with 0·04% trifluoroacetic acid.

RNA isolation and Scd1 gene expression analysis

Colletotrichum acutatum conidia were inoculated in 5 ml of PDB with 1 × 105 spores ml−1 concentration and incubated under the four different light wavelengths or in the dark at 25°C as previously described. After 48 h of incubation, RNA was extracted from the culture using RNAiso Plus (Takara, Japan), following the manufacturer's instructions. The isolated RNA was treated with DNase (Takara, Japan) to avoid genomic DNA contamination. cDNA was synthesized by the combination of oligo-dT and random primers (PrimScript, Takara, Japan). Real-time PCR (RT-PCR) was performed with 50 ng of cDNA and 5 pmole of each forward and reverse primer, using TOPreal™ qPCR 2× PreMIX (SYBR Green) RT-PCR master mix (Enzynomics, Daejeon, Korea). The thermal profile followed was initial denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 10 s, 60°C for 20 s and 72°C for 30 s. Scytalone dehydratase-specific primers (5′-gacccagcacttcatcggcggc-3′ and 5′-gttgtagctgtgcgcgtggccc-3′) were used to study the scd1 expression by the relative quantification method. Glyceraldehyde-3-phosphate dehydrogenase (gpd), a housekeeping gene, was used as an internal control and was amplified with the primers, 5′-cccctcgccaaggtcatcaacg-3′ and 5′-ggagggaccgtcaacggtcttc-3′.

Statistical analysis

Statistical analysis was carried out with SAS software (Statistical Analysis System 9.2, SAS Institute Inc., Cary, NC, USA) using Tukey's test. Mean ± standard deviation of the pooled values of the replicates and repeated experiments was calculated, and the means were compared by the least significant difference (LSD); values were considered statistically significant at P < 0·05.

Results

Effect of light intensity and wavelength on mycelial growth of Colletotrichum acutatum

In a preliminary study, the effect of light quantities and wavelengths on the mycelial growth was investigated under white, red, green and blue light sources at a range of intensities, from 40 to 360 PPFD (Fig. S1). Light intensities of 40 PPFD did not affect the culture morphology significantly, irrespective of the wavelengths, while light with 120 and higher PPFD produced detectable and clear differences in colony morphology, and, hence, 120 PPFD light intensity was selected for further experiments. At this light intensity, reduced mycelial growth was observed under red and green light wavelengths, in comparison with cultures incubated under other light wavelengths. The cultures incubated under white and blue light and in the dark all showed similar growth in colony diameter (Fig. 1).

Figure 1.

Effect of light wavelengths on mycelial growth of Colletotrichum acutatum. A mycelial plug (8 mm diameter) from the margin of a 7- to 10-day-old PDA culture was placed at the centre of each PDA plate and incubated at 25°C with exposure to white, red, green and blue light at 120 PPFD, or in the dark. (a) The diameter of the colony was measured at 7 DAI, and (b) the upper and lower panels show the top and bottom views of the fungal cultures, incubated under different light wavelengths. The same letters are not significantly different at P < 0·05 according to Tukey's test. Vertical bars indicate the mean ± standard deviation of the replicates.

Conidial germination and germ tube length

Conidial germination, observed after 6 h of incubation under different light wavelengths, indicated that light wavelengths affected the rate of conidial germination and germ tube growth. The least percentage of conidial germination was observed under blue light, while the variation in conidial germination among white, red and green light wavelengths and the dark was insignificant (Fig. 2). A similar effect was also observed in the case of conidial germ tube growth; blue light inhibited the germ tube growth significantly, followed by green light. White and red light wavelengths and the dark showed comparable effects on germ tube length.

Figure 2.

Effect of light wavelength on the conidial germination of Colletotrichum acutatum. A conidial suspension (1 × 105 spores ml−1) in 10% PDA was incubated at 25°C with white, red, green and blue light illumination at an intensity of 120 μmol m−2 s−1 or in the dark. The numbers of germinated conidia, identified by a doubling in the length of conidia size, were counted, and their lengths were measured within a micrometre 6 h after inoculation. The same letters are not significantly different at P < 0·05 according to Tukey's test. The experiments were repeated three times. Vertical bars indicate the mean ± standard deviation of the replicates.

In vivo disease severity assay on hot pepper fruits

In vivo disease severity of C. acutatum was assessed by measuring lesion size following the artificial infection of hot pepper fruits under different light wavelengths. Surprisingly, the light wavelength had a varying effect on the virulence of C. acutatum. The disease severity was significantly higher in pepper fruits incubated under all the light sources than those incubated in the dark. The largest lesion area (3·93 cm2) developed on the pepper fruits incubated under blue light (Fig. 3), which was two times greater than under other light wavelengths. The lesion sizes in the pepper fruits incubated under white, red and green light sources were significantly larger (2·62, 2·57 and 2·25 cm2, respectively) than those on the pepper fruits incubated in the dark 0·02 cm2).

Figure 3.

Effect of light wavelength on anthracnose incidence in pepper fruits. Pepper fruits were inoculated with 10 μl of conidial suspension (1 × 105 conidia ml−1) of Colletotrichum acutatum at each wound and incubated with white, red, green and blue light at 120 μmol m−2 s−1 at a 10 cm distance. The diseased area (cm2) was calculated as [3·14 ×  width of the diseased area × length of the diseased area] after 5 days of incubation at 25°C and 95% RH. The same letters are not significantly different at P < 0·05 according to Tukey's test. Five fruits with five wounds per fruit were used for each light treatment. The experiment was repeated five times. Vertical bars indicate the mean ± standard deviation of the replicates.

Pigmentation and production of scytalone

Higher pigment depositions were visually observed in the cultures incubated under blue and white light wavelengths, while the least quantity of pigment deposition was observed in the control culture, which was incubated in the dark (Fig. 1b). Following the effect of light wavelength on conidial germination, lesion size and pigmentation, melanin production was assayed by scytalone quantification. The highest quantity of scytalone was observed in the culture incubated under blue light with 10·9 mAU at a 25·3 retention time (RT), which is 4·7 times greater than the scytalone produced in the dark (2·3 mAU). The lowest scytalone production was observed in the culture incubated in green light (1·5 mAU). The cultures incubated under white and red light wavelengths produced 4·6 and 6·7 mAU, respectively, which are 2 and 2·9 times greater than those produced in the dark (Fig. 4). Interestingly, the quantities of scytalone produced were in close agreement with the disease severity, incubated under the respective light wavelengths, which indicated the positive correlation among them.

Figure 4.

Scytalone quantification by RP-HPLC. The fractions having scytalone were separated and purified by RP-HPLC, using a Zorbax C18 column. Intermediates were eluted with a linear gradient of 10–100% (v/v) acetonitrile/water with 0·04% trifluoroacetic acid. Vertical bars indicate the mean ± standard deviation of the replicates.

Expression pattern of melanin biosynthesis gene, scd1

The expression pattern of scd1, involved in melanin biosynthesis, was analysed to check whether the light wavelength affects the melanin production at the transcription level. Higher expression was observed in the cultures incubated under white and blue light wavelengths, with a nearly 40-fold increase in scd1 expression in comparison with the control culture, which was incubated in the dark. The cultures incubated under green and red light wavelengths had 15- and threefold increase in scd1 expression, respectively, over the control culture. The expression of scd1 in the dark was significantly lower in comparison with that of white, green and blue light exposure (Fig. 5). This analysis provided the strong evidence that different light wavelengths affect the genes involved in melanin/scytalone production in C. acutatum.

Figure 5.

Expression pattern of the scd1 gene under different light wavelengths. Colletotrichum acutatum conidia were inoculated in PDB and incubated under green, red, blue and white light wavelengths or in the dark at 25°C. After 48 h of incubation, RNA was extracted, and real-time PCR was performed with scd1 and gpd-specific primers. Vertical bars indicate the mean ± standard deviation of the replicates.

Discussion

Light can be used as a fundamental energy source or as a signal from the surrounding environment. In the fungal kingdom, light is used for growth regulation, direction of growth (phototropism), asexual and sexual reproduction and pigment production (Idnurm and Heitman 2005). As light affects many metabolites and the physiology of fungi, fungal interactions with light have garnered much attention recently, and the topic has been reviewed by many researchers (Idnurm and Heitman 2005; Purschwitz et al. 2006; Corrochano 2007; Herrera-Estrella and Horwitz 2007; Idnurm et al. 2010; Tisch and Schmoll 2010). However, not all fungi react to light, and it is difficult to detect the effect of light in many fungi (Idnurm et al. 2010).

Fungi can recognize a wide range of light wavelengths from ultraviolet to far-red light. Blue light is recognized by a phototropin-like protein, White Collar 1 (Wc1), and many other metabolic pathways are sensitive to this blue light (Purschwitz et al. 2006). Red light is recognized by the FphA receptor, which induces the fragmentation of plasmodium and sporulation in Physarum polycephalum (Starostzik and Marwan 1995). Although reactivity to green light was stated in a few reviews (Purschwitz et al. 2006; Tisch and Schmoll 2010), the actual mechanism has yet to be elucidated. More studies on the effects of different wavelengths on fungi will enhance the understanding of light–pathogen interactions.

In the present study, although the light intensity of 40 PPFD showed insignificant effect on colony structure and growth of the pathogen, 120 PPFD and other evaluated light intensities showed varying effect on mycelial growth (Figs 1 and S1). We analysed the effect of different light wavelengths at 120 PPFD on select physiological characters and virulence of Colletotrichum acutatum. The effect of light on mycelia growth has been observed in a few fungi, such as Trichoderma atroviride, in which normal growth was observed under blue and white light wavelengths, while the inhibitory effect was observed under red light (Casas-Flores et al. 2004). The observations of the present study are in agreement with that report, in that higher growth was observed under blue and white light wavelengths, as well as under the dark, than under red and green light wavelengths. In the case of conidial germination and germ tube length, white, red and green light wavelengths, as well as the dark, did not significantly affect the conidial germination, while the least percentage of germination and germ tube length were observed under blue light. However, after 10 h of incubation, nearly 100% of conidial germination was observed under all light wavelengths, and also in the dark (data not shown). In addition, no major differences were observed in mycelial growth and conidial germination, when grown under different light wavelengths at 120 PPFD light after 7 days of growth.

Although many studies have reported that light is an important regulator of melanin, which is highly related to the pathogenicity of fungi, there have been no investigations on the effect of light wavelength on fungal melanin production in connection with pathogenicity. In this study, analysis of C. acutatum pathogenicity on pepper fruits under different light wavelengths revealed that light had a significant influence on disease severity. Melanization, which is also tied to pathogenicity, was also affected by the light wavelength.

The most common phenomena resulting from light effects on fungi are sporulation and pigmentation, the latter of which helps fungi to protect themselves from harmful light, such as ultraviolet light (Moline et al. 2009). In this current study, light wavelength induced different quantities of pigmentation in C. acutatum; higher pigmentation was visually observed under white and blue light, while the least pigmentation was observed in mycelia incubated in the dark. Melanin is a macromolecule produced by many kinds of organisms, from bacteria to human beings. It plays many important roles in fungal life cycles; it protects cells from environmental stresses such as heat, radiation and free radicals, acts as a cross-linker in the fungal cell wall, acts as chelating agent and binds with many metals (Jacobson 2000). Melanin is also involved in conidial attachment to the host cell wall, and hence, it is responsible for the expression of adhesins and other virulence factors during the initial stage of host cell penetration (Pihet et al. 2009). It is particularly important for pathogenic fungi, such as Colletotrichum lagenarium (Kubo et al. 1982) and Magnaporthe grisea, (Woloshuk et al. 1983) to create high pressure within appressoria, which can be translated into a mechanical force to penetrate the host plant cuticle layer. Melanin is produced by 1,8-DHN pathways in C. lagenarium (Tsuji et al. 2003). Scytalone is a significant intermediate product of melanin, and scd1 (codes for scytalone dehydratase) is one of the important and major genes, which converts scytalone to 1,3,8-trihydroxynaphthalene. A C. lagenarium scd1 mutant strain could not infect tobacco leaves on artificial infection, which illustrated the significance of scd1 in melanin production and fungal pathogenicity (Kubo et al. 1996).

Light affects many genes at the transcriptional level, but the percentages varies among species, with 2·8% affected in the case of Trichoderma atroviride (Rosales-Saavedra et al. 2006) and 5·6% in Neurospora crassa (Chen et al. 2009). To clarify the effect of different light wavelengths on melanin production at the transcriptional level, expression of the scd1 gene was analysed in the present study. Interestingly, higher gene expression was observed in all mycelial samples incubated under light wavelengths evaluated than the dark control, which clearly indicates light-induced scd1 expression in C. acutatum. Among them, significantly, higher expression was observed under blue and white light wavelengths, and thus, scd1 expression supported the pigmentation pattern and virulence observed in the fungus in previous components of this study. To validate this association, scytalone, a significant intermediate of fungal melanin, was isolated, purified and quantified using chromatography techniques. Scytalone production was also highly associated with fungal pathogenicity, in that the highest quantity of scytalone was observed in mycelium incubated under blue light, while the least quantity was observed from that incubated in the dark, indicating that C. acutatum requires light for active production of melanin. The results clearly illustrate that the influence of light wavelength on melanin production affects the pathogenicity of C. acutatum. Blue light enhances melanin production and, consequently, the virulence of C. acutatum. Moreover, the stimulation effect of green and red light on the melanin production is lower than that of blue light, and, hence, they lead to the reduced disease severity.

In conclusion, this study provides evidence that light wavelength has a major effect on fungal pathogenicity by influencing melanin production. The findings of this study could potentially be used to manage fungal pathogens in which melanin plays major role for host plant cuticle layer penetration.

Acknowledgements

This work was supported by the Industrial Technology Research Infrastructure Programme (N0000004), funded by the Ministry of Knowledge Economy (MKE, Korea). This research was also partly supported by the Basic Science Research Programme through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science and Technology (No. 2010-0002677).

Conflict of Interest

The authors declare that they have no conflicts of interest in this study.

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