*Corresponding author. Phone) +82-42-821-5762, FAX) +82-42-823-8679, E-mail) Chili pepper (Capsicum), which is produced mainly in tropical and subtropical regions, is one of the most important vegetables worldwide. The grown areas include Pakistan, India, China, Mexico, Korea, Myanmar, Turkey, and Ghana (Olatunji and Afolayan, 2018). Global production was approximately 34.5 million tons of fresh chili and 3.92 million tons of dry chili in 2016 (Ridzuan et al., 2018). Chili plants face a lot of biotic and abiotic stresses. Anthracnose, caused by Colletotrichum spp., is one of the key biotic stresses of chili worldwide. Anthracnose can infect chili peppers and cause economic loss during the pre-harvest and post-harvest stages (Ali et al., 2016). According to the report, the yield loss of chili fruits caused by anthracnose was considered to be around 40% in China, 50% in Thailand, 10% in Korea, 50 % in Malaysia, and 20% to 80% in Vietnam (Chowdhury et al., 2020; Gao et al., 2018). Colletotrichum acutatum species complex can infect a broad range of host plants, such as strawberries, peaches, peppers, almonds, citrus, apples, blueberry, tomato, and mango (Damm et al., 2012; Liao et al., 2012). C. scovillei, belonging to the C. acutatum species complex, is one of common and aggressive species that can cause enormous economic loss (Jayawardena et al., 2016). C. scovillei was the major causal agent of anthracnose of chili in Korea (Oo et al., 2017). In Bali, Khalimi et al. (2019) identified six Colletotrichum spp. that caused anthracnose on chili pepper. Among them, C. scovillei, with rates of more than 55%, was the most prevalent species (Khalimi et al., 2019).
At present, the application of synthetic fungicides, such as propiconazole, difenoconazole, carbendazim, azoxystrobin, and trifloxystrobin, have been widely used traditionally in the control of anthracnose (Ali et al., 2016). Unfortunately, the widespread use of chemical fungicides would lead to the evolution of fungicide resistance (Chechi et al., 2019). In addition, the over-application synthesis of chemicals would cause environmental pollution, affect the no-target biology, and damage human health (Alengebawy et al., 2021). Botanical pesticides are regarded as a key solution for advancing sustainable agriculture. They offer several advantages, including reducing crop losses, being environmentally friendly, readily biodegradable, suitable for organic farming, and cost-effective (Gurjar et al., 2012). Many reports indicate that various plant extracts possess antibacterial activity and can be used as potential biopesticides. For example, crude extracts derived from Rhizophora mangle demonstrated antifungal properties against Fusarium guttiforme (Sales et al., 2016). Plant extracts such as Solanum indicum, Azadirachta indica, and Oxalis latifolia indicated significant antifungal efficacy against Fusarium oxysporum f. sp. Lycopersici (Onaran and Yanar, 2016). Additionally, extracts from Zingiber officinale rhizomes, as well as leaves of Polyalthia longifolia and Clerodendrum inerme, have shown inhibitory effects on the growth of Colletotrichum musae, the causative agent of banana anthracnose (Bhutia et al., 2016). Therefore, developing safe and effective natural compounds for plant disease management is urgently required.
This research found that Cestrum tomentosum L.f. crude extracts (CTCE) had antifungal activity against Colletotrichum scovillei. The objectives of this study were to investigate the antifungal activity of CTCE in vitro on C. scovillei, to evaluate the protective and curative activity of CTCE on detached chili peppers, and to study the potential mode of action of CTCE against C. scovillei.
Materials and Methods
Pathogen and plant extract
The pathogen Colletotrichum scovillei was provided by the molecular plant fungal pathogen lab at the College of Agriculture & Life Science, Chungnam National University (Oo et al., 2017). The plant extract of Cestrum tomentosum L.f. (FBM247-052) used in this research was obtained from the International Biological Material Research Center at the Korea Research Institute of Bioscience and Biotechnology (Daejeon, Republic of Korea). The plant was collected La Amistad National Park, Costa Rica in June 2015. A voucher specimen (KRIB 0069866) is kept in the herbarium of the Korea Research Institute of Bioscience and Biotechnology. The dried leaves and shoots (100 g) in the shade were extracted with 700 ml of 95% ethanol for 30 min by ultrasonic-assisted maceration, three times. The extract was percolated through filter paper, condensed using a rotary evaporator, and lyophilized, C. tomentosum extract (14.4 g) was obtained.
Effect of CTCE on C. scovillei different development stages
Spore germination and germ tube elongation
The 96 well cell culture plates (SPL Life Sciences, Pocheon, Korea) were used for spore germination and germ tube elongation evaluated. Spore suspension (1 × 106 spores/ml) of C. scovillei was harvested from 7-day cultured potato dextrose agar (PDA) plates by filtering through 3 layers of sterile gauze, and spore suspension was cultured at 96-well cell culture plates.
The 96-well cell culture plate was incubated at 25°C for 6 h for spore germination inhibition analysis. Different concentrations of CTCE (1,000, 500, 250, 125, and 62.5 μg/ml) were added to the cell, and the dimethyl sulfoxide (DMSO) concentration was limited to 0.5% (v/v). Distilled water (DW) and 0.5% DMSO were used as control and incubated for another 24 h. The spore germination was observed using the optical microscope (BX50, Olympus, Tokyo, Japan), and germination was defined as the length of the germ tube being longer than or equal to the diameter of the spore. More than 200 spores were counted, and the spore germination rate was calculated as germination rate (%) = (Number of germinated spores/Number of total spores) × 100%.
In addition, the plate was incubated at 25°C for 12 h after the spores germinated, and different concentrations of CTCE were added to the cells described above. After incubating for another 12 h, the phenotype of spores was recorded by photo using the optical microscope, and Image J (National Institutes of Health, Bethesda, MD, USA) was used to measure the length of the germ tube.
Mycelium growth
The mycelium growth inhibition analysis was conducted as described before but slightly modified (Li et al., 2018). The PDA medium was cooled down to 50-60°C and mixed with the CTCE, and the final concentration of CTCE was 1000, 500, 250, 125, 62.5, 31.25, 15.625, and 7.8125 μg/ml. The final concentration of DMSO was limited to 0.5% (v/v). DW and 0.5% DMSO were used as controls. Mycelia plugs (5 mm in diameter) from the leading edge of an actively growing colony were transferred to a series of PDA plates containing the different concentrations of CTCE and incubated at 25°C for 5 days. The mean colony diameter was measured for each treatment. The effectiveness of CTCE against C. scovillei was calculated based on the percentage of mycelium growth inhibition.
Cell viability analysis
The cell viability activity was determined by a tetrazolium salt XTT reduction assay. The C. scovillei spore suspension (1 × 106 spores/ml) was treated with the CTCE, 0.5% DMSO, and DW incubated at 25°C for 24-48 h. The activated-XTT solution was prepared as described by previous research (Reddy and Nancharaiah, 2020). The specimens were washed gently twice in phosphate buffered saline (PBS), then re-suspended in 200 μl PBS buffer, and dipped with 25 μl XTT working solution, kept in the dark at 37°C for 2 h. The optical density was measured at a wavelength of 450 nm by using the microplate spectrophotometer.
In vivo analysis
A previous method was used to evaluate the efficiency of the CTCE against C. scovillei on detached chili pepper fruits (Gao et al., 2018). The detached chili pepper fruits (Pyongyang chili pepper) were surface sterilized at 1% NaOCl for 3 min, then washed in sterile distilled water 3 times. The CTCE was sprayed on the fruits, and 0.5% DMSO and DW were used as a negative control. The sterilized fruits were treated with CTCE before and after inoculation with the spore suspension (1 × 106 spores/ml) of C. scovillei to investigate the protective and curative activity. And the samples were placed in a stealing box to keep the humidity. The symptoms were recorded by the digital camera.
Observation of morphology
Optical microscope observation
Optical microscope observation was performed as described in the previous research with some modifications (Yan et al., 2020). The mycelium plugs were cut from 7-day cultured PDA plate containing the CTCE and DW, 0.5% DMSO. Then they were incubated on the sterilized glass slide at 25°C for 24 h; the plugs were observed using the optical microscope (BX50, Olympus).
Scanning electron microscopy observation
Scanning electron microscopy (SEM) analysis of the samples was performed using the methods described early study with some modifications (Yang et al., 2020). The C. scovillei spore suspension was prepared and treated with the CTCE at 100 μg/ml concentration, and 0.5% DMSO and DW were used as control. The samples were collected by centrifuge (12,000 rpm, 4°C, 5 min) and washed in PBS (0.1 M) buffer twice. After dehydration, preparations were made by fixing with 2.5% glutaraldehyde for 4 h, using graded aqueous ethanol solution (30%, 50%, 70%, 90%, and 100%) for 15 min at each concentration. SEM was used for cell ultrastructure observation.
Plasma membrane integrity analysis
To determine if lesions in the plasma membrane exist in the fungal cells, propidium iodide (PI) was used (Souza et al., 2020). The mycelium of C. scovillei cultured in potato dextrose broth (PDB) medium was washed with PBS buffer twice by centrifuge, re-suspended in CTCE, DW, and 0.5% DMSO, and kept at 25°C for 24 h. Following stained by PI with the final concentration of 10 μg/ml, keep at 25°C for 10 min. Then mycelium was washed using PBS buffer three times and photographed using a fluorescence microscope (DM 2500, Leica, Wetzlar, Germany) at excitation and emission wavelengths of 480 and 580 nm, respectively.
Reactive oxygen species accumulation analysis
The 2′,7′-dichlorofluorescein diacetate (DCFH-DA) was used for checking the reactive oxygen species (ROS) level in the mycelium of C. scovillei (Pan et al., 2019). The prepared C. scovillei spore suspension was incubated at 25°C for 24 h, then treated with the CTCE at 25°C for 6 h. After this process, the mycelium was stained with 10 μmol/l (DCFH-DA) dye at 25°C for 40 min in the dark, washed the samples three times with PBS buffer. The fluorescence intensity of the re-suspended mycelium was measured using a fluorescence microscope.
Release of cellular content
The release of DNA from the intracellular of C. scovillei was investigated using the method described by Chen et al. (2023) with modification. The C. scovillei spore suspension was incubated in PDB at 25°C for 48 h. The aliquot was removed by centrifuge at 10,000 rpm for 5 min. Then treated mycelium with DW, 0.5% DMSO, and CTCE and kept at 25°C for 24 h. Then, the supernatant was collected by centrifuge, and the 500 μl into a column in a collection tube. Centrifuge 13,000 rpm for 1 min. Transfer the flow through into a new tube, the 50 μl of 3 M sodium acetate was added and mixed, following adding 3 times the volume of 100% ethanol, kept at −20°C for 24 h. Centrifuge 13,000 rpm for 15 min at 4°C. Pour off the supernatant and let the tube air-dry. Add 10 μl DW and DNA concentration was determined by the SimpliNano Spectrophotometers. In addition, internal transcribed spacer (ITS) and Actin genes were used to perform the PCR, following running the 1% agarose gel electrophoresis to confirm the DNA in the supernatant.
Results
CTCE affects C. scovillei at different development stages
To investigate the effect of plant extract on spore germination and germ tube elongation, the spore suspension was treated with CTCE, DW and DMSO after keeping in the incubator for 6 and 12 h, respectively. After 6 h incubation and then treated with CTCE, results indicated that the spore germination and germ tube elongation had no significant difference at 8 μg/ml concentration compared with control; they were inhibited at 40 μg/ml and up to 100% inhibition at 200 μg/ml (Fig. 1). Spores already germinated after incubating for 12 h, then treated with CTCE; results showed that germ tube elongation was significantly inhibited at the concentration of 40 and 200 μg/ml (Fig. 1).
Compared with the control, the C. scovillei mycelium growth was significantly inhibited in the PDA medium containing CTCE. The colony growth rate was strongly decreased in the plate treated with CTCE, especially with a high concentration of 1,000 μg/ml showing almost completely inhibited mycelium growth (Fig. 2A and B). Dose-response fitting in Origin 8.1 was plotted, and the half-maximal effective concentration (EC50) of CTCE against C. scovillei mycelium growth was 18.81 μg/ml (Fig. 2C).
CTCE reduced the cell viability of C. scovillei
The XTT Cell Viability Assay Kit (Sigma, St. Louis, MO, USA) provides a simple method for determining cellular proliferation and cell viability by absorbance on a microplate reader. XTT is a tetrazolium derivative turned into a water-soluble orange product after being reduced by mitochondrial enzymes only present in metabolically active live cells. The amount of orange product generated was proportional to the number of living cells in the sample. The result indicated that the cell viability was high in the control and decreased after treatment with the CTCE, especially at high concentrations of 100 and 200 μg/ml with a significant difference from the control (Fig. 3).
CTCE showed both protective and curative activities in pepper fruit against anthracnose disease
The results indicated that the 500 μg/ml CTCE provides protective and curative activity, with control efficacies of 93% and 86.4% after 10 days of inoculation of C. scovillei. After 7 days of inoculation with the spores of C. scovillei, the symptoms appeared in the control group, and the fruits of the treatment group were barely infected. However, the difference was not significant (Fig. 4). After 10 days, for protective activity, the average area of symptoms on fruits was 6.7, 6.45, 3.89, and 0.46 cm2 on DW, DMSO, 100, and 500 μg/ml CTCE treatment, respectively (Fig. 4A and B). For curative activity, the average area of symptoms on fruits was 6.88, 6.97, 3.15, and 0.94 cm2 on DW, DMSO, 100, and 500 μg/ml treatment, respectively (Fig. 4C and D). According to the findings, the protective activity of each treatment was higher than the curative activity.
CTCE affected the mycelial morphology and ultrastructure of C. scovillei
The impact of CTCE on the morphology of C. scovillei mycelia was evaluated through both optical microscopy and SEM. As illustrated in Fig. 5, the mycelia in the control group appeared smooth, uniform, and structurally intact. In contrast, the mycelia exposed to CTCE exhibited significant morphological alterations, including bending, shrinkage, collapse, and deformation, along with pronounced hyphal branching. These observations, derived from both optical microscopy and SEM, demonstrate that CTCE induces structural damage to the mycelia and spores of C. scovillei.
CTCE induced membrane disruption and ROS accumulation
PI is a nucleic acid-binding fluorescent dye used to determine if the lesions in the plasma membrane exist in fungal cells. Damaged cell membranes can be penetrated by PI and emit red fluorescence. In the control group, the mycelium exhibited almost no red fluorescence. However, red fluorescence became clearly visible after treatment with 50 μg/ml CTCE (Fig. 6A). These results indicated that the CTCE could destroy the cell membrane integrity of C. scovillei.
The endogenous ROS was determined by using the fluorescent dye DCFH-DA, which is well used for investigating the changes of ROS level in the fungal cells. DCFH-DA would generate DCFH when hydrolyzed by intracellular esterase enzymes of the cell, and the oxidation of DCFH by ROS converts the molecule to fluorescent dichlorofluorescein (DCF), which emits green fluorescence. The intensity of fluorescence can directly represent ROS level. After being treated for 24 h, the mycelium demonstrated negligible green fluorescence in the control cohort. Conversely, after 50 μg/ml CTCE was administered, green fluorescence became distinctly observable (Fig. 6B). The data declared that the CTCE could induce the ROS level in the mycelium of C. scovillei.
Release of intracellular content
The cell membrane permeability was investigated by checking DNA and protein amounts in the supernatant of mycelium. The results showed that after 24 h treatment, the DNA concentration in the supernatant was increased after the CTCE treatment with a concentration of more than 50 μg/ml (Fig. 7A). 1% agarose gel electrophoresis showed that the band only appeared in the supernatant treated with CTCE (Fig. 7B).
Discussion
Colletotrichum spp. was rated as one of the top 10 fungal plant pathogens (Dean et al., 2012) and was primarily reported as the causal agent of anthracnose causing severe yield losses (Cannon et al., 2012). There are 24 Colletotrichum spp. that have been reported to cause chili anthracnose; among them, C. scovillei is one of the most aggressive species based on the pathogenicity test (de Silva et al., 2019; Mongkolporn and Taylor, 2018). Infection of C. scovillei initiates when conidia attach and adhere to the surface of pepper fruits. Upon recognition of host signals, conidia germinate and extend their germ tubes, then the germ tubes form appressoria and penetrate the cuticle and mesophyll cells by the penetration peg. After successful penetration, the pathogen starts producing infectious hyphae, resulting in cellular necrosis (Fu et al., 2022). In our study, we evaluated the effect of CTCE on spore germination, germ tube elongation, and mycelium growth which play crucial roles in the infection of the host, maintaining fungal viability and life cycle in C. scovillei. At 40 μg/ml, the germ tube elongation and mycelium growth were significantly inhibited. However, there was no significant difference in the spore germination rate (Figs. 1 and 2). The present results demonstrated that germ tube elongation and mycelium growth were more sensitive to CTCE compared to spore germination.
Many compounds from the plant extract inhibit the pathogens by changing the morphology of the spore or hyphal (Shao et al., 2013; Yang et al., 2020). Our study also found that CTCE changed the morphology and ultrastructure of C. scovillei in the hyphal and spores, which usually indicate damage to the cell membrane. The cell membrane is one of the most important targets of nature products (Seyedjavadi et al., 2019; Wang et al., 2019; Xu et al., 2021). To determine whether the plasma membrane was damaged, the PI staining and leakage of cell components measurements were performed (Figs. 6A and 7). The results suggested that the CTCE disrupted the membrane of C. scovillei and further caused DNA to flow into the supernatant from the intracellular.
Fungi form ROS during metabolic activity and are primarily generated in the mitochondria, which play important roles in fungal development (Gessler et al., 2007). Under stress conditions, cells would produce more ROS, and the extra ROS could cause cell dysfunction once the ROS level exceeds the critical value (Li et al., 2015). Anethum graveolens L essential oil treated Colletotrichum gloeosporioides spores induced ROS accumulation, resulting in mitochondria dysfunction (Tian et al., 2012). Tea tree oil treatment also stimulates ROS levels in B. cinerea mycelium, leading to mitochondria dysfunction (Li et al., 2017). In our study, the ROS level was increased after CTCE treatment (Fig. 6B). The mitochondrial enzyme complex contributes substantially to the XTT response, and its activity is a general indicator of mitochondrial health (Chang and Doering, 2018). Our data indicated that the CTCE might decrease the activity of mitochondria in C. scovillei hyphal (Fig. 3). Our results also showed that CTCE treatment induces ROS accumulation in C. scovillei, which may be responsible for mitochondria dysfunction.
In conclusion, we found CTCE with antifungal activity against C. scovillei, inhibiting spore germination, germ tube elongation, and mycelium growth. In addition, the CTCE also showed strong protective and curative activity on detached chili pepper fruits. Mode of action analysis results indicated that the CTCE might control the C. scovillei by damaging the cell membrane and inducing ROS accumulation. The CTCE can be a candidate for botanical fungicide against anthracnose caused by C. scovillei. Further, the main active compound should be identified from the crude CTCE, and molecular experiments should be performed to confirm the mode of action analysis.
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