https://orcid.org/0000-0001-6874-0791
Figures
Abstract
Colletotrichum is a major plant-pathogenic fungus responsible for anthracnose, a disease that significantly reduces crop yield and quality, thereby hindering commercial production. This study aimed to isolate, identify, and characterize Colletotrichum species associated with anthracnose in various fruits and ornamental plants in Thailand, using both morphological and molecular approaches. Subsequently, the medicinal plant extracts were evaluated for their antifungal activity against the isolated Colletotrichum species, and their bioactive compounds were profiled using GC-MS and LC-MS analyses. Eleven Colletotrichum isolates were obtained and identified as nine distinct species, namely C. asianum, C. brasiliense, C. fructicola, C. musae, C. nymphaeae, C. okinawense, C. orchidearum, C. pandanicola, and C. truncatum. Among the ten medicinal plants tested, only the ethanolic extract of clove (Syzygium aromaticum) exhibited strong antifungal activity against all fungal isolates. The extract exhibited minimum inhibitory concentrations as low as 12.5 mg/mL against C. okinawense and C. orchidearum, and 25 mg/mL against the remaining species. GC-MS profiling of the ethanolic clove extract revealed that 2-methoxy-4-prop-2-enylphenol (eugenol, 72.417%) was the predominant compound, followed by (1R,4E,9S)-4,11,11-trimethyl-8-methylenebicyclo [7.2.0] undec-4-ene (β-caryophyllene, 12.125%), and (2-methoxy-4-prop-2-enylphenyl) acetate (eugenyl acetate, 8.121%). Additionally, LC-MS profiling indicated that the extract contained several antifungal constituents, including quercetin, kaempferol, isorhamnetin, myricetin, gallic acid, ellagic acid, catechol, caffeic acid, p-coumaric acid, methyl trans-cinnamic acid, and resveratrol. These findings highlight the diversity among pathogenic Colletotrichum species in Thailand and establish that clove extract, which contains flavonoids, phenolics, terpenoids, and alkaloids, holds potential as an eco-friendly alternative for agricultural disease management.
Citation: Pitiwittayakul N, Niyomvong N, Wongsorn D, Kumla J, Suwannarach N (2025) Phytochemical characterization and antifungal activity of medicinal plant extracts against Colletotrichum species associated with anthracnose in Thailand. PLoS One 20(12): e0339399. https://doi.org/10.1371/journal.pone.0339399
Editor: Abhijeet Shankar Kashyap, ICAR National Bureau of Agriculturally Important Microorganism, INDIA
Received: July 18, 2025; Accepted: December 5, 2025; Published: December 30, 2025
Copyright: © 2025 Pitiwittayakul et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All nucleotide sequence files are available from the NCBI database (accession numbers: PV478438, PV548285, PV548306, PV548295, PV478439, PV548286, PV548307, PV548296, PV478440, PV548287, PV548308, PV548297, PV478437, PV548284, PV548305, PV478433, PV548280, PV548301, PV548291, PV478431, PV548278, PV548299, PV548289, PV478432, PV548279, PV548300, PV548290, PV478434, PV548281, PV548302, PV548292, PV478436, PV548283, PV548304, PV548294, PV548298, and PV5482880).
Funding: This research project is supported by the Science Research and Innovation Fund. Agreement No. FF67/P1-027. This work was partially supported by Chiang Mai University, Thailand. There was no additional external funding received for this study.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Colletotrichum is a genus of economically significant ascomycetous fungi that collectively cause anthracnose and leaf blight diseases in major agricultural crops and ornamental plants worldwide [1–4]. Infections primarily occur in tropical and subtropical regions, while they are less common in temperate areas due to their dependence on warm temperatures and high relative humidity [5]. Anthracnose is a severe disease affecting climacteric fruits such as avocados, bananas, guavas, mangoes, papayas, and pears, largely due to the physiological and biochemical changes that occur during ripening. These changes including cell wall remodeling and degradation create favorable conditions for fungal growth and pose a significant threat to non-climacteric fruits such as dragon fruit, oranges, and strawberries [6]. In fruit crops, this disease can lead to significant reductions in yield and quality, ultimately causing customer dissatisfaction and economic losses.
Traditional methods of controlling Colletotrichum, the causal agent of anthracnose in several fruits, have involved the use of various synthetic fungicides, such as mancozeb, carbendazim, thiram, ziram, captan, prochloraz, and tiabendazole [7]. However, the extensive use of chemical fungicides has contributed to the emergence of fungicide-resistant strains in Colletotrichum species, reducing treatment efficacy and complicating disease management [8,9]. In addition, prolonged fungicide application poses risks to food safety, the environment, and human health. In response to these concerns, green agricultural technologies for plant pathogen control are being increasingly promoted due to their ecological, safety, and societal benefits. Plants serve as rich sources of bioactive compounds, including terpenes, phenolics, essential oils, and alkaloids [10,11]. Thailand, in particular, possesses a high diversity of medicinal plants, many of which have been reported for their therapeutic properties [12]. Several studies have demonstrated that plant extracts contain a wide array of bioactive compounds with antifungal properties, making them promising alternatives to synthetic fungicides [13–16]. Numerous essential oils, alkane hydrocarbons, and fatty acids from various plant extracts have been identified using gas chromatography-mass spectrometry (GC-MS), while polyphenols and triterpenoids have been identified through liquid chromatography-mass spectrometry (LC-MS) analyses [17–19]. For a comprehensive phytochemical characterization, GC-MS and LC-MS are frequently employed in tandem, as their complementary analytical capabilities enable the detection of a broad spectrum of compounds in plant extracts. Therefore, this study aims to isolate Colletotrichum from tropical fruits and ornamental plants exhibiting anthracnose symptoms in Thailand, and to identify the isolates using both morphological characteristics and multi-gene molecular techniques. Furthermore, the antifungal activity of selected medicinal plant extracts was evaluated, and the most effective extract was characterized for its chemical composition using GC-MS and LC-MS analyses.
Materials and methods
Anthracnose sample collection
A total of 11 diseased plant samples showing anthracnose symptoms, representing nine plant species, were surveyed and collected in Thailand between 2023–2024. These included two samples from mango fruits (Mangifera indica L. cv. ‘Nam Dok Mai’ and M. indica L. cv. ‘Kaew Kamin’), one sample from a mango leaf, two samples from avocados (Persea americana Mill), and one sample each from chili [Capsicum annuum L. (Syn. Capsicum frutescens L. var. frutescens)], strawberry (Fragaria × ananassa Duchesne), passion fruit (Passiflora edulis Sims), Holland papaya (Carica papaya L.), banana (Musa × sapientum L. cv. ‘Nam Wa’), and a Strawberry Shake philodendron leaf (Philodendron erubescens cv. ‘Strawberry Shake’). The samples were collected from local markets in Chiang Mai Province, Thailand, except for the ornamental plant Strawberry Shake philodendron, which was collected in Nakhon Ratchasima Province.
Fungal isolation and morphological study
Naturally infected plant parts and fruits showing typical anthracnose lesions were incubated at 25°C for 1–2 days in a plastic container lined with moist filter paper to encourage sporulation. When spore masses or conidiomata appeared, single-spore isolation was performed according to Choi et al. [20]. Using a sterile Pasteur pipette, a drop of the conidial suspension in sterile 0.85% NaCl solution was dragged across the surface of 2% water agar to separate individual conidia. After 24–48 h of incubation at 25°C, germinating spores were observed under a microscope, and single germinated spores were transferred to potato dextrose agar (PDA; Corda, Madrid, Spain) supplemented with 0.5 mg/L streptomycin. Pure cultures of each fungal isolate were obtained through successive subculturing on PDA. Pure fungal isolates were maintained short-term on PDA slants and preserved long-term in 20% glycerol at −80°C. Colony morphology including colony shape, color, and pigmentation was determined. The following micromorphological characteristics were examined: conidiomata and conidiophores. The mean lengths and width of 50 randomly selected conidia from each isolate were measured using 100X magnification in a microscope (Nikon Ti-S inverted microscope, Japan). Each pure fungal culture was deposited in the Culture Collection of the Sustainable Development of Biological Resources (SDBR) Laboratory, Faculty of Science, Chiang Mai University (CMU), Thailand.
DNA extraction and PCR amplification
DNA was extracted from a one-week-old pure culture grown on PDA. Genomic DNA was obtained using the DNA Extraction Mini Kit (FAVORGEN, Taiwan), following the manufacturer’s instructions. The targeted gene regions, primers, and PCR thermal cycle programs used for amplification are detailed in Table 1. Each PCR amplification reaction had a total volume is 20 μL, consisting of 6 μL ddH2O, 10 μL 2x Quick TaqTM HS DyeMix (TOYOBO, Japan), 2 μL DNA template, and 1 μL each of forward and reverse primers [21]. PCR products were purified using the PrimeWay Gel Extraction/ PCR Extraction Kit (1stBase, Malaysia) and directly sequenced via Sanger sequencing by 1st Base Company (Kembangan, Malaysia) using the same primers mentioned above.
Phylogenetic analysis
The resulting sequences were used to query GenBank via BLAST (http://blast.ddbj.nig.ac.jp/top-e.html). Sequences obtained in this study and from previous works, along with entries from the GenBank database, were used for phylogenetic analysis (Table 2). Multiple sequence alignment for each locus was performed using MUSCLE [22] with manual adjustments in BioEdit v6.0.7. The aligned sequences of the four loci, including the internal transcribed spacer (ITS) of ribosomal DNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), and beta-tubulin (TUB2) genes were concatenated into a single dataset using BioEdit v6.0.7 for phylogenetic analysis. Maximum likelihood (ML) phylogenetic trees were constructed using RAxML-HPC2 v8.2.12 on the CIPRES Science Gateway with the GTRCAT model and 1000 bootstrap replicates for statistical support [23]. Phylogenetic trees were visualized using TreeView v32 and edited in Adobe Illustrator v25.2.3.
Preparation of medicinal plant extracts
The extraction method using ethanol was modified from the procedure described in Al-Otibi et al. [24]. Ethanol is widely used as a plant extraction solvent because it effectively solubilizes a broad range of bioactive constituents, including flavonoids, phenolics, and other antimicrobial metabolites [24,25]. Ten different medicinal plants, using various parts, were collected: lemongrass (Cymbopogon citratus), clove (Syzygium aromaticum), sea holly (Acanthus ebracteatus), heart-leaved moonseed (Tinospora cordifolia), turmeric (Curcuma longa), black pepper (Piper nigrum), cassumunar ginger (Zingiber montanum), Indian gooseberry (Phyllanthus emblica), cinnamon (Cinnamomum verum), and kariyat (Andrographis paniculata). The plant materials were dried at 50°C and ground into a fine powder. Briefly, 100 g of dried plant powder was soaked in approximately 500 mL of absolute ethanol (1:5 w/v) and incubated for 72 hours. The mixture was then filtered using Whatman No.1 filter paper. After filtration, the extracts were evaporated using a rotary evaporator (Buchi Rota vapor, Switzerland) at a temperature below 50°C and a rotation speed of 90 rpm to obtain semi-solid products. The extracts were then weighed to determine the percentage extraction yield using the equation from Akwongo et al. [26]: Extract yield (%) = (weight of dried extract/weight of dried plant sample) × 100
The crude extracts were dissolved in absolute ethanol and stored in a refrigerator for further use in antifungal tests.
The crude extracts obtained from the respective plants were dissolved in absolute ethanol to achieve a final concentration of 100 mg/mL. The potential antifungal activity of selected crude plant extracts was evaluated using the minimum inhibitory concentration (MIC) method, with concentrations adjusted to 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, and 0.78125 mg/ml. Pure methyl eugenol (Sigma Aldrich, USA) was diluted two-fold with ethanol to a final concentration of 250 mg/mL for comparison with the plant crude extracts in antifungal activity tests. Methyl eugenol, a compound with known antifungal properties that is commonly extracted from various plant species, was selected as a positive control for comparison with the plant extracts. However, methyl eugenol can exhibit lower antifungal efficacy, as methylation of the phenolic hydroxyl group has been reported to reduce antifungal activity [27]. Consequently, methyl eugenol was applied at a higher concentration than the plant extracts.
Evaluation of antifungal activity of plant extracts against Colletotrichum species
The paper disc diffusion method was used to screen the antifungal activity of the plant extracts on PDA. Fungal spores were collected from the spore masses of each isolate and suspended in sterile distilled water to achieve a concentration of approximately 1.0 × 107 spores/mL. The spore suspension was evenly spread over the surface of agar plates using a sterile cotton swab. Sterile filter paper discs (6 mm diameter) were impregnated with crude extract in absolute ethanol, air-dried at room temperature (25 °C) under aseptic conditions in a laminar flow hood, and placed on the agar surface. Antifungal activity was assessed after 24–48 hours of incubation at 28°C. The diameters (cm) of the inhibition zones were measured, and antifungal activity was expressed as the mean inhibition zone based on three replicates per treatment.
GC-MS analysis
GC-MS analysis was performed using an Agilent 7890A gas chromatograph coupled with an Agilent 5975C mass spectrometer. The column used was an HP-5MS UI capillary column (30 m in length × 250 μm in diameter × 0.25 μm in thickness). The oven temperature was programmed to increase from 40°C to 200°C at a rate of 6°C/min, then from 200°C to 280°C at 30°C/min, and was finally held at 300°C for 10 minutes. A post-run was conducted at 280°C for 10 minutes. Helium was used as the carrier gas at a constant flow rate of 1 mL/min. The injector and detector temperatures were both set to 250°C. GC-MS analysis was carried out by injecting 1 μL of the sample (0.1% in absolute methanol) in splitless mode, using scan mode detection. Quinaldine was used as an external standard. The chemical constituents of the clove extract were identified by comparing retention times and mass spectra with those in the Wiley mass spectral library (W8N08, John Wiley & Sons, Inc., USA).
Phytochemical constituents by LC-MS analysis of selected plant extract
Plant extracts exhibiting antifungal activity were selected for this experiment. High-performance liquid chromatography (HPLC) separation was performed using a Poroshell 120 EC-C18 column (100 mm × 2.1 mm, 2.7 μm, Agilent Company, USA), maintained at 50°C. A 10 μl aliquot of crude plant extract, prepared in 70% methanol containing 25 ng/ml sulfadimethoxine, was injected for analysis. The mobile phase consisted of solvent A (deionized water with 0.1% formic acid, v/v) and solvent B (acetonitrile with 0.1% formic acid, v/v). An exploratory gradient elution was applied as follows: 55–75% solvent B from 10.5 to 12.5 minutes, followed by 100% solvent B from 14.0 to 17.0 minutes, at a constant flow rate of 0.4 mL/min. Samples were analyzed in all-ion MS/MS acquisition modes, with a collision energy of 20 eV in positive ion mode and 10 eV in negative ion mode.
Mass spectrometry analysis was performed using an LC-QTOF 6545XT system (Agilent Technologies, USA) equipped with an electrospray ionization (ESI) source utilizing Jet Stream technology. The instrument was operated under the following conditions: drying gas (N2) flow rate, 13 L/min; drying gas temperature, 325°C; nebulizer pressure, 45 psi; sheath gas temperature, 275°C; sheath gas flow, 12 L/min; and capillary voltage, 4000 V (positive mode) or 3000 V (negative mode). Each sample was analyzed in both positive and negative ionization modes over an m/z range of 40–1700 for MS1 and 25–1000 for MS2 acquisitions. Data processing was performed using MS-DIAL version 5.3, utilizing ESI (±) MS/MS data. Compound annotation was conducted using authentic standards and referenced against the Fiehn/Vaniya Natural Product Database and the BMDMS-NP library [28].
Results
Isolation and morphological characterization of Colletotrichum species from anthracnose samples
In total, eleven Colletotrichum isolates were obtained from eleven diseased samples: two from mango fruits (cv. “Num Dok Mai” and “Kaew Khamin”), one from a mango leaf, one from a strawberry, one from a passion fruit, one from a papaya, one from a banana, one from a chili, two from avocados (cv. “Hass” and “Buccaneer”), and one from an ornamental philodendron. All isolates were cultivated on PDA. Most Colletotrichum isolates exhibited white to grey aerial mycelia, while some displayed yellow to pale brown mycelia (Fig 1, Table 3). Orange spore masses were visible on the aerial mycelia of some isolates. On the reverse side, nearly all fungal colonies were pale yellow to yellowish gray, while a few showed grayish olive to olive black coloration at the center. None of the isolates produced pigmentation in the PDA medium. The conidia of nearly all isolates were cylindrical or oval with varying lengths and widths, except for isolate CHL1, which exhibited curved or sickle-shaped conidia. All Colletotrichum isolates were identified through multi-gene molecular phylogenetic analyses.
Colletotrichum orchidearum PH1 (A), C. okinawense PY1 (B), C. nymphaeae SB1 (C), C. brasiliense PF1 (D), C. truncatum CHL1 (E), C. fructicola ADH1 (F), C. pandanicola MGL1 (G), C. pandanicola AD1 (H), C. musae BN1 (I), C. asianum MGK1 (J), and C. asianum MGN1 (K). From left to right, each panel shows the upper surface of colonies grown on potato dextrose agar (PDA) at 14 days after inoculation, the reverse side of the colonies, and the conidia or ascospores. Scale bars: 1 cm for culture plate images; 10 μm for spore micrographs.
Multi-gene molecular phylogenetic analyses
Based on the maximum likelihood (ML) tree, the dataset comprising four genes (ITS, ACT, TUB2, and GAPDH) was divided into six Colletotrichum species complexes: Orchidearum, Magnum, Acutatum, Boninense, Truncatum, and Gloeosporioides clades, in accordance with the percentage identity results of the ACT, TUB2, and GAPDH genes. Phylogenetic trees obtained from the ML analysis are shown in Figs 2 to 4. The results revealed that isolates PH1, PY1, SB1, PF1, and CHL1 were identified as Colletotrichum orchidearum (Orchidearum clade), C. okinawense (Magnum clade), C. nymphaeae (Acutatum clade), C. brasiliense (Boninense clade), and C. truncatum (Truncatum clade), respectively (Fig 2 and 3). Six additional isolates were placed within the Gloeosporioides clades (Fig 4). Among these, isolates ADH1 and BN1 were identified as C. fructicola and C. musae, respectively. Both AD1 and MGL1 were identified as C. pandanicola, while MGK1 and MGN1 were identified as C. asianum. All isolates clustered with the type strains of their respective species. Therefore, the eleven Colletotrichum isolates obtained in this study were identified as belonging to nine different species, based on both morphological characteristics and multi-gene molecular phylogenetic analyses. All fungal isolates were deposited in the SDBR Laboratory’s Culture Collection under numbers SDBR-CMUPH1, SDBR-CMUPY1, SDBR-CMUSB1, SDBR-CMUPF1, SDBR-CMUCHL1, SDBR-CMUADH1, SDBR-CMUMGL1, SDBR-CMUAD1, SDBR-CMUBN1, SDBR-CMUMGK1, and SDBR-CMUMGN1.
Strains isolated in this study are shown in blue. The bar indicates substitution per site. Colletotrichum asianum strain ICMP 18580 and C. gloeosporioides strain ICMP 17821 were used as outgroups. Type strains are shown in bold.
Strains isolated in this study are shown in blue. The bar indicates substitution per site. Colletotrichum asianum strain ICMP 18580 and C. gloeosporioides strain ICMP 17821 were used as outgroups. Type strains are shown in bold.
Strains isolated in this study are shown in blue. The bar indicates substitution per site. Colletotrichum boninense strain CBS 123755 and C. brasilliense strain CBS 128501 were used as outgroups. Type strains are shown in bold.
Yield of plant crude extracts
The extraction yields of the ten medicinal plants varied, with the percentage of ethanolic extracts ranging from 0.711% to 23.281% (Table 4). The highest yield was obtained from cinnamon, while the lowest was from sea holly. The second to fifth highest extraction yields were from Indian gooseberry (7.44%), turmeric (7.34%), cassumunar ginger (5.40%), and clove (5.29%), respectively.
Evaluation of antifungal activity of plant extracts against Colletotrichum species
The effect of various ethanol-based plant extracts on Colletotrichum growth was evaluated using the paper disc diffusion method. Fungi were cultured on PDA in the absence (control) or presence (treatment) of each plant extract. At a concentration of 100 mg/mL, the clove ethanolic extract strongly inhibited mycelial growth in all Colletotrichum isolates, producing inhibition zones ranging from 2.70 to 4.50 cm (Fig 5). Among the 10 plant extracts tested, only the clove extract demonstrated significant antifungal activity and was therefore selected for further MIC analysis.
Colletotrichum orchidearum PH1 (A), C. okinawense PY1 (B), C. nymphaeae SB1 (C), C. brasiliense PF1 (D), C. truncatum CHL1 (E), C. fructicola ADH1 (F), C. pandanicola MGL1 (G), C. pandanicola AD1 (H), C. musae BN1 (I), C. asianum MGK1 (J), and C. asianum MGN1 (K). Scale bars = 1 cm.
Determination of minimum inhibitory concentrations (MICs) of selected plant extract
MICs were determined for the plant extract that exhibited antifungal activity against nine different Colletotrichum species. The antifungal activities of the clove ethanolic extract are summarized in Table 5 as inhibition-zone diameters. For most isolates, the MIC of the clove extract was 25 mg/mL; however, C. orchidearum and C. okinawense showed MIC values of 12.5 mg/mL (Fig 6). For comparison, purified methyl eugenol (50% v/v in ethanol) also inhibited the growth of all isolates, producing inhibition zones of varying diameters. The largest zones produced by methyl eugenol were observed for C. okinawense (4.03 cm), followed by C. orchidearum (3.80 cm). Microscopic examination at the same magnification revealed that the inhibition zone produced by eugenol completely prevented mycelial growth in C. okinawense and C. orchidearum (Fig 6A and I), in contrast to other Colletotrichum species.
Colletotrichum orchidearum PH1 (A), C. okinawense PY1 (B), C. nymphaeae SB1 (C), C. brasiliense PF1 (D), C. truncatum CHL1 (E), C. fructicola ADH1 (F), C. pandanicola MGL1 (G), C. musae BN1 (H), and C. asianum MGK1 (I). Scale bars = 1 cm.
GC-MS analysis of bioactive compounds in selected plant extract
GC-MS analysis of the ethanolic clove extract revealed the presence of various bioactive chemical compounds (Table 6). The major constituent identified was 2-methoxy-4-prop-2-enylphenol (eugenol, 72.417%), followed by (1R,4E,9S)-4,11,11-trimethyl-8-methylenebicyclo[7.2.0]undec-4-ene (β-caryophyllene, 12.125%), (2-methoxy-4-prop-2-enylphenyl) acetate (eugenyl acetate, 8.121%), and (1E,4E,8E)-2,6,6,9-tetramethylcycloundeca-1,4,8-triene (α-humulene, 3.268%).
Phytochemical constituents identified by LC-MS analysis of selected plant extract
LC-MS analysis of the clove ethanolic extract (Table 7) revealed a diverse array of bioactive metabolites. The flavonoids detected included quercetin, kaempferol, hispidulin, isosakuranin, myricetin, and delphinidin, while the phenolic compounds comprised gallic acid and pyrogallol. The terpenoid profile was dominated by pygenic acid and ursolic acid. Additional metabolites identified included alkaloids, catechol, resveratrol, and coumaric acid.
Discussion
Anthracnose disease, caused by Colletotrichum species, is a serious problem affecting many tropical plants and results in significant crop losses, predominantly in the postharvest period when fruits are highly susceptible [2,4,29]. In this study, nine Colletotrichum species (C. asianum, C. brasiliense, C. fructicola, C. musae, C. nymphaeae, C. okinawense, C. orchidearum, C. pandanicola, and C. truncatum) from six species complexes were isolated from various hosts, including tropical fruits such as papaya, banana, mango, passion fruit, avocado, and chili; the temperate fruit strawberry; and the ornamental plant philodendron. Some of the isolated Colletotrichum species exhibited strong host preferences, such as C. musae in banana [30], C. asianum in mango [31,32], and C. truncatum in chili [33]. However, multiple Colletotrichum species have been found to infect or colonize the same host plant [34]. For instance, Fuentes-Aragón et al. [35] reported that C. karsti, C. godetiae, C. siamense, C. fioriniae, and C. nymphaeae were isolated from avocados in Mexico. In contrast, C. fructicola and C. pandanicola were isolated from avocados in the present study. Furthermore, some species such as C. okinawense, isolated from Holland papaya were reported for the first time in Thailand. Previously, this species had only been documented in Japan, Brazil, and Taiwan [36,37]. In this study, C. nymphaeae was isolated from strawberries. To date, there have been no documented reports of C. nymphaeae in Thailand; however, it has been identified as a pathogen in various other regions, including Brazil, South Korea, and China, affecting multiple hosts such as strawberry and walnut [38–40].
Nowadays, research on the discovery of antifungal agents from natural sources, including plants, has gained increasing attention as a sustainable strategy to control and manage anthracnose caused by Colletotrichum species, while also reducing the reliance on chemical fungicides. For example, extracts from allspice (Pimenta dioica) [41], Indian borage (Plectranthus amboinicus) [41], garlic (Allium sativum) [42], ginger (Zingiber officinale) [42], and camphor tree (Cinnamomum camphora) [43] have demonstrated antifungal activity by inhibiting the mycelial growth and spore germination of Colletotrichum species. In this study, ten medicinal plants including lemongrass, clove, sea holly, heart-leaved moonseed, turmeric, black pepper, cassumunar ginger, Indian gooseberry, cinnamon, and kariyat were screened for antifungal activity against nine Colletotrichum species at a concentration of 100 mg/mL. Among these, only the ethanolic extract of clove showed antifungal activity against all nine Colletotrichum species isolated in this study. Further research at increased extract concentrations is required to fully evaluate the antifungal potential of the remaining nine medicinal plants against all Colletotrichum species identified in this study. Previous studies have reported that clove essential oil significantly inhibited the mycelial growth and conidia germination of C. gloeosporioides, likely due to its ability to damage the fungal cell wall and membrane, resulting in leakage of intracellular contents [44,45]. Moreover, clove essential oil treatment has been shown to impair cell membrane integrity and biological function by downregulating genes involved in membrane components and transmembrane transport [46].
Several studies have demonstrated that the plant source, plant part, cultivation conditions, extraction solvent, and extraction method can significantly affect the chemical composition of plant extracts [47–49]. Although the chemical composition of clove has been reported previously, it was necessary to identify it in this study. Therefore, GC-MS and LC-MS analyses were performed to identify the chemical composition of the ethanolic clove extract. In this study, eugenol was identified as the predominant compound in the ethanolic clove extract via GC-MS analysis, consistent with previous reports [46,50]. To confirm the antifungal activity of eugenol, methyl eugenol (at a final concentration of 250 mg/mL) was tested, and the results showed that it inhibited all isolated Colletotrichum species. In addition, β-caryophyllene, eugenyl acetate, and α-humulene also detected in the clove extract have been reported to exhibit antifungal activity against phytopathogenic fungi, primarily by inhibiting hyphal growth and spore germination [51–53]. Beyond the well-known essential oil component eugenol, bioactive compounds in the clove extract were further analyzed using LC-MS in this study. Several flavonoids, including quercetin, kaempferol, isorhamnetin, and myricetin, were identified, along with phenolics such as gallic acid, ellagic acid, and catechol. Hydroxycinnamic acids, including caffeic acid, p-coumaric acid, and methyl trans-cinnamic acid, as well as stilbenes like resveratrol, were also detected in the ethanolic clove extract, consistent with previous reports [54,55]. These flavonoids and phenolic compounds have demonstrated notable antifungal properties against phytopathogenic fungi such as Fusarium, C. gloeosporioides, Botrytis cinerea, and Alternaria alternata [56–59]. Their mechanisms of action typically involved disrupting fungal cell walls, compromising membrane integrity, and inhibiting essential fungal enzymes. Additionally, they interfere with organic acid secretion and the biosynthesis of mycotoxins such as fumonisin B1 and beauvericin produced by Fusarium. Moreover, bioactive compounds from the triterpenoids group such as pygenic acid, cucurbitacin, ganoderic acid, and ursolic acid as well as alkaloids and their derivatives, including piperine, reserpine, and piperanine, were identified in the clove phytoextract in this study. These compounds have also been reported in other medicinal plants in previous studies [60–64]. Triterpenoids and alkaloids found in plants exhibit antifungal, antibacterial, and antioxidant activities, as well as anticancer and anti-inflammatory properties [60,65]. However, antifungal activity observed in vitro may not fully reflect in vivo responses due to environmental factors and plant metabolic processes. Therefore, future studies will involve controlled in vivo assays on fruit and plant tissues to evaluate the efficacy of the ethanolic clove extract in controlling anthracnose disease caused by Colletotrichum species. Lesion development, spore germination, and disease progression will then be monitored to validate the in vitro findings. Furthermore, future studies will determine a suitable control concentration that is non-toxic to the target fruits and plants.
Conclusion
This study highlighted the diversity of Colletotrichum species associated with fruits and ornamental plants in Thailand, identifying eleven isolates across nine species: C. asianum, C. brasiliense, C. fructicola, C. musae, C. nymphaeae, C. okinawense, C. orchidearum, C. pandanicola, and C. truncatum. The findings also demonstrate the promising antifungal potential of clove ethanolic extract, which showed strong inhibitory activity against all isolated Colletotrichum species, with MICs ranging from 12.5 to 25 mg/mL. GC-MS and LC-MS analyses revealed a rich profile of bioactive compounds in the clove extract, including eugenol, flavonoids, phenolics, terpenoids, and alkaloids. These results support the potential use of clove phytoextracts as a natural and sustainable alternative for fungal management in agriculture. Overall, the findings of this study enhance our understanding of Colletotrichum species associated with fruits and ornamental plants in Thailand and offer valuable insights for developing effective alternative management strategies using plant-based extracts.
Acknowledgments
We are grateful to Ms. Martha Maloi Eromine for the English editing of our manuscript.
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