Antifungal activity of 8-methoxynaphthalen-1-ol isolated from the endophytic fungus Diatrype palmicola MFLUCC 17-0313 against the plant pathogenic fungus Athelia rolfsii on tomatoes

Plant material

Healthy leaves of P. fruticosa were collected in January 2017 from Mae Chan District, Chiang Rai Province, Thailand (99°70′56″E, 20°10′36″N) for endophytic fungi isolation. The botanical specimens of this plant (MFL10011) were deposited at the Mae Fah Luang Botanical Garden of Mae Fah Luang University, Thailand.

Isolation of endophytic fungi

The isolation procedures were performed following the method described by Atiphasaworn et al. (2017). Healthy leaves of P. fruticosa were washed with running water for 2 min followed by 70% ethanol for 10 s and then sterilized with 1% sodium hypochlorite for 30 s and washed twice with sterile water. Sterilized leaf tissues were sectioned into 0.5 cm² pieces. Approximately 100 pieces were randomly selected and evenly distributed between 30 Petri dishes containing potato dextrose agar (PDA) mixed with chloramphenicol (30 µg/mL) to prevent bacterial growth. Petri dishes were sealed with sterile Parafilm prior to incubation at room temperature (27 °C) for 5 days. Petri dishes were observed daily, and the individual hypha tips of the emerging fungal colonies were sub-cultured on fresh PDA plates at room temperature (27 °C). All isolates were deposited at the Center of Excellence in Fungal Research, Mae Fah Luang University, Thailand (Voucher specimen no. MFLUCC 17-0280-MFLUCC 17-0313).

Isolation and molecular identification of the phytopathogen A. rolfsii

For antifungal activity testing, the pathogen A. rolfsii was isolated from infected S. lycopersicum leaves following a slightly modified method described by Adesegun et al. (2013). The infected S. lycopersicum leaves were collected in July 2016 from a tomato farm in Sansai district, Chiang Mai province, Thailand (98°95′58″E, 19°10′13″N). The infected leaves were washed with sterile distilled water and cut into five mm² segments. Their surface was sterilized using 1% sodium hypochlorite for 1 min and rinsed with sterilized distilled water three times. The segments were placed on Petri dishes to dry at room temperature (27 °C) and further placed on 10 Petri dishes containing PDA mixed with chloramphenicol (30 µg/mL) to prevent bacterial growth. The dishes were incubated at room temperature (27 °C) and observed for 5 days. The isolate was sub-cultured to obtain a pure culture of A. rolfsii, which was stored at room temperature. The pathogen was identified based on its morphological characteristics (Adesegun et al., 2013). The pathogenicity of this A. rolfsii isolate was confirmed by Koch’s postulates. The aerial mycelium of A. rolfsii isolate was scraped from the PDA surface and pulverized with a mortar and pestle to obtain a mycelium pulp. The genomic DNA was extracted following the cetyltrimethyl ammonium bromide method (Elias et al., 2018). Two universal primers, ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) and ITS5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′), were used. Polymerase chain reactions (PCR) were performed at 95 °C for 5 min, followed by 40 cycles of 95 °C for 50 s, 52 °C for 50 s, 72 °C for 50 s, and 72 °C for 10 min on a PeqSTAR 2×  thermal cycler (Peqlab, Germany). The PCR-amplified product was analyzed by gel electrophoresis on 1% agarose gels stained with ethidium bromide under UV light and purified using NucleoSpin^® Gel and PCR Clean-up Kit (Macherey-Nagel, Germany). DNA sequencing was performed on an automated sequencing system ¹ST Base (Malaysia). The obtained sequence was used as a query to search for sequences in GenBank using the BLAST program (Basic Local Alignment Search Tool, https://blast.ncbi.nlm.nih.gov/Blast.cgi).

In vitro antifungal screening by dual culture assay

Endophytic fungal isolates were screened for their antifungal activity against the growth of A. rolfsii using the in vitro dual culture assay. PDA was used as a medium. For each combination of the pathogen and endophytic fungus, a mycelial plug (five mm in diameter) of A. rolfsii and fungal isolate were placed on Petri dishes containing PDA. The plates were incubated at 30 °C. Petri dishes inoculated only with A. rolfsii were used as negative controls. All plates were incubated for seven days until the negative control plates were completely covered with A. rolfsii mycelia The growth inhibition of each endophytic fungus against the tested pathogen was expressed as the inhibition percentage of a radical growth relative to the controls, using the following formula: Inhibition percentage (%) = 100 × [1 −(growth area of the treatment/growth area of the control)]. All experiments were conducted in triplicate.

Fungal cultivation and extraction

From the results of the dual culture assay, the endophytic fungus MFLUCC 17-0313, which showed the strongest in vitro antifungal activity against A. rolfsii, was selected for further cultivation, extraction, isolation, and characterization of the active compound. This isolate was cultured in potato dextrose broth (PDB) using a method described by Atiphasaworn et al. (2017). Five mycelial agar plugs ( five mm diameter, seven days old) of the endophytic fungus MFLUCC 17-0313 were placed in each 1,000 mL Erlenmeyer flask (40 flasks total) containing 250 mL of PDB. The flasks were incubated at room temperature (27 °C) for 28 days without shaking. The culture broth was separated from fungal mycelia by filtration through filter paper. The culture broth was extracted with an equal volume of ethyl acetate three times. The extracts were concentrated under vacuum using a rotary evaporator to produce dried broth extract. The mycelial fraction was macerated with methanol for two days and further concentrated using a rotary evaporator. They were dissolved in 200 mL of distilled water before being partitioned with an equal volume of hexane and ethyl acetate three times, respectively. The obtained hexane and ethyl acetate extracts were collected, and combined. Residue water was eliminated using anhydrous sodium sulfate. The extracts were evaporated using a rotary evaporator to obtain dried extracts from the hexane and ethyl acetate fraction, respectively. In addition, the same mycelia was macerated in dichloromethane for 2 days. The solution was filtered to obtain the dichloromethane extract. Residue water was eliminated using anhydrous sodium sulfate. The extract was evaporated using a rotary evaporator to obtain the dried dichloromethane extract. All extracts were stored at 4 °C until use.

In vitro antifungal activity by disc diffusion assay

The antifungal activity of each crude extract obtained from the endophytic fungus MFLUCC 17-0313 was screened against A. rolfsii using the disc diffusion assay, as described by Tanapichatsakul et al. (2019). The dried crude extracts were dissolved using the individual extraction solvent to yield a concentration of 5 mg/mL. A plug of pathogenic fungal culture (five mm diameter, 5 days old) was placed on the center of the sterilized PDA plates and incubated at 30 °C for 2 days. A 6-mm filter paper disc (no. 3; Whatman, Maidstone, UK) moistened with 30 µL of crude extract solution was placed onto the surface of culture plates. Each crude extract solution was tested individually. The plates were incubated at 30 °C for 7 days and observed daily. Ethyl acetate, hexane, and dichloromethane were used as negative controls, while an amphotericin B solution (dissolved in distilled water to yield a concentration of 5 mg/mL) was used as a positive control (Sigma-Aldrich, USA). The growth inhibition of all endophytic fungal extracts against the tested fungal pathogen was expressed as the inhibition percentage of a radical growth relative to the control, using the following formula: inhibition percentage (%) = 100 × [1 −(radical growth of the treatment (mm)/radical growth of the control (mm))]. All experiments were conducted in triplicate.

Isolation of antifungal compound from the endophytic fungus MFLUCC 17-0313

According to the results of the disc diffusion assay, only the crude hexane extract of fungal mycelia displayed antifungal activity against A. rolfsii. Therefore, it was fractioned using Sephadex LH-20 (GE Health Care Bio-Sciences AB, USA) gel filtration chromatography (3 × 84 cm column) and eluted with a mixture of methanol and dichloromethane (1:1 ratio) to yield a total of 24 fractions (F1–F24). All fractions were evaporated to dryness. The dried residue was dissolved with hexane to yield a solution with a concentration of 5 mg/mL. Each solution was tested for antifungal activity using the disc diffusion method. The F19 solution exhibited the strongest antifungal activity. Thin layer chromatography (TLC) was used to determine the purity of this solution. The TLC result indicated that the F19 solution contains only one compound. Therefore, the F19 fraction was subject to structural identification using ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy. ¹H- and ¹³C-NMR spectra were recorded at 300 and 75 MHz, respectively, using a Bruker AVANCE 300 spectrometer with CDCl₃ as an internal standard. The obtained NMR data was compared to those found in the report of Emre (2018). A mass spectrum of the isolated compound was obtained using a Bruker MicroTOF_LC mass spectrometer (Bruker, United Kingdom).

Determination of the minimum inhibitory concentration

The minimum inhibitory concentration (MIC) value of the isolated antifungal compound against A. rolfsii was determined. Serial dilution was used to prepare seven concentrations (1,000, 500, 250, 125, 62.5, and 31.25 µg/mL) of the active compound in hexane. MIC was defined as the lowest concentration of the active compound that displayed visible growth inhibition of the pathogen on the agar plate. All experiments were performed in triplicate and the results were compared with the amphotericin B solution prepared at a concentration of 1,000 µg/mL.

Phytotoxic assay

The phytotoxicity of the active compound was assayed using a leaf disk and absorption assay according to a method by Reveglia et al. (2019) with modifications.

The leaf disk assays were performed using fourteen-day old S. lycopersicum leaves. Disks of the lamina of plant leaves (six mm diameter) were prepared using a sharp corkborer. The compound was dissolved in a mixture of 0.25% tween 20 with 4% methanol into final concentrations of 1,000, 500, 250, and 125 µg/mL. The leaf disks were immersed in 1.0 mL of different compound solutions. After 18 h, the disks were washed with distilled water. The symptoms were visually observed using a 0 to four scale, where a 0 was no effect, 1 was area of necrotic spots <50%, 2 was area of necrotic spots 50–70%, 3 was area of necrotic spots 70–90%, and 4 was area of necrotic spots >90%. Distilled water and a mixture of 0.25% tween 20 with 4% methanol were used as negative controls whereas glyphosate dissolved in distilled water was used as a positive control. The experiment was carried out in triplicate.

In the leaf absorption assays, 14-day old S. lycopersicum leaves were cut and then placed in a tube containing 1.0 mL of compound solutions. The compound solutions were prepared by dissolving in a mixture of 0.25% tween 20 with 4% methanol and tested at four different concentrations: 1,000, 500, 250, and 125 µg/mL. After 48 h, the leaves were moved to a new tube containing distilled water. The symptoms were visually observed after 48 h using a 0 to four scale, where a 0 was no effect, 1 was area of necrotic spots <50%, 2 was area of necrotic spots 50–70%, 3 was area of necrotic spots 70–90%, and 4 was area of necrotic spots >90%. Distilled water and a mixture of 0.25% tween 20 with 4% methanol were used as negative control whereas glyphosate dissolved in distilled water were used as positive control. The experiment was carried out in triplicate.

Identification of the endophytic fungus MFLUCC 17-0313

The molecular identification of endophytic fungus MFLUCC 17-0313 was preliminarily performed using the method as described in section of molecular identification of the phytopathogen A. rolfsii. The sequences of representative taxa in Diatrypaceae used in our phylogenetic analyses were selected from GenBank based on the BLASTn searches and recently published data (Senwanna et al., 2017; Konta et al., 2020). ITS and β-tubulin sequence datasets were selected to construct the phylogenetic tree. The combined ITS and β-tubulin sequence data were initially aligned by using MAFFT version 7 (Katoh, Rozewicki & Yamada, 2017; http://mafft.cbrc.jp/alignment/server/). The alignment was checked and improved in BioEdit v. 7.0.5.3 (Hall, 1999). The final alignment of the combined ITS and β-tubulin sequence datasets was analyzed and inferred the phylogenetic tree based on maximum likelihood (ML) and Bayesian inference analyses (BI). The estimated evolutionary model of Bayesian inference and maximum likelihood were performed independently for each locus using MrModeltest v. 2.3 (Nylander, 2004). The best-fit model is resulted as GTR+I+G model for each locus under the Akaike Information Criterion (AIC). Maximum likelihood (ML) analyses was performed by using the RAxML-HPC2 on XSEDE (v. 8.2.12) (Stamatakis, 2014) via the CIPRES Science Gateway platform (Miller, Pfeiffer & Schwartz, 2010). Bayesian inference (BI) analysis was performed by MrBayes on XSEDE, MrBayes 3.2.6 (Huelsenbeck & Ronquist, 2001) via the CIPRES Science Gateway platform (Miller, Pfeiffer & Schwartz, 2010). Bayesian posterior probabilities (BYPP) (Rannala & Yang, 1996; Zhaxybayeva & Gogarten, 2002) were evaluated by Markov Chain Monte Carlo sampling (BMCMC). Two parallel runs were conducted using the default settings, but with the following adjustments: Six simultaneous Markov chains were set up at 5,000,000 generations. Trees were sampled every 100th generation. The first 10% of generated trees representing the burn-in phase were discarded and the remaining trees were used to calculate posterior probabilities of the majority rule consensus tree. The phylogenetic trees were figured in FigTree v.1.4.3 (Rambaut, 2016) and edited using Microsoft PowerPoint 2013 and converted to jpeg file in Adobe Photoshop CS6 version 13.0 (Adobe Systems. USA). The sequence of the fungus MFLUCC 17-0313 was also submitted to the NCBI GenBank (accession number MK329053).

Statistical analysis

All experiments were performed in triplicate. Results were expressed as means with error bars for standard deviations. One-way analysis of variance (ANOVA), followed by a t-test, was used to determine the statistically significant difference in the experiments. A P-value of less than 0.05 was considered to indicate statistically significant differences.

Isolation of endophytic fungi

Thirty-four fungal isolates were obtained from P. fruticosa leaves. Morphology of the endophytic fungi isolates are shown in Fig. S1.

Screening of antifungal activity

The antifungal activity of each isolated endophytic fungi against the pathogen A. rolfsii was observed. Figure 1 and Table S1 show the inhibition percentage of each endophytic fungus against the pathogen A. rolfsii from dual culture assay. Antifungal activity of some endophytic fungi against A. rolfsii for seven days using dual culture assay is shown in Fig. 2. The percentage of growth inhibition ranged from 6.32% to 49.98%. The endophytic MFLUCC 17-0313 isolate possessed the highest inhibition percentage (49.98%). The mean inhibition percentage obtained from the endophytic fungus MFLUCC 17-0313 was significantly different from the other 33 isolates.

Antifungal activity of crude extracts of the endophytic fungus MFLUCC 17-0313

The endophytic fungus MFLUCC 17-0313 was further cultured in PDB before its bioactive compound was extracted using different organic solvents. The crude extracts were tested for their antifungal activity against the pathogen A. rolfsii. Only the crude hexane extract of the fungal mycelium was found to exhibit a significant antifungal activity with an inhibition of 64.71%, while amphotericin B (prepared at 5 mg/mL) showed an inhibition percentage of 67.53% (Fig. S2). No antifungal activity against A. rolfsii was found in the other tested broth or mycelium extracts.

Isolation and identification of the active compounds

The crude hexane extract of fungal mycelium was fractioned to obtain the one containing the bioactive compound. Results from the disc diffusion method indicated that F19 fraction contains a bioactive compound with an inhibition percentage of 76.42 ± 0.04%. The results of antifungal activity of some fractions are shown in Fig. S3. The bioactive compound was concentrated and subjected to structural identification. The ¹H NMR (400 MHz, CDCl₃) results are as follows: δ 9.32 (1H, s), 7.42 (1H, dd, J = 0.5, 8.3 Hz), 7.35 (1H, t, J = 8.1 Hz), 7.33–7.28 (2H, m), 6.88 (1H, dd, J = 1.3, 7.4 Hz), 6.78 (1H, d, J = 7.6 Hz), and 4.06 (3H, s), while the ¹³C NMR (CDCl₃, 100 MHz) results are also follows: δ 156.13, 154.45, 136.71, 127.70, 125.58, 121.84, 118.84, 115.04, 110.40, 103.86, and 56.08. These NMR data are in good agreement with those reported in Emre (2018). In addition, the mass spectrum of this compound showed an m/z of 175.0759, with a predicted chemical formula of C₁₁H₁₁O₂. This compound was identified as 8-methoxynaphthalen-1-ol (Fig. 3). All NMR and mass spectral data are depicted in supplement information.

MIC value

The MIC value of the isolated compound, 8-methoxynaphthalen-1-ol, was determined. The results revealed moderate antimicrobial activity of the compound against A. rolfsii with an MIC of 250 µg/mL. The result of antifungal activity of this compound is shown in Fig. S4.

Phytotoxicity test

In the present study, 8-methoxynaphthalen-1-ol, which showed a moderate antifungal activity against A. rolfsii, was further tested for its phytotoxic effect on plant leaves. Using the leaf disk and absorption assays, the development of symptoms after treatments with 8-methoxynaphthalen-1-ol at various concentrations were observed and compared to those obtained from glyphosate at the same concentrations. Distilled water and a mixture of 0.25% tween 20 with 4% methanol were used in control experiments. The effects of 8-methoxynaphthalen-1-ol on S. lycopersicum leaves from the leaf disk are shown in Table 1. Symptoms caused by compounds toward S. lycopersicum leaf disks at different concentrations after 18 h are shown in Fig. S5 while leaf absorption assay is shown in Fig. 4. From the results, 8-methoxynaphthalen-1-ol and the negative controls showed no visual effects on the leaves, while the positive control glyphosate exhibited phytotoxic effects on S. lycopersicum leaves at all concentrations.

Phylogenetic analyses

The dataset consisted of 68 taxa including our strain and the outgroup taxa. RAxML analysis of the combined ITS and β-tubulin dataset had 1,246 distinct alignment patterns and 64.54% of undetermined characters or gaps. The best scoring of RAxML analysis shown in Fig. 5, with the final ML optimization likelihood value of −17386.379663. Bayesian posterior probabilities (BY) from MCMC were evaluated with the final average standard deviation of split frequencies = 0.010983. Phylogenetic analysis from ML, and BI gave trees with similar overall topologies of the generic placement and in agreement with previous studies (Senwanna et al., 2017; Konta et al., 2020). In the phylogenetic tree (Fig. 5), our taxon was clustered with Diatrype palmicola (MFLUCC 11-0018 and MFLUCC 11-0020) with 90% ML and 1.00 BY bootstrap support.