Production of eugenol from fungal endophytes Neopestalotiopsis sp. and Diaporthe sp. isolated from Cinnamomum loureiroi leaves

Plant material and chemicals

Cinnamomum loureiroi were collected from Mae Fah Luang University Botanical Garden, Mae Fah Luang University, Chaing Rai province, Thailand (20°2′42″N, 99°53′41″E) during May 2015. A voucher herbarium specimen (MFLU No. 10021) was identified and deposited at the Mae Fah Luang University Botanical Garden, Chiang Rai, Thailand. Standard C₁₁–C₁₉ n-alkanes were purchased from Fluka, Germany. Ethyl acetate solvent (analytical reagent grade) was purchased from Merck (Darmstadt, Germany).

Isolation, culturing, and morphology of fungal endophytes

The surface of healthy Cinnamomum loureiroi leaves was pre-sterilized by washing with deionized water. The surface was further sterilized by immersing in 70% ethanol for 60 s, 1% sodium hypochlorite (NaClO) for 60 s prior washing with sterile deionized water for 60 s. The sterilized leaves were cut into 0.5 cm² size and then immediately placed on potato dextrose agar (PDA) medium plate mixed with chloramphenicol antibiotic (100 μg/mL) to suppress the growth of bacteria. The culture medium plates were incubated at room temperature (27 °C) for three days until growth of the fungal hyphae was visible. All endophytic fungi that formed colonies were separated and cultured on new PDA plates and then incubated at room temperature (27 °C) for a week. Seven mycelial agar plugs of endophytic strain were placed into 500 mL Erlenmeyer flask containing 150 mL of potato dextrose broth. The cultures were incubated at room temperature (27 °C) for a month prior to the extracting process. The isolated fungal endophytes were deposited at the Center of Excellence in Fungal Research, Mae Fah Luang University under strain accession MFLUCC15-1112, MFLUCC15-1113, and MFLUCC15-1130–MFLUCC15-1138. The morphological characteristics, for example, colony morphology, growth pattern, hyphae, and spore of isolated fungal endophytes were studied for preliminarily identification.

Mycelial culture extraction

After incubating for 1 month, the broth culture was filtrated with Whatman™ filter paper No. 3 under vacuum using Buchner funnel to separate the culture broth and mycelia. The medium broth and ground mycelia were portioned and macerated with ethyl acetate with ratio of 1:1 and 1:1.5 v/v, respectively, using modified method of Monggoot et al. (2017). Mycelial fraction was further sonicated using ultrasound-assisted extraction (Crest/690DAE; GuangDong GT Ultrasonic, Guangdong, China) at room temperature for 5 h. All fractions were combined and concentrated by using a rotary evaporator (Buchi Rotavapor Model R114, BUCHI) under reduced pressure. The obtained extract was subsequently stored at 4 °C until further use.

Screening of antibacterial activity

Antibacterial activity of 11 isolated endophytic fungal extracts was screened against pathogenic bacteria obtained from Department of Medical Sciences, Bangkok, Thailand. Antibacterial activity was evaluated against three Gram-positive (Bacillus cereus ATCC 11778, Staphylococcus aureus ATCC 25923 and Staphylococcus epidermidis ATCC 12228) and three Gram-negative (E. coli ATCC 25922, Pseudomonas aeruginosa ATCC 9027, Salmonella typhimurium ATCC 13311) bacterial pathogens. Antibacterial activities were determined by using a disc diffusion assay. Initially, all crude extracts from isolated fungal endophytes were prepared with a concentration of 1,000 μg/mL using ethyl acetate as a solvent. The obtained solution was then serially diluted using the twofold dilution method until obtaining the concentration of 500, 250, 125, 62.50, 31.25, 7.81, and 3.91 μg/mL, respectively. A single colony of each pathogenic bacterium was activated by culturing in tryptic soy agar medium followed the 0.5 McFarland standard. All pathogenic strains were further cultured on Mueller Hinton agar medium plate. A total of 20 microliters of different diluted solutions were individually loaded into sterilized six mm-diameter filter paper discs (Whatman™, Sigma-Aldrich Corp., St. Louis, MO, USA) and placed onto agar medium plates suppressing bacterial pathogens. Chloramphenicol (20 μg/disc; Sigma-Aldrich, St. Louis, MO, USA) and ethyl acetate solvent was used as positive and negative control, respectively. Plates were then incubated at 37 °C for 24 h. All experiments were performed in triplicate. The inhibition diameter zone values of all endophytic fungal extracts at a concentration of 1,000 μg/mL and chloramphenicol were reported as mean ± standard deviation. The minimum inhibitory concentration (MIC) values were also determined. The variation of inhibition zone values was examined by one-way ANOVA (at P < 0.05).

Screening of antifungal activity

The antifungal activity of all crude endophytic fungal extracts was investigated against Candida albicans ATCC 90028 and Aspergillus niger ATCC 11414 using disc diffusion assay. All crude extracts were prepared as used in antibacterial activity screening. Both pathogenic fungi were cultured on Sabouraud dextrose agar media and incubated at 30 °C for a week. A plug of 1-week old fungal culture (six mm diameter) was then placed on the center of the sterilized plates and incubated at 30 °C for 5 days. The six mm filter paper discs (no. 3; Whatman, Maidstone, UK) moistened with 20 microliters of each crude extract solution were placed onto the surface of culture plates. The plates were incubated at 30 °C for a week in while the fungal growth was also monitored. The growth inhibition of all endophytic fungal extracts against both fungal strains was measured as the percentage of inhibition of a radical growth relative to the control following formula: Percentage of inhibition (%) = 100 × (1 − (radical growth of treatment (mm)/radical growth of control (mm))). All experiments were performed in triplicate. Ethyl acetate was used as negative control. In addition, MIC value of each sample were evaluated for their antifungal activity against lowest concentration of fungal extract inhibiting the visible growth of each pathogen on the agar plate. The growth inhibition and MIC are shown with the value of mean ± SD.

Screening of antioxidant activity

The electron or hydrogen atom donation or ability of endophytic fungal extracts was measured from the decolorizing of the purple colored solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH) dissolving in methanol solvent. All endophytic fungal solutions were prepared at various concentrations (1,000, 500, 250, 125, 62.50, 31.25, 7.81, and 3.91 μg/mL) in methanol. One mL of various solutions of fungal extract was added to a one mL of 60 mM DPPH methanolic solution. The solution was mixed vigorously and incubated in dark place at room temperature for 30 min. After incubation time, the absorbance of all solutions was measured at 517 nm with a Perkin–Elmer–Lamda 25 UV/vis spectrophotometer. The antioxidant activity of endophytic fungal extracts was determined as percentage inhibition of DPPH (I%) using following equation: I% = 100 × (Acontrol − Asample)/Acontrol where Acontrol is the absorbance of the control solution (containing all solutions except the fungal extract), and Asample is the absorbance of the crude extract solution. The antioxidant activity of all endophytic fungal extracts was expressed as IC₅₀ value (μg/mL) and shown in Table 1. Trolox was used as standard for comparison.

Genomic DNA extraction and polymerase chain reaction

MFLUCC15-1130 and MFLUCC15-1131 were selected for genomic DNA extraction and polymerase chain reaction (PCR) due to their notable antibacterial properties. The selected fungal endophytes were grown on PDA for 1 week and the aerial mycelium of each isolate was scraped from the PDA surface. The fungal biomass was then freeze-dried and ground into a fine powder with a pestle and mortar. A modified SDS-CTAB method (Suwannarach et al., 2013) was applied for this experiment. The internal transcribed spacer (ITS) 1-5.8S-ITS2 region of ribosomal DNA was amplified by PCR using ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) and ITS5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′) primers (White et al., 1990) under the following thermal cycling conditions: initial denaturation at 95 °C for 2 min, 30 cycles of 95 °C for 30 s, 50 °C for 30 s 72 °C for 1 min, and final extension at 72 °C for 10 min on a PeqSTAR 2× thermal cycler (Peqlab, Erlangen, Germany). Negative controls lacking fungal DNA were run in parallel. PCR products were checked on 1% agarose gels stained with ethidium bromide under UV light and purified using NucleoSpin® Gel and PCR Clean-up Kit (Macherey-Nagel, Düren, Germany). The purified PCR products were directly sequenced at 1ST Base Company (Malaysia) using the same PCR primers mentioned above. Sequences were used to query GenBank via BLAST (http://blast.ddbj.nig.ac.jp/top-e.html).

Phylogenetic analysis

The assembled contigs were subjected to BLAST search against the publicly available NCBI database to exclude bacterial contamination and to get a preliminary identification of the genera. Two ITS1-5.8S-ITS2 datasets were assembled. MFLUCC15-1130 and MFLUCC15-1131 were evaluated in a group of Xylariales and Diaporthales, respectively. Nucleotide sequences were aligned using MAFFT (default parameters) (Katoh & Toh, 2010). The alignment was further subjected to minor manual adjustments and then trimmed with trimAl (automated option) (Capella-Gutiérrez, Silla-Martínez & Gabaldón, 2009). After trimming, MFLUCC15-1130 contained 656 sites while MFLUCC15-1131 had 670 sites. A maximum likelihood phylogenetic tree was constructed using RAxML (Stamatakis, 2006) and the general time reversible model of nucleotide substitution. Bootstrap values were obtained from 1,000 replicates. A Bayesian inference (BI) (Ronquist & Huelsenbeck, 2003) analysis, available on CIPRES Science Gateway (http://www.phylo.org/portal2/home.action) (Miller, Pfeiffer & Schwartz, 2010), was also performed. Four Monte Carlo Markov chains were run for 1,000,000 generations. Convergence was declared when all retention indices (RI) parameters converged toward zero.

Analysis of volatile compounds by gas chromatography-mass spectrometry

The volatile constituents of crude extracts from strains MFLUCC15-1130 and MFLUCC15-1131 were identified by using a Hewlett Packard model HP6890 gas chromatograph (GC) (Agilent Technologies, Palo Alto, CA, USA), equipped with a HP-5MS (5% phenylpolymethylsiloxane) capillary column (30 m × 0.25 mm × 0.25 μm; Agilent Technologies, Santa Clara, CA, USA). Helium was used as a carrier gas with a flow rate one mL/min, whereas the split ratio of column was operated to 1:50. The oven temperature program was started at 60 °C and increased up to 200 °C, at intervals of 2 °C/min. The GC was coupled to a mass spectrometer (MS) with HP model 5973 (Agilent Technologies, Palo Alto, CA, USA). The electronic ionization energy was set up at 70 eV, while mass range was scanned from 33 to 600 Da with scan rate at 10 spectra/s. Each crude extract was diluted 1:50 in ethyl acetate solvent. Automatically, one μL of solution was injected to the injection port of GC with split mode. The temperature of injector and detector were set at 250 and 280 °C, respectively. Data analysis was performed with ChemStation software (Agilent, Santa Clara, CA, USA). The volatile compounds of crude extract were tentatively identified by comparing the obtained fragmentation patterns of mass spectra with those of verified standards in the National Institute of Standards and Technology database. Linear RI to homologous series of n-alkanes, C₈-C₁₇, were calculated to those obtained by Kovat retention index method. Different RI databases of volatile compounds were obtained from Adams (2017), http://www.pherobase.com and http://www.flavornet.org/f_kovats.html.

Isolation of fungal endophytes of Cinnamomum loureiroi leaves

A total of 11 fungal endophytes were isolated from Cinnamomum loureiroi leaves and then deposited at Center of Excellence in Fungal Research, Mae Fah Luang University, Thailand to obtain a strain accession number. Six genera were characterized based on morphological characteristics including conidiophore, growth rate, colony color, and texture. Colonies and morphologies of all isolated endophytic fungi are shown in the Supplemental Material (Fig. S1). Colletotrichum genus was detected in stains MFLUCC15-1113, MFLUCC15-1134, and MFLUCC15-1137, Diaporthe genus was observed in strains MFLUCC15-1131, MFLUCC15-1135, and MFLUCC15-1136, Pestalotiopsis was found in strains MFLUCC15-1112 and MFLUCC15-1133. Neopestalotiopsis, Botryosphaeria, and Fusarium genus was obtained in a single strain from MFLUCC15-1130, MFLUCC15-1132, and MFLUCC15-1138, respectively. The isolated endophytic fungi were cultured in PDA plates with colonies being approximately 6.0–7.0 cm in diameter after incubating at room temperature (27 °C) for a week. Colonies of Colletotrichum strain MFLUCC15-1113 were light gray at the border of culture, while the conidial mass of MFLUCC1115-1134 was pale gray at the center and MFLUCC1115-1137 was slightly white and their hyphae were swollen in only half the culture, the pattern of growth layer by Diaporthe strains MFLUCC15-1131, MFLUCC15-1135, and MFLUCC15-1136 was grown from mass center. The colony color of MFLUCC15-1131, MFLUCC15-1135 was white, whereas MFLUCC15-1136 presented pastel gray color. Pestalotiopsis strain MFLUCC15-1112 was pale gray color with thick-walled and smooth hyphae distribution, while the hyphae of strains MFLUCC15-1130 and MFLUCC15-1133 were septate and formed a white ring colony. Moreover, the sporulation of Pestalotiopsis strain was identified by its spore, which consisted of four to five conidia and appendages being dark brown. Colonies of Fusarium strains were grayish, but slightly yellow in the center. The growth rate of this genus was rather slow comparing to all other strains in this study. The colony of Botryosphaeria strains was white with abundant aerial mycelia.

Screening of antimicrobial and antioxidant activities

The in vitro antimicrobial activity of 11 endophytic fungal extracts against pathogenic strains from both Gram-positive, Gram-negative bacteria, and fungi was screened using the disc diffusion method by determining inhibition zone diameters. All crude extracts showed antibacterial activities at a concentration of 1,000 μg/mL based on these inhibition zone diameters (Fig. 1). Most fungal extracts were effective on all tested bacteria, with inhibition zones ranging from 7.04 to 14.37 mm. Interestingly, crude extracts of strain MFLUCC15-1130 and MFLUCC15-1131 produced inhibition zone diameter against B. cereus much larger than those obtained of chloramphenicol, suggesting that they were more active than this antibiotic. Crude extracts of strains MFLUCC15-1113, MFLUCC15-1132, MFLUCC15-1134, MFLUCC15-1137, and MFLUCC15-1138 presented lower antibacterial activity against test bacterial pathogens than other crude extracts, while broad spectrum antimicrobial activity with the greatest inhibition was observed by crude extracts of fungus MFLUCC15-1130 and MFLUCC15-1131 against all tested pathogens. Crude extracts of fungus MFLUCC15-1113 and MFLUCC15-1132 had no antibacterial activity against E. coli, while MFLUCC15-1134 and MFLUCC15-1137 fungal extracts had no effect on P. aeruginosa. In addition, MFLUCC15-1113 and MFLUCC15-1134 fungal crude extracts presented less antibacterial activity against Salmonella typhimurium. As noticed, Gram-positive bacterial strains were much more sensitive to crude extracts and chloramphenicol than Gram-negative strains. The MIC of antibacterial activity assay was also measured for these 11 endophytic fungal extracts by using the disc diffusion method. The MIC results are shown in Fig. 1. These antibacterial activity assays indicated that crude extracts of strain MFLUCC15-1130 and MFLUCC15-1131 have a very strong activity with MIC ranging from 3.91 to 500 μg/mL and 3.91 to 250 μg/mL, respectively, while the crude extract of strain MFLUCC15-1132 had less antibacterial activity with MIC of 1,000 μg/mL against all tested bacteria. All crude extracts inhibited the growth of Gram-positive bacteria including B. cereus, Staphylococcus aureus, and Staphylococcus epidermidis.

A disc diffusion assay was also used to investigate the antifungal activity of 11 endophytic fungal extracts at a concentration of 1,000 μg/mL against two fungal strains by determining the percentage of radical growth inhibition. Most crude extracts at concentrations of 1,000 μg/mL showed antifungal activity inhibiting the mycelial growth of Candida albicans and Aspergillus niger as shown in Fig. 2 ranging from 28.09% to 77.89%. MFLUCC15-1112 and MFLUCC15-1138 crude fungal extracts exhibited a lower inhibition percentage. MFLUCC15-1137 crude extract had no fungicidal activity against Candida albicans while no fungicidal activity against Aspergillus niger was detected in the crude extracts of fungus MFLUCC15-1113 and MFLUCC15-1134, respectively. The MIC value of all crude extracts against Candida albicans and Aspergillus niger are also summarized in Fig. 2. The greatest antifungal activity against both tested strains was presented by MFLUCC15-1130 and MFLUCC15-1131 fungal extracts which had MICs of 62.50 and 31.25–62.50 μg/mL, respectively.

The antioxidant activity using DPPH radical scavenging assay was investigated for the 11 fungal extracts. Table 1 shows IC₅₀ values of the fungal crude extracts ranging from 22.92 ± 0.67 to >1,000 μg/mL. The crude extracts exhibiting the greatest scavenging effects was obtained from fungus MFLUCC15-1131 with IC₅₀ of 22.92 ± 0.67 μg/mL while MFLUCC15-1131 exhibited the second best IC₅₀ of 37.61 ± 0.49 μg/mL as compared to the positive control, trolox which was 4.47 ± 0.02 μg/mL, respectively.

Molecular phylogenetic analysis of MFLUCC15-1130 and MFLUCC15-1131

Given the great antimicrobial and antioxidant activities of crude extracts of MFLUCC15-1130 and MFLUCC15-1131, molecular phylogenetic analysis was used to complement morphology. The phylogenetic trees of the endophytic fungal strain MFLUCC15-1130 and MFLUCC15-1131 are shown in Figs. 3 and 4, respectively. Strain MFLUCC15-1130 grouped in the family Sporocadaceae consisting of the genus Neopestalotiopsis and Pestalotiopsis with a supporting value of 91BS/0.99BI. The new strain sequence was also close to Neopestalotiopsis ellipsospora KM199343 with strong supported value of 99BS/0.99BI. Strain sequences of MFLUCC15-1131 grouped in the family Diaporthaceae and within genus Diaporthe with a supporting value of 92BS/0.98BI. The new isolate was sister to Diaporthe raonikayaporum (86BS/0.97BI). Gene sequences of the Neopestalotiopsis sp. MFLUCC15-1130 and strain Diaporthe sp. MFLUCC15-1131 were submitted to GenBank database under accession numbers KY646065 and KY646066, respectively.

Volatile profiles of fungal crude extract of MFLUCC15-1130 and MFLUCC15-1131

The fungal extracts obtained from Neopestalotiopsis sp. MFLUCC15-1130 and Diaporthe sp. MFLUCC15-1131 were subjected to detailed GC–MS analysis. Identified chemical constituents of crude extracts of fungus Neopestalotiopsis sp. MFLUCC15-1130 and Diaporthe sp. MFLUCC15-1131 are presented in Table 2. Similar profiles of chemical constituents were obtained among both crude fungal extracts. Exactly 34 constituents were identified in two samples. In Neopestalotiopsis sp. MFLUCC15-1130, 34 constituents were identified, representing 97.38% of the total crude extract. Eugenol, lauric acid, myristaldehyde, and caprylic acid were found as the main representatives. A total of 34 constituents were also identified in the Diaporthe sp. MFLUCC15-1131, representing 98.70% of the total crude extract. Similarly with Neopestalotiopsis sp. MFLUCC15-1130, this sample contained eugenol as the major representative with high content of 59.68%.