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.

Thirty-four endophytic fungal isolates were obtained from the leaves of the medicinal plant Polyscias fruticosa, and their antagonistic activities against the growth of the common tomatoes plant pathogenic fungus Athelia rolfsii were initially screened using a dual culture assay. The endophytic fungus MFLUCC 17-0313, which was later molecularly identified as Diatrype palmicola, displayed the highest inhibition percentage (49.98%) in comparison to the others. This fungus was then chosen for further evaluation. Its culture broth and mycelia from a 10 L scale were separated and extracted using ethyl acetate, methanol, and hexane. Each extract was tested for antifungal activity against the same pathogen using a disc diffusion assay. Only the crude hexane extract of fungal mycelium showed antifungal activity. The hexane extract was fractioned using sephadex gel filtration chromatography and each fraction was tested for antifungal activity until the one with the highest inhibition percentage was obtained. The bioactive compound was identified as 8-methoxynaphthalen-1-ol using nuclear magnetic resonance spectroscopy and mass spectrometry. The minimum inhibition concentration of 8-methoxynaphthalen-1-ol was demonstrated at 250 µg/mL against the selected pathogen. Using the leaf assay, the solution of 8-methoxynapthalen-1-ol was tested for phytotoxic activity against A. rolfsii and was found to have no phytotoxic effects. These results showed that 8-methoxynaphthalen-1-ol has the potential for controlling the growth of A. rolfsii, the cause of Southern blight disease on tomatoes. This study may provide the foundation for future use of this compound as a biofungicide.

Essential Oil Compositions and Antibacterial and Antioxidant Activities of Five Lavandula stoechas Cultivars Grown in Thailand.

The essential oils of five Lavandula stoechas cultivars grown in Thailand were characterized for their volatile compounds using GC-FID and GC/MS methods as well as screened for antibacterial and antioxidant activities. Dried aerial parts, including flowers and stems from each cultivar, were subjected to hydrodistillation for 4 h. The essential oil yields were 0.18 %-0.82 % w/w. Of the 95 compounds detected and identified, 1,8-cineole, fenchone, and camphor were considered the major compounds. Essential oil from each cultivar demonstrated different patterns of antibacterial activity and a variety of antioxidant properties. The highest antibacterial activity, MIC=0.39 mg mL-1 , was observed from the essential oil of L. stoechas 'major' (against Klebsiella pneumoniae and Salmonella typhimurium) and the essential oil of L. stoechas 'white lavender' (against S. typhimurium). The essential oil of L. stoechas×viridis 'St. Brelade' possessed the highest antioxidant capacity, as determined by the DPPH and ABTS assays (IC50 of 67.65 and 89.26 mg mL-1 , respectively). The results indicated that some of these essential oils could be used as key ingredients in lavender oil products in Thailand to increase their therapeutic efficacy, depending on their intended application.

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

Endophytic fungi, which colonize within a host plant without causing any apparent diseases, have been considered as an important source of bioactive secondary metabolites containing antimicrobial and antioxidant activities. The aim of this research was to isolate the endophytic fungi of Cinnamomum loureiroi and then to screen their antimicrobial and antioxidant activities. A total of 11 fungal endophytes were isolated from healthy leaves of Cinnamomum loureiroi belonging to six genera: Botryosphaeria, Colletotrichum, Diaporthe, Fusarium, Neopestalotiopsis, and Pestalotiopsis. All isolated strains were cultured and further extracted with ethyl acetate solvent. Antimicrobial activity of all crude endophytic fungal extracts was analyzed using disc diffusion assay against six bacterial and two fungal pathogens. Crude extracts of strains MFLUCC15-1130 and MFLUCC15-1131 showed broad-spectrum antimicrobial activity against all tested pathogens. Activity against Bacillus cereus and Staphylococcus epidermidis was notable, showing the lowest minimum inhibitory concentration at 3.91 µg/mL. Antioxidant activity of all crude endophytic fungal extracts was also evaluated based on 2,2-diphenyl-1-picrylhydrazyl assay. Significant antioxidant activity was detected in the crude extracts of fungus MFLUCC15-1130 and MFLUCC15-1131 with IC50 of 22.92 ± 0.67 and 37.61 ± 0.49 µg/mL, respectively. Using molecular identification, MFLUCC15-1130 and MFLUCC15-1131 were identified as Neopestalotiopsis sp. and Diaporthe sp., respectively. The major chemical constituents produced by both crude extracts were identified by gas chromatography-mass spectrometry. Eugenol, myristaldehyde, lauric acid, and caprylic acid were the primary antimicrobial and antioxidant compounds in both crude extracts. This is the first report of eugenol being a biologically active compound of Neopestalotiopsis sp. and Diaporthe sp. fungal endophytes. Eugenol has been reported as antimicrobial and antioxidant agents with agronomic applications. Thus the two newly-isolated endophytes may be used for eugenol production, which in turn can be used in a variety of applications.

Incubation of Aquilaria subintegra with Microbial Culture Supernatants Enhances Production of Volatile Compounds and Improves Quality of Agarwood Oil.

Incubation with microbial culture supernatants improved essential oil yield from Aquilaria subintegra woodchips. The harvested woodchips were incubated with de man, rogosa and sharpe (MRS) agar, yeast mold (YM) agar medium and six different microbial culture supernatants obtained from Lactobacillus bulgaricus, L. acidophilus, Streptococcus thermophilus, Lactococcus lactis, Saccharomyces carlsbergensis and S. cerevisiae prior to hydrodistillation. Incubation with lactic acid bacteria supernatants provided higher yield of agarwood oil (0.45% w/w) than that obtained from yeast (0.25% w/w), agar media (0.23% w/w) and water (0.22% w/w). The composition of agarwood oil from all media and microbial supernatant incubations was investigated by using gas chromatography-mass spectrometry. Overall, three major volatile profiles were obtained, which corresponded to water soaking (control), as well as, both YM and MRS media, lactic acid bacteria, and yeast supernatant incubations. Sesquiterpenes and their oxygenated derivatives were key components of agarwood oil. Fifty-two volatile components were tentatively identified in all samples. Beta-agarofuran, α-eudesmol, karanone, α-agarofuran and agarospirol were major components present in most of the incubated samples, while S. cerevisiae-incubated A. subintegra provided higher amount of phenyl acetaldehyde. Microbial culture supernatant incubation numerically provided the highest yield of agarwood oil compared to water soaking traditional method, possibly resulting from activity of extracellular enzymes produced by the microbes. Incubation of agarwood with lactic acid bacteria supernatant significantly enhanced oil yields without changing volatile profile/composition of agarwood essential oil, thus this is a promising method for future use.

Antibacterial potential of secondary metabolites produced by Aspergillus sp., an endophyte of Mitrephora wangii.

Mitrephora wangii, an ethnomedicinal plant, has been used as a natural antibiotic and immunity booster in Thailand. A total of 22 fungi were isolated from M. wangii flowers. The fungal isolates were categorized into six genera including Agrocybe, Aspergillus, Colletotrichum, Nigrospora, Puccinia and Ustilago. Most extracts exhibited antibacterial activity against at least one of the test bacteria. Aspergillus sp. MFLUCC16-0845 was identified as the most bioactive fungus. Chemical composition of Aspergillus sp. MFLUCC16-0845 investigated using gas chromatography-mass spectrometry indicated that the major antibacterial compound was ß-thujaplicin. Moreover, the newly isolated Aspergillus sp. MFLUCC16-0845 could be exploited as a potential source of bioactive compounds and plant defense activators. In addition, it is the first time that strain of Aspergillus sp. isolated and cultured from M. wangii flowers could produce ß-thujaplicin at high yield with strong antimicrobial spectrum, which may lead to wide utilization in producing cosmetics and clinical products.

Antibacterial and Antioxidant Metabolites of Diaporthe spp. Isolated from Flowers of Melodorum fruticosum.

Fifty-two strains of endophytic fungi were isolated from flowers of the medicinal plant Melodorum fruticosum. Seven genera were identified including Alternaria, Aspergillus, Colletotrichum, Diaporthe, Fusarium, Greeneria and Nigrospora. All strains were cultured for 30 days and further macerated in ethyl acetate solvent for 3 days. The obtained fungal extracts were examined for antibacterial activity using agar disc diffusion against nine pathogenic bacteria: Staphylococcus aureus, Bacillus subtilis, B. cereus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Shigella flexneri, Vibrio cholerae and V. parahaemolyticus. Forty-three fungal extracts exhibited antibacterial activity against at least one tested pathogen. The antioxidant properties of all extracts were also investigated by DPPH scavenging assay. Sixteen extracts displayed high antioxidant capacity (IC50 ranging from 10 to 50 µg/mL) when compared to the gallic acid and trolox standards (IC50 of 12.46 and 2.55 µg/mL, respectively). The crude extracts of Diaporthe sp. MFLUCC16-0682 and Diaporthe sp. MFLUCC16-0693 exhibited notable antibacterial and antioxidant activities. Analysis of chemical composition using gas chromatography-mass spectrometry suggested that the observed antibacterial activity of the two Diaporthe spp. was possibly due to the presence of abienol, 4-methoxy stilbene, phenethyl cinnamate and 2Z,6Z-farnesal, while their potential antioxidant activity could be attributed to phenolic compounds, such as benzene acetaldehyde, benzyl benzoate, salicylaldehyde, benzoin and benzyl cinnamate. The results suggest that the genus Diaporthe is a potential source of metabolites that can be used in a variety of applications.

Antibacterial and Antioxidant Constituents of Extracts of Endophytic Fungi Isolated from Ocimum basilicum var. thyrsiflora Leaves.

Fourteen fungal endophytes were isolated from the Ocimum basilicum var. thyrsiflora leaves collected from Northern Thailand. Eight genera were identified including Aspergillus, Ascochyta, Nigrospora, Blastomyces, Colletotrichum, Exidia, Clitopilus, and Nomuraea. The antibacterial activity of crude extracts from all endophytic fungi was tested against nine human bacterial pathogens: Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Bacillus cereus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Vibrio cholerae, and Vibrio parahaemolyticus. All crude extracts showed some degree of antibacterial activity, but the crude extract from Nigrospora MFLUCC16-0605 exhibited broad spectrum activity with MIC values ranging from 7.81 to 250 µg/mL. The antioxidant activity of all crude extracts was also investigated by DPPH radical scavenging assay. Crude extract from MFLUCC16-0605 had high antioxidant activity (IC50 value of 15.36 µg/mL) comparable to the trolox and gallic acid standards showing IC50 values of 2.56 and 12.89 µg/mL, respectively. The chemical composition of the crude extract from MFLUCC16-0605 was determined using GC-MS. Sixty-two compounds were identified representing 92.09% of crude extract with six major components including 5E,9E-farnesyl acetone, columellarin, totarene, laurenan-2-one, and 8S,13-cedranediol. PCR amplification and sequencing of the barcoding region identified MFLUCC16-0605 as belonging to Nigrospora genus. The notable activities of MFLUCC16-0605 indicate that the endophyte is a potent natural resource and its use as an antibacterial/antioxidant agent should be further explored.

Fungal Endophytes: an Alternative Source for Production of Volatile Compounds from Agarwood Oil of Aquilaria subintegra.

Fungal endophytes are microorganisms that are well-known for producing a diverse array of secondary metabolites. Recent studies have uncovered the bioprospecting potential of several plant endophytic fungi. Here, we demonstrate the presence of highly bioactive fungal endophytic species in Aquilaria subintegra, a fragrant wood plant collected from Thailand. Thirty-three fungal endophytic strains were isolated and further identified to genus level based on morphological characteristics. These genera included Colletotrichum, Pestalotiopsis, Fusarium, Russula, Arthrinium, Diaporthe and Cladosporium. All strains were cultured on potato dextrose broth for 30 days prior to partitioning with ethyl acetate. The volatile compounds of all extracts were investigated by gas chromatography-mass spectrometry (GC-MS). Four strains-Arthrinium sp. MFLUCC16-0042, Colletotrichum sp. MFLUCC16-0047, Colletotrichum sp. MFLUCC16-0048 and Diaporthe sp. MFLUCC16-0051-produced a broad spectrum of volatile compounds, including ß-agarofuran, α-agarofuran, δ-eudesmol, oxo-agarospirol, and ß-dihydro agarofuran. These compounds are especially important, because they greatly resemble those originating from the host-produced agarwood oil. Our findings demonstrate the potential of endophytic fungi to produce bioactive compounds with applications in perfumery and cosmetic industries. Antioxidant activity of all extracts was also evaluated by using 2,2-diphenyl-2-picrylhydrazyl radical scavenging assays. The ethyl acetate extract of Diaporthe sp. MFLUCC16-0051 demonstrated superior antioxidant capacity, which was comparable to that of the gallic acid standard. Our results indicate that the MFLUCC16-0051 strain is a resource of natural antioxidant with potential medicinal applications.

Antibacterial Activities of Endophytic Fungi Isolated from Mentha cordifolia Leaves and Their Volatile Constituents.

A total of 17 endophytic fungal isolates were obtained from the leaves of Mentha cordifolia Opiz (Lamiaceae). Seven isolates were identified to the level of genus by using taxonomically relevant morphological traits. Colletotrichum and Phoinopsis species were dominant among these strains. All strains were separated from M cordifolia leaf for the first time. The ethyl acetate extracts of all endophytic fungi were tested for antibacterial activity against Salmonella typhimurium TISTR 1166 and Pseudomonas aeruginosa TISTR781. Most endophytes exhibited antibacterial activity. Ustilago sp. MFLUCC15-1024 presented the highest inhibition zone diameter with a MIC of 31.25 µg/mL against the tesfed pathogens. The chemical composition of the ethyl acetate extract of this strain was investigated using gas chromatography-mass spectrometry. Twenty-one components were identified. 2-Phenylethanol (38.7%), E-ligustilide (12.4%), a-eudesmol (10.2%), ß-vetivone (4.6%), ß-ylangene (3.7%) and verbanol (3.4%) were the major components of the extract. The strong antibacterial activity of Ustilago sp. MFLUCC15-1024 ethyl acetate extract may be attributed to the presence of a high concentration of bioactive compounds including phenyl ethyl alcohol, E-ligustilide and a-eudesmol. The results indicate that there is high diversity of endophytic fungi in M cordifolia leaf, and that Ustilago sp. MFLUCC l5-1024 strain could be an excellent resource of natural antibacterial compounds.