Phenyl Ethers from the Marine-Derived Fungus Aspergillus tabacinus and Their Antimicrobial Activity Against Plant Pathogenic Fungi and Bacteria.

Marine fungi produce various secondary metabolites with unique chemical structures and diverse biological activities. In the continuing search for new antifungal agents from fungi isolated from marine environments, the culture filtrate of a fungus Aspergillus tabacinus SFC20160407-M11 exhibited the potential to control plant diseases caused by fungi. From the culture filtrate of A. tabacinus SFC20160407-M11, a total of seven compounds were isolated and identified by activity-guided column chromatography and spectroscopic analysis: violaceol I (1), violaceol II (2), diorcinol (3), versinol (4), orcinol (5), orsellinic acid (6), and sydowiol C (7). Based on in vitro bioassays against 17 plant pathogenic fungi and bacteria, violaceols and diorcinol (1-3) showed a broad spectrum of antimicrobial activity with minimum inhibitory concentration values in the range of 6.3-200 μg mL-1. These compounds also effectively reduced the development of rice blast, tomato late blight, and pepper anthracnose caused by plant pathogenic fungi in a dose-dependent manner. Our results suggest that A. tabacinus SFC20160407-M11 and its phenyl ether compounds could be used for developing new antimicrobial agents to protect crops from plant pathogens.

Introduction

Plant diseases caused by fungi, bacteria, viruses, and nematodes result in significant losses in the yield and quality of crops, fruits, and vegetables. Synthetic pesticides have been widely used to control plant diseases in the agricultural field; however, current concerns about the exposure of synthetic chemicals to humans, animals, and the environment and the occurrence of pesticide resistance in the field have led to an effort to discover and develop new ecofriendly crop protection agents.[1,2]

Natural products derived from the marine environment have been represented as one of the remarkable sources for agrochemical agents.[3] For example, meridianin alkaloids isolated from tunicate Aplidium meridianum exhibited a broad spectrum of biological activities, and these compounds have been used as lead compounds in developing new fungicide agents.[4] Pulmonarin alkaloids were isolated from the sub-Arctic ascidian Synoicum pulmonaria, and their synthetic derivatives exhibited high antiviral activity against tobacco mosaic virus and potent fungicidal activities.[5] Furthermore, several research papers present that secondary metabolites isolated from marine-derived fungi exhibit promising antimicrobial activity against plant pathogens.[6] Therefore, marine-derived resources have gained much attention in discovering bioactive compounds with benefits for crop protection.[6]

Aspergillus is a large genus with more than 180 different anamorphic species distributed in various ecological niches.[7] This genus has been recognized as a rich source of secondary metabolites, which have been classified as alkaloids, terpenoids, polyketides, sterols, and anthraquinones with various biological activities, including anti-inflammatory, anticancer, antibacterial, and antiviral activities.[8−10] Of the marine-derived Aspergillus species, novel skeletons of natural products have been identified with a variety of interesting bioactivities, such as antimicrobial and cytotoxic activity.[11−14] New diphenyl ether derivatives, diorcinols D and E, isolated from Aspergillus versicolor were cytotoxic to tumor cell lines.[11] A new diphenyl ether 3-methylpentyl-2,4-dichloroasterrate with antitumor activity was discovered from the marine-derived Aspergillus flavipes.[12] Aspergillusether E produced by the marine sponge-derived Aspergillus unguis exhibited strong antimicrobial activity against Staphylococcus aureus and Microsporum gypseum.[13] In addition, two novel prenylated diphenyl ethers, diorcinol L and (R)-diorcinol B, were isolated from the marine algal-derived Aspergillus tennesseensis, and these compounds exhibited in vitro antimicrobial activities against some human and plant pathogenic microbes.[14] Despite efforts to find secondary metabolites showing antimicrobial activity, relatively few Aspergillus species and their active metabolites have been considered as biological agents for crop protection.[6]

In the screening course for the discovery of antagonistic microorganisms, a marine-derived fungus Aspergillus tabacinus SFC20160407-M11 was found to have strong antimicrobial activity against plant pathogens. Here, the present study aimed to isolate and identify the antimicrobial compounds through chromatographic and spectroscopic techniques and evaluate their antimicrobial activity against plant pathogenic fungi and bacteria by in vitro and in vivo assessments.

Results and Discussion

In Vivo and In Vitro Antimicrobial Activity of the Culture Filtrate and Extracts of A. tabacinus SFC20160407-M11

In the continuing search for a new antifungal agent from marine microbes, we found that the culture filtrate of the marine fungus A. tabacinus SFC20160407-M11 exhibited a potent activity that controlled the development of fungal plant diseases such as rice blast caused by Magnaporthe oryzae, tomato late blight caused by Phytophthora infestans, and wheat leaf rust caused by Puccinia triticina (Table ). To find the organic solvent extracts exhibiting the antifungal activity, the culture filtrate of A. tabacinus SFC20160407-M11 was successively partitioned with ethyl acetate (EtOAc) and n-butanol, yielding EtOAc (5.0 g), n-butanol (3.5 g), and water (8.0 g) extracts (Figure S1). When each extract was sprayed onto plants at a concentration of 1000 μg mL–1, the EtOAc extract effectively suppressed the development of rice blast, tomato late blight, and wheat leaf rust with control values of 83, 88, and 90%, respectively (Table ). Furthermore, based on the in vitro antifungal assay, we found that the plant pathogenic fungi M. oryzae and P. infestans were most sensitive to the EtOAc extract with a MIC value of 63 μg mL–1 (Table ). The EtOAc extract also showed a potent inhibitory effect on the bacterial growth of Acidovorax avenae subsp. cattleyae, Dickeya chrysanthemi, Pectobacterium carotovorum subsp. carotovorum, and Ralstonia solanacearum with MIC values of 16, 31, 31, and 63 μg mL–1, respectively (Table ). In contrast to EtOAc extract, n-butanol and water extracts were inactive in both the in vivo and in vitro assays (Tables and 2). Therefore, the EtOAc extract was subjected to column chromatography to isolate the bioactive constituents.

Table 1

In Vivo Disease Control Efficacy of the Culture Filtrate and Extracts of A. tabacinus SFC20160407-M11 Against Six Plant Diseases Caused by Plant Pathogenic Fungia

		control value (%)
treatment	concentration (μg mL–1)	RCB	TGM	TLB	WLR	BPM	PAN
culture filtrate	1-fold dilution	83 ± 0b	17 ± 14ce	92 ± 0b	80 ± 0b	17 ± 0c	23 ± 11b
ethyl acetate extract	1000	83 ± 0b	17 ± 14ce	88 ± 6b	80 ± 0b	17 ± 0c	25 ± 7b
n-butanol extract	1000	50 ± 24cd	0e	17 ± 14de	0d	0d	0c
water extract	1000	25 ± 12de	22 ± 16ce	0e	0d	0d	0c
blasticidin-S	1	71 ± 6bc	–	–	–	–	–
	50	100	–	–	–	–	–
fenhexamid	20	–	92 ± 6b	–	–	–	–
	100	–	100	–	–	–	–
dimethomorph	2	–	–	70 ± 10bc	–	–	–
	10	–	–	100	–	–	–
flusilazole	2	–	–	–	64 ± 5c	–	–
	10	–	–	–	100	–	–
benomyl	1	–	–	–	–	74 ± 9b	–
	100	–	–	–	–	100	–
dithianon	10	–	–	–	–	–	18 ± 6b
	50	–	–	–	–	–	95 ± 2

Disease control values (%) represent the mean of three replicates. Values with different letters are significantly different at p < 0.05 according to Duncan’s multiple range test. RCB, rice blast caused by Magnaporthe oryzae; TGM, tomato gray mold caused by Botrytis cinerea; TLB, tomato late blight caused by Phytophthora infestans; WLR, wheat leaf rust caused by Puccinia triticina; BPM, barley powdery mildew caused by Blumeria graminis f. sp. hordei; PAN, pepper anthracnose caused by Colletotrichum coccodes; −, not tested.

Table 2

In Vitro Antimicrobial Activities of Ethyl Acetate, n-Butanol, and Water Extracts of A. tabacinus SFC20160407-M11 Against Plant Pathogenic Fungi and Bacteria

		MIC (μg mL–1)
plant pathogen		ethyl acetate extract	n-butanol extract	water extract
fungus	Alternaria brassicicola	250	>500	>500
	Botrytis cinerea	500	>500	>500
	Cladosporium cucumerinum	125	>500	>500
	Colletotrichum coccodes	500	>500	>500
	Cylindrocarpon destructans	500	>500	>500
	Fusarium oxysporum	250	>500	>500
	Magnaporthe oryzae	63	>500	>500
	Phytophthora infestans	63	>500	>500
bacterium	Acidovorax avenae subsp. cattleyae	16	>500	>500
	Agrobacterium tumefaciens	125	>500	>500
	Burkholderia glumae	250	>500	>500
	Clavibacter michiganensis subsp. michiganensis	250	>500	>500
	Dickeya chrysanthemi	31	>500	>500
	Pectobacterium carotovorum subsp. carotovorum	31	>500	>500
	Pseudomonas syringae pv. actinidiae	>500	>500	>500
	Ralstonia solanacearum	63	>500	>500
	Xanthomonas arboricola pv. pruni	500	>500	>500

Identification of the Isolated Compounds from the EtOAc Extract of A. tabacinus SFC20160407-M11

Under activity-guided fractionation by a combination of column chromatography, thin-layer chromatography (TLC), medium-pressure liquid chromatography (MPLC), and high-pressure liquid chromatography (HPLC) methods, seven compounds were isolated from the EtOAc extract of A. tabacinus SFC20160407-M11 shown in Figure . The structures of these compounds were identified as violaceol I (1), violaceol II (2), diorcinol (3), versiol (4), orcinol (5), orsellinic acid (6), and sydowiol C (7) by comparing their nuclear magnetic resonance (NMR) spectroscopic data with the literature (Figures S2–S8 and Table S1).[15−19]

Figure 1

Chemical structures of compounds 1–7 isolated from A. tabacinus SFC20160407-M11. 1, violaceol I; 2, violaceol II; 3, diorcinol; 4, versiol; 5, orcinol; 6, orsellinic acid; and 7, sydowiol C.

Violaceols I and II (1 and 2) were previously reported as phenyl ethers produced by Aspergillus species, such as A. versicolor, Aspergillus sydowii, and Aspergillus falconensis.[15] Violaceols I and II (1 and 2) are dimeric ethers consisting of 3,4,5-trihydroxytoluene units.[20] Violaceol I (1) was invariably isomerized into a mixture of violaceol I and II (1 and 2) in methanol.[20] In the present study, violaceols I and II (1 and 2) were successfully separated from each other by preparative TLC on RP-18 F245s (Merck, Darmstadt, Germany), developing in 55% aqueous acetonitrile without isomerization.

In Vitro Antimicrobial Activity of the Isolated Compounds against Plant Pathogens

The antimicrobial activity of all of the isolated compounds was evaluated against plant pathogenic fungi and bacteria based on the MIC values. Violaceols and diorcinol (1–3) exhibited vigorous antifungal activity against the rice blast fungus M. oryzae and the tomato late blight oomycete P. infestans with MIC values in the range of 6.3–25 μg mL–1. However, these compounds exhibited a moderate activity against the other fungal species (Table ). In addition to the antifungal activity, the violaceols (1 and 2) exhibited strong antibacterial activities against A. avenae subsp. cattleyae, D. chrysanthemi, P. carotovorum subsp. carotovorum, and R. solanacearum with MIC values in the range of 6.3–25 μg mL–1; however, diorcinol (3) showed a less potent antibacterial activity compared to violaceols I and II (1 and 2) (Table ). Of the tested bacteria, X. arboricola pv. pruni did not exhibit susceptibility to violaceols and diorcinol (1–3) up to a concentration of 200 μg mL–1 (Table ).

Table 3

In Vitro Antimicrobial Activities of Compounds 1–7 against Plant Pathogenic Fungi and Bacteria

		MIC (μg mL–1)
plant pathogen		1	2	3	4	5	6	7	PCa
fungus	A. brassicicola	200	200	100	200	>200	>200	>200	3.1
	B. cinerea	200	200	100	>200	>200	>200	>200	25
	C. cucumerinum	100	100	100	>200	>200	>200	>200	50
	C. coccodes	200	200	100	>200	>200	>200	>200	6.3
	C. destructans	200	200	100	>200	>200	>200	>200	100
	F. oxysporum	200	200	100	>200	>200	>200	>200	25
	M. oryzae	12.5	12.5	6.3	200	>200	>200	>200	6.3
	P. infestans	25	25	25	200	>200	>200	>200	1.6
bacterium	A. avenae subsp. cattleyae	6.3	6.3	25	>200	>200	>200	>200	6
	A. tumefaciens	50	50	100	>200	>200	>200	>200	1
	B. glumae	100	100	200	>200	>200	>200	>200	0.4
	C. michiganensis subsp. michiganensis	200	200	200	>200	>200	>200	>200	0.4
	D. chrysanthemi	25	25	200	>200	>200	>200	>200	0.4
	P. carotovorum subsp. carotovorum	25	25	200	>200	100	200	>200	0.4
	P. syringae pv. actinidiae	100	100	>200	>200	>200	>200	>200	1
	R. solanacearum	25	25	50	>200	>200	>200	>200	0.4
	X. arboricola pv. pruni	>200	>200	>200	>200	>200	>200	>200	13

Blasticidin-S and oxytetracycline were used as positive controls (PCs) for fungi and bacteria, respectively. 1, violaceol I; 2, violaceol II; 3, diorcinol; 4, versiol; 5, orcinol; 6, orsellinic acid; 7, and sydowiol C.

As with our observations, violaceol I (1), violaceol II (2), and diorcinol (3) are known as broad-spectrum antimicrobial compounds.[19,21] Violaceol I (1) was reported to inhibit the growth of Mycobacterium tuberculosis, S. aureus, Pseudomonas aeruginosa, Bacillus subtilis, and Candida albicans with MIC values in the range of 12.5–50 μg mL–1; violaceol II (2) was less active than violaceol I (1).[19] However, in this study, there was no difference in the antimicrobial activity between violaceol I (1) and violaceol II (2). Diorcinol (3), a dimeric ether of two 3,5-dihydroxytoluenes (orcinols), exhibited antibacterial activity against the Gram-positive bacteria S. aureus and B. subtilis with a MIC value of 4.35 mmol L–1 and also showed antifungal activity against the yeast fungus C. albicans with a MIC value of 3.45 mmol L–1.[21] In addition to the antimicrobial activity, it has been reported that diorcinol (3) has a moderate cytotoxicity in A549 and HeLa cells with IC50 values of 15.5 ± 1.6 and 83.8 ± 1.6 μM, respectively.[22,23] To the best of our knowledge, there have been no reports of the inhibitory activities of violaceols and diorcinol (1–3) against plant pathogenic fungi and bacteria.

Versiol (4) is a polyketide metabolite produced by fungi, such as A. versicolor, Penicillium striatisporum, and Spororamia affinis.[16] Although versiol (4) has been identified from various microorganisms, there is little known about the biological activities of this compound. Our results showed that versiol (4) exhibits a moderate antifungal activity against A. brassicicola, M. oryzae, and P. infestans with MIC values of 200 μg mL–1 (Table ), which is the first discovery of the biological activity of this compound. Orcinol (5) and orsellinic acid (6) were previously isolated from the culture broths of Aspergillus sp. and Penicillium sp., and these compounds were known to possess antibiotic properties against several human pathogenic bacteria, such as S. aureus and P. aeruginosa.[18,24] Similarly, orcinol (5) and orsellinic acid (6) in this study exhibited a moderate antibacterial activity against P. carotovorum subsp. carotovorum with MIC values of 100 and 200 μg mL–1, whereas orcinol (5) and orsellinic acid (6) had no antifungal activity up to a concentration of 200 μg mL–1 (Table ). Sydowiol C (7) was first reported from the marine-derived fungus A. sydowii with a strong inhibitory effect on the growth of Bacillus cereus with a MIC value of 6.25 μg mL–1.[19,25] However, sydowiol C did not exhibit antifungal activity against plant pathogenic fungi Colletotrichum capsici, Colletotrichum gloeosporioides, and A. brassicicola at a concentration of 25 μg mL–1.[25] In our study, there was no significant activity of sydowiol C (7) against all tested plant pathogenic fungi and bacteria up to a concentration of 200 μg mL–1 (Table ).

Disease Control Efficacy of the Isolated Compounds

Considering the in vitro antifungal activity of the isolated compounds, we examined the control efficacy of violaceol I (1), violaceol II (2), and diorcinol (3) against plant diseases caused by fungi. When plants were treated with each compound at concentrations of 125, 250, and 500 μg mL–1, violaceols and diorcinol (1–3) suppressed the disease development of rice blast, tomato late blight, and pepper anthracnose (Figure and Table S2). In particular, the treatments exhibited disease control values of 64–71% against the rice blast disease at concentrations of 250 and 500 μg mL–1. However, at a low concentration of 125 μg mL–1, diorcinol (3) exhibited a weak disease control efficacy against rice blast compared to the violaceols (1 and 2), even though diorcinol (3) had a better in vitro antifungal activity than that of the violaceols (1 and 2) (Figure and Table ). With regard to the disease control of tomato late blight, diorcinol (3) reduced the disease development with a control value of 96% at a concentration of 500 μg mL–1, followed by violaceol II (2) and violaceol I (1) with control values of 62 and 50%, respectively. At a concentration of 250 μg mL–1, violaceols and diorcinol (1–3) showed a similar pattern of control values to those of the plants treated with 500 μg mL–1. Diorcinol (3) also exhibited promising disease control values of 83 and 89% against pepper anthracnose at concentrations of 250 and 500 μg mL–1, respectively, whereas the violaceols (1 and 2) exhibited weak or no disease control efficacies (Figure ). Together, our results showed that diorcinol (3) exhibits the most potent disease control efficacy against tomato late blight and pepper anthracnose diseases at 250 and 500 μg mL–1 (Figure ). In addition to the control efficacy for rice blast, tomato late blight, and pepper anthracnose, we also observed that violaceol II (2) and diorcinol (3) suppressed the development of wheat leaf rust by over 60% compared to the nontreatment control at concentrations of 250 or 500 μg mL–1 (Table S2).

Figure 2

Effects of violaceols and diorcinol (1–3) on plant disease development. (A) Disease control efficacy of violaceols and diorcinol (1–3) against rice blast, tomato late blight, and pepper anthracnose. The bars represent the mean ± standard deviation of two runs with three replicates. The bars with different letters are significantly different from each other at p < 0.01 according to Duncan’s new multiple range test. (B) Representatives of plants treated with diorcinol (3). Photos were taken 5 days post-inoculation (dpi) for rice blast, 4 dpi for tomato late blight, and 3 dpi for pepper anthracnose. NC, treatment with the 0.025% Tween 20 solution containing 1% DMSO as a negative control; PC, fungicides blasticidin-S (50 μg mL–1), dimethomorph (10 μg mL–1), and dithianon (50 μg mL–1) were used as positive controls for rice blast, tomato late blight, and pepper anthracnose, respectively.

Because violaceol and diorcinol (1–3) are often reported as mycotoxins,[26−28] the possibility that these compounds may cause some damage to plants cannot be excluded entirely. Violaceols (1 and 2) were isolated from the fungus Aspergillus violacea as toxic metabolites with an uncoupling effect on oxidative phosphorylation of the rat liver mitochondria.[26] Diorcinol (3) was first isolated from an oak pathogenic fungus Diplodia corticola, which was toxic to the leaves of oak trees (Quercus spp.) and European nettle trees (Celtis australis) at 1000 μg mL–1, causing necrotic lesions.[27] Diorcinol (3) also exhibited an inhibitory effect on the root and shoot growth of Lolium perenne and Poa crymophila seedlings and was as active as the positive control glyphosate.[28] Nevertheless, the plants treated with violaceols and diorcinol (1–3) did not exhibit any phytotoxic symptoms beyond the disease control effects in this study (Figure ).

Conclusions

Herein, we described the isolation and identification of seven compounds from the A. tabacinus SFC20160407-M11 culture filtrate that exhibited inhibitory effects on fungal disease development such as rice blast, tomato late blight, and wheat leaf rust. Of the seven substances, violaceols and diorcinol (1–3) exhibited a vigorous antimicrobial activity in vitro against plant pathogenic fungi and bacteria. In particular, violaceols and diorcinol (1–3) effectively reduced the disease development of rice blast, tomato late blight, and pepper anthracnose compared to the untreated control. Therefore, the culture filtrate of A. tabacinus SFC20160407-M11 and its active constituents can be valuable for developing new natural fungicides used in agriculture.

Materials and Methods

Microbial Strains

A fungal strain A. tabacinus SFC20160407-M11 was isolated from an intertidal mudflat on the western and southern coasts of Korea,[29] which was kindly provided by Dr. Myung Soo Park (Marine Fungal Resource Bank, Seoul National University). For the antifungal assay, the following plant pathogenic fungi provided by the Korea Agricultural Culture Collection (KACC) were used: A. brassicicola (KACC 40036), B. cinerea (KACC 48736), C. cucumerinum (KACC 40576), C. coccodes (KACC 48737), C. destructans (KACC 41077), F. oxysporum (KACC 40043), M. oryzae (KACC 46552), and P. infestans (KACC 48738). These fungal species were maintained on a potato dextrose agar (PDA; BD Difco, Sparks, MD).[30] The two obligate parasitic fungi P. triticina and B. graminis f. sp. hordei were prepared in our laboratory and maintained on their host plants.[31] Plant pathogenic bacteria A. avenae subsp. cattleyyae SL4351, A. tumefaciens SL2434, B. glumae SL4269, C. michiganensis subsp. michiganensis SL4135, D. chrysanthemi SL3218, X. arboricola pv. pruni SL4370, P. carotovorum subsp. carotovorum SL290, P. syringae pv. actinidiae CJW7, and R. solanacearum SL1944 were provided by the National Academy of Agricultural Sciences (Wanju, Korea) and Dr. Seon-Woo Lee of Dong-A University.[32,33] All bacterial strains were maintained on a tryptic soy agar (TSA; BD Difco).

Extraction and Purification of Active Compounds from the Culture Broth of A. tabacinus SFC20160407-M11

The mycelial disks (8 mm in diameter) of a 7-day-old culture of A. tabacinus SFC20160407-M11 grown on a PDA medium at 25 °C were prepared as an inoculum. Twenty mycelial disks were inoculated into 400 mL of potato dextrose broth (PDB; BD Difco) in a baffled 2 L Erlenmeyer flask and incubated at 25 °C for 14 days with an agitation of 150 rpm. A total of 10 L of culture broth was centrifuged at 10 000g for 30 min and filtered through Whatman No. 1 filter paper. The culture filtrates were successively extracted with equal volumes of EtOAc and n-butanol using a separatory funnel. EtOAc, n-butanol, and water layers were concentrated using a rotary evaporator (Rotavapor, Büchi Labortechnik AG, Flawil, Switzerland) to yield EtOAc (5.0 g), n-butanol (3.5 g), and water (8.0 g) extracts, respectively.

The EtOAc extract was applied onto silica gel column chromatography eluting with a stepwise gradient of methanol in dichloromethane (4, 5, 7, 10, 15, 30, and 50%, v/v). Based on the TLC profile, similar fractions were pooled together into three fractions E1 (190 mg), E2 (1.6 g), and E3 (305 mg). TLC plates (Silica gel 60 F254; Merck) were developed with chloroform–methanol (10:1, v/v) and stained with p-anisaldehyde solution (6 mL of p-anisaldehyde, 10 mL of sulfuric acid, 2 mL of acetic acid, and 180 mL of ethanol). MPLC was performed using the Biotage Isolera One system (Uppsala, Sweden) equipped with Biotage SNAP cartridges. Fraction E1 was applied to the Biotage SNAP KP-Sil (25 g) cartridge and eluted with n-hexane-EtOAc (5:1, v/v) to yield compound 4 (40 mg). Fraction E2 was applied to the Biotage SNAP KP-Sil cartridge (50 g) column and eluted with a linear gradient of EtOAc in n-hexane (15, 20, 30, and 50%, v/v) to yield compounds 3 (60 mg) and 5 (8.2 mg), along with two fractions E23 (600 mg) and E24 (100 mg). A part of fraction E23 (200 mg) was purified by preparative TLC (RP-18 F245s; Merck), developing with 55% aqueous acetonitrile to yield compounds 1 (80 mg; Rf value of 0.3) and 2 (85 mg; Rf value of 0.5). HPLC was performed using the Shimadzu Prominence LC-20AR system equipped with a Polaris RP C18-A column (250 mm × 21.2 mm, 5 μm; Agilent Technologies, Santa Clara, CA). Fraction E24 was loaded to preparative HPLC and eluted with a linear gradient of aqueous methanol (50–70%, v/v) at 2 mL min–1 to yield compound 7 (10 mg). Fraction E3 was applied to the Biotage SNAP Ultra C18 cartridge (60 g) column and eluted with a stepwise gradient of methanol in water (10, 30, 50, and 70%, v/v) to yield compound 6 (115.1 mg). The isolation scheme for compounds 1–7 is presented in Figure S1.

Spectroscopic Analysis

Chemical structures of the purified compounds were determined by NMR spectroscopic analyses and comparison with previous literature data. The 1H and 13C NMR spectra were recorded on a Bruker Advance 400 MHz spectrometer (Burker BioSpin, Rheinstetten, Germany) in chloroform-d or methanol-d4 (Cambridge Isotope Laboratories, Andover, MA). Chemical shifts were referenced to the solvent peaks (δH 7.26 and δC 77.2 for chloroform-d; δH 3.31 and δC 49.0 for methanol-d4).

In Vitro Antimicrobial Assay

The MIC values of the crude extracts or purified compounds were determined by the broth microdilution method using a 96-well microtiter plate.[30] Fungal spores or bacterial cells were added to the wells at a final concentration of 1 × 104 cells mL–1. The crude extracts (50 mg mL–1) and purified compounds (20 mg mL–1) dissolved in dimethyl sulfoxide (DMSO) were serially 2-fold diluted at initial concentrations of 500 and 200 μg mL–1, respectively. The fungicide blasticidin-S and antibacterial agent oxytetracycline were used as positive controls. Additionally, 1% DMSO was used as a negative control. All treatments contained no more than 1% DMSO (v/v). The 96-well plates were incubated for 2–3 days, and the MIC values were determined by visual inspection of complete growth inhibition. Assays were performed two times with three replicates for each treatment.

Disease Control Efficacy Assay

The in vivo antifungal activity of the culture filtrate, culture extracts, and purified compounds 1–3 was evaluated against rice blast (caused by M. oryzae), tomato gray mold (caused by B. cinerea), tomato late blight (caused by P. infestans), barley powdery mildew (caused by B. graminis f. sp. hordei), wheat leaf rust (caused by P. triticina), and pepper anthracnose (caused by C. coccodes). For the treatments, the culture filtrate containing 0.025% (w/v) Tween 20 solution was sprayed onto the plants. The culture extracts and purified compounds were dissolved in DMSO and then resuspended in 0.025% (w/v) Tween 20 solution. The final concentration of DMSO used in each treatment did not exceed 1% (v/v) of the solution volume. Positive controls were plants treated with chemical fungicides (blasticidin-S, fenhexamid, dimethomorph, flusilazole, benomyl, and dithianon). Negative controls were plants treated with 0.025% (w/v) Tween 20 solution containing 1% (v/v) DMSO. As hosts for the pathogens, tomato (Solanum lycopersicum cv. Seokwang), wheat (Triticum aestivum cv. Geumgang), barley (Hordeum sativum cv. Hanyoung), and pepper (Capsicum annuum cv. Bugang) were used, which were grown in a greenhouse at 25 ± 5 °C for 1–5 weeks. After the treatment with the culture filtrate, extracts, or purified compounds, the plants were inoculated with each fungal pathogen and incubated as described previously.[30] The experiment was conducted twice with three replicates for each treatment, and the disease control efficacy was calculated with the following equation: control value (%) = 100 × [1 – B/A], where A is the mean lesion area (%) on the leaves or sheaths of the control plants, and B is the mean lesion area (%) on the leaves or sheaths of the treated plants.