Coprinolide, a novel antifungal tricyclic polyketide with a rare furanone-fused chromene skeleton isolated from the mushroom Coprinus comatus

Abstract

A search for antifungal compounds from the mushroom Coprinus comatus using a bioassay-guided chromatographic fractionation approach led to the discovery of a novel polyketide harboring a rare 3,3a,9,9a-tetrahydro-1H-furo[3,4-b]chromen-1-one skeleton. The novel compound was named coprinolide. The inhibitory activity and fungicidal potential of coprinolide were evaluated against five economically important plant-pathogenic fungi. Coprinolide showed inhibitory effects on conidial germination and germ tube elongation of all tested fungi. The strongest effect was observed for Colletotrichum orbiculare with half-maximal inhibitory concentrations of 7.1 ppm and 8.2 ppm for conidial germination and germ tube elongation, respectively. Furthermore, coprinolide exhibited fungicidal activity against the tested fungi by inhibiting conidial germination to conidial death as confirmed by fluorescence microscopy using fluorescein diacetate and propidium iodide. These findings showed the potential of the mushroom as a source of a novel bioactive compound with promising agricultural application as an antifungal agent against different plant-pathogenic fungi.

Introduction

Diseases caused by plant pathogenic fungi are one of the main contributors to significant economic losses in the agricultural industry. The most prevalent plant pathogenic fungi include Colletotrichum species, which cause anthracnose disease in different fruits and crops,¹⁾ Pyricularia oryzae, the causal agent of rice blast disease,²⁾ and Fusarium oxysporum f. sp. cubense, which causes Fusarium wilt and the most devastating fungal pathogen of banana, worldwide.³⁾ Plant pathogenic fungi employ diverse strategies and mechanisms for a successful infection of their hosts. The attachment of fungal spores to the host plant is considered the primary requirement for spore germination, germ tube elongation, secretion of cell wall-degrading enzymes, up to the formation of specialized structure such as appressorium.^4–6) Some plant pathogenic fungi such as Colletotrichum spp. and Pyricularia oryzae produce highly specialized melanized appressoria, which are important features for the successful penetration of their hosts.⁷⁾ However, other plant pathogenic fungi particularly F. oxysporum f. sp. cubense do not produce appressoria or penetration pegs to invade its host successfully.^8,9) F. oxysporum f. sp. cubense undergoes asexual reproduction, and produces three different types of spores: microconidia, macroconidia and chlamydospores.¹⁰⁾ Conidia and chlamydospores are involved in the infection of host plant, leading to wilting,¹¹⁾ and should therefore be one the targets of any infection management strategies. However, the elimination of these plant pathogenic fungi is a huge challenge for farmers and agricultural producers. Currently, commercially available fungicides are the mainstay of treatment for the prevention of diseases caused by some plant pathogenic fungi. However, the continuous and excessive use of commercially available fungicides has disadvantages such as pollution to the natural environment and adverse effects on the other organisms associated with cultivated plants,¹²⁾ and could lead to the emergence of fungicide-resistant fungal pathogens. Therefore, the discovery of bio-eco-friendly solutions that can inhibit plant pathogenic fungi, particularly their conidial germination, is essential. The search for novel bioactive compounds from natural sources has gained considerable attention as it will lead to the discovery of new antifungal agents to combat various plant diseases caused by fungi.

Among the vast number of natural sources, mushrooms have been proven as essential producers of novel bioactive compounds. For instance, a new β-carboline alkaloid, 1-acetyl-7-hydroxy-β-carboline-3-carboxylic acid, from Sarcomyxa edulis exhibited an anti-inflammatory activity against lipopolysaccharide (LPS)-induced NO production in RAW 264.7 macrophage.¹³⁾ Nineteen new sesquiterpenoids, including bergamotane sesquiterpenes (craterodoratins A–R), and a victoxinine derivative (craterodoratin S), were isolated from the edible mushroom Craterellus odoratus. Of these new compounds, seven (craterodoratins C, J, L, M, N, O, and S) exhibited potent inhibitory activity against LPS-induced proliferation of B lymphocyte cells.¹⁴⁾ Additionally, eight new compounds, furanopaxins A–F, deoxybisinvolutone and coumarinvol were isolated from the wild mushroom Paxillus involutus. Furanopaxins E and F, deoxybisinvolutone, and coumarinvol showed significant α-glucosidase inhibitory activities.¹⁵⁾ In addition, cyclocircadins A and B, isolated from Cyclocybe erebia showed circadian rhythm-regulating activity.¹⁶⁾ Recently, a phenolic compound inaoside A, isolated from the edible mushroom Laetiporus cremeiporus, was shown to exhibit significant antioxidant activity.¹⁷⁾ These studies also highlighted the diverse biological activities of these novel compounds. While various species of mushrooms have been studied leading to the isolation of novel compounds, many species remain unexplored. The isolation of novel compounds from mushrooms for agricultural applications, specifically as antifungals against different plant pathogenic fungi is highly desirable.

Cop. comatus, a basidiomycete of the Agaricaceae family under the order Agaricales has been widely studied for its nutritional values, chemical composition, and functional properties.^18,19) Previous works reported that Cop. comatus may contain compounds with antimicrobial activities^18,19) and therefore, can be a target for the isolation of compounds for agricultural application, specifically as antifungals. Recently, we isolated a potent antifungal compound orsellinaldehyde, from the culture filtrate of Cop. comatus against anthracnose pathogens. Orsellinaldehyde showed significant inhibitory and fungicidal activity against the conidial germination and germ tube elongation of different Colletotrichum species, and suppressed anthracnose symptoms on mango and cucumber fruits infected with Colletotrichum gloeosporioides and Col. orbiculare, respectively.²⁰⁾

During the isolation of the antifungal compound from Cop. comatus, we detected strong antifungal activity against Col. gloeosporioides in another fraction. In the present study, this fraction was further purified, leading to the isolation of a novel antifungal compound, together with a known compound. The isolated novel compound was identified through spectroscopic analyses. The inhibitory activity of the isolated compound was evaluated against the conidial germination and germ tube elongation of five economically important plant pathogenic fungi. The findings of this study provide insights into the potential of mushrooms as a source of novel biologically active compounds against plant pathogenic fungi for agricultural application and will serve as additional baseline information for future studies on the development of bio-eco-friendly solutions against the problems caused by plant pathogenic microorganisms.

Materials and methods

1. General experimental procedures

The high-resolution mass spectra (HRMS) were measured in an Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) while electrospray ionization (ESI) mass spectra were recorded using a Quattro Micro API mass spectrometer (Waters, Milford, MA, USA) connected with an Acquity UPLC (Waters). The ¹H NMR (600 MHz), ¹³C NMR (150 MHz), and 2D NMR spectra (COSY, HMQC, HMBC and NOESY) were recorded using a JEOL JNM-ECZ600 (Tokyo, Japan). Preparative high-performance liquid chromatography (HPLC) was performed using a 10A HPLC system (Shimadzu, Kyoto, Japan). The specific rotation was measured using a SEPA-500 polarimeter (HORIBA, Kyoto, Japan) while the circular dichroism (CD) spectrum was measured using J-820 circular dichroism spectrometer (JASCO, Tokyo, Japan).

2. Mushroom strain

The strain of Cop. comatus (TUFC 30838) used in this study was obtained from the Fungus/Mushroom Resource and Research Center (FMRC), Tottori University, Japan. It was immediately sub-cultured on potato dextrose agar (PDA) (DB Difco, Sparks, MD, U.S.A.) slants and incubated at 25°C for further use.

3. Plant pathogenic fungi

The strains of Col. gloeosporioides MAFF 243178, Col. orbiculare MAFF 726522, and P. oryzae MAFF 101512 were obtained from the the National Agriculture and Food Research Organization (NARO) Genebank (www.gene.affrc.go.jp/index_en.php). F. oxyporum f. sp. cubense race 1 (R1) 160527 and F. oxyporum f. sp. cubense race TR4 (TR4) MSK1-1 are strains preserved in Plant Pathology Laboratory in Tokyo University of Agriculture and Technology, Tokyo. All fungal strains were sub-cultured on PDA slant and incubated at 25°C for further use.

4. Cop. comatus culture filtrate preparation and extraction

Cop. comatus was first grown on malt extract agar (MEA) (50 g malt grain, 30 g glucose, 3 g peptone, 15 g agar, and 1 L tap water) plate at 25°C for two months. To prepare the liquid culture of Cop. comatus, three mycelial disks approximately 6 mm in diameter from the MEA plate were inoculated to a 500-mL flask containing 200 mL of malt extract broth (MEB, 50 g malt grain, 30 g glucose, 3 g peptone and 1 L tap water). In total, we cultured Cop. comatus in 5 L of MEB. The culture was incubated at 25°C under stationary condition for five months. The contents of the 25 flasks were combined and filtered through layers of Whatman filter paper no. 1 and cheesecloth. The culture filtrate was extracted three times with ethyl acetate (EtOAc), and the resulting EtOAc layer was dried over Na₂SO₄ overnight and evaporated to dryness in a rotary evaporator. The EtOAc extract yielded 2.6 g.

5. Bioassay-guided isolation of antifungal compounds

The Cop. comatus culture filtrate extract (2.6 g) was subjected to silica gel column chromatography (130 g silica gel, Daisogel IR- 60-63/210; Daiso, Osaka, Japan) using 1300 mL each of acetone and hexane mixtures as eluting solvent (0–100% acetone, 20% increment, v/v). The eluted fractions were evaporated to dryness and subjected to a bioassay for the inhibition of conidial germination against Col. gloeosporioides. The strongest inhibitory activity was observed in the 20% acetone fraction (479.4 mg). This fraction was applied again onto a silica gel column (24 g silica gel) using 280 mL of EtOAc and hexane mixtures (0–100% EtOAc, 20% increment, v/v). The eluted fractions were concentrated to dryness and subjected to a bioassay to inhibit the conidial germination of Col. gloeosporioides. The activity was observed in the 20% EtOAc fraction (67 mg), which was subjected to further fractionation by ODS column chromatography (3.3 g; Cosmosil 75C₁₈-OPN, Nacalai Tesque, Kyoto, Japan) using 60 mL mixtures of methanol (MeOH) and water as eluting solvent (20–100% MeOH, 20% increment, v/v). The strongest inhibitory activity was observed in 40% MeOH fraction from which orsellinaldehyde was previously isolated. Furthermore, another fraction (60% MeOH fraction) had also shown strong inhibitory activity against Col. gloeosporioides and was subjected to preparative HPLC under the following conditions: column: Cosmosil 5C₁₈ AR-II 10 ID×250 mm; temperature: 40°C; flow rate: 1.5 mL/min; solvent: 55% acetonitrile; detection: 210 nm. Two peaks at retention times of 20.5 min and 22.5 min were isolated. The peak at retention time of 20.5 min was identified as 4,6-dimethoxyphthalide upon confirmation of the NMR data with the NMR data of 4,6-dimethoxyphthalide in our previous study.²¹⁾ We focused on identifying the compound corresponding to the peak at a retention time 22.5 min, and denoted as compound 1 (3.4 mg). To obtain a higher yield of 1, Cop. comatus was cultured again in MEB for six months. We purified 1 following the purification process described above. Successfully, 1 was isolated again with 3.1 mg yield.

Spectroscopic data of compound 1: [α]_D²⁰=−30.3° (c 0.08, CHCl₃); ECD (MeOH) λ[nm](Δε): 234 (–2.1); ESIMS positive: m/z 279.3 [M+H]⁺, negative: m/z 277.2 [M−H]⁻, m/z 323.2 [M+HCOO]⁻ (Fig. S1); HRESIMS positive: m/z 279.1223 [M+H]⁺ (calcd for C₁₅H₁₉O₅, 279.1233), m/z 301.1042 [M+Na]⁺ (calcd for C₁₅H₁₈O₅Na, 301.1052), negative: m/z 277.1083 [M–H]⁻ (calcd for C₁₅H₁₇O₅, 277.1076), m/z 313.0853 [M+³⁵Cl]⁻ (calcd for C₁₅H₁₈O₅³⁵Cl, 313.0843) and m/z 315.0824 [M+³⁷Cl]⁻ (calcd for C₁₅H₁₈O₅³⁷Cl, 315.0819). The ¹H and ¹³C NMR data are presented in Table 1 (Figs. S2).

Table 1. ¹H NMR (600 MHz) and ¹³C NMR (150 MHz) data of coprinolide (1)

Position	1H NMR						13C NMR
	DMSO-d6			CDCl3			CDCl3
	δΗ, ppm	multiplicity		δΗ, ppm	multiplicity		δc, ppm, type
1							177.4, C
3	4.64	q (J=6.6 Hz)	H	4.65	q (J=6.6 Hz)	H	80.0, CH
3a							102.2, C
4a							152.6, C
5	6.24	d (J=2.4 Hz)	H	6.32	d (J=2.4 Hz)	H	100.1, CH
6							159.1, C
7	6.39	d (J=2.4 Hz)	H	6.43	d (J=2.4 Hz)	H	110.4, CH
8							137.9, C
8a							110.9, C
9	2.83	d (J=16.2 Hz)	2H	2.82	d (J=15.6 Hz)	2H	30.3, CH2
	2.56	d (J=15.6 Hz)		2.76	d (J=16.2 Hz)
9a							44.9, C
10	1.32	d (J=6.6 Hz)	3H	1.49	d (J=6.6 Hz)	3H	12.8, CH3
11	3.64	s	3H	3.74	s	3H	55.3, CH3
12	2.13	s	3H	2.21	s	3H	19.3, CH3
13	1.10	s	3H	1.23	s	3H	16.0, CH3
OH	7.38	brs	1H

6. Bioassays for inhibitory activities on the conidial germination and germ tube elongation

Col. gloeosporioides MAFF 243178 was grown on PDA plates, while Col. orbiculare MAFF 726522 was grown in rice bran agar (RBA) plates (50 g rice bran, 20 g sucrose, 20 g agar, 1 L distilled water). F. oxyporum f. sp. cubense R1 and F. oxyporum f. sp. cubense TR4 were grown on V8 juice medium plates [200 mL V8 juice (Del Monte Quality Vegetable Juice, Nagano, Japan), 3 g CaCO₃, 15 g agar at 1.5%, and 1 L distilled water]. The plates were incubated at 25–27°C for 7–10 days. Meanwhile, P. oryzae MAFF 101512 was grown on oat meal agar (OMA) plates [50 g oatmeal (Melvit Secret Whole Rolled Oats, Warszawa, Poland), 20 g agar, 1 L distilled water], and incubated at 20°C for 14 days under a black light lamp (FL-15BLB, Hitachi, Tokyo, Japan). The aerial hyphae were removed by flooding the plate with distilled water containing 0.01% glucose by gently rubbing the surface with a spatula, and the plate was further incubated for another 3 days at 25°C. In order to collect conidia of all the test fungi, the surface of the fungal colony was flooded with distilled water containing 0.25% Tween 20 and gently scraped using a spatula. The collected conidial suspension was filtered through 4 layers of cheesecloth and microcentrifuged twice at 1200 rpm for 3 min. The conidia were resuspended in distilled water containing 0.25% Tween 20 and the number of conidia was adjusted to 5×10⁵ conidia/mL using a cell counting chamber and used immediately for the inhibition of the conidial germination assay.

The assay for the inhibition of conidial germination and germ tube elongation was based on the study of Morimoto et al. (2018).²²⁾ Initially, 1 was dissolved in DMSO and added to the conidial suspensions to achieve the following compound concentrations: 1, 3, 10, 30, 100, 300 and 1000 ppm, with a final concentration of DMSO at 1%. As a control, DMSO was added to the fungal suspension. The mixture was vortexed and three drops (25 µL) were placed on a microscope glass slide coated with collodion. The glass slides were placed in a container with a cover lined with tissue paper moistened with distilled water and incubated at 25°C for 24 hr in the dark. After incubation, the germinated and ungerminated conidia were observed under a compound microscope (Olympus BX43, Tokyo, Japan). The number of germinated and ungerminated conidia was counted at a limit of 100 conidia in each drop. Germ tube elongation was then measured using the ImageJ software (https://imagej.nih.gov/ij/index.html). The inhibition rate on conidial germination was calculated using the following formula:

  

where Gc is the number of germinated conidia in the control, and Gt is the number of germinated conidia in the sample treated with the compound. Moreover, the inhibition rate on germ tube elongation was computed using the same formula using the length (µm) of the germ tube.

For the determination of the half maximal inhibitory concentration (IC₅₀) of 1 on each fungus, a dose-response curve was generated using the log (conc.) of the compound on the x axis and % inhibition on the y axis. Then, the IC₅₀ value was calculated from a linear line drawn between the two points that encompass 50% inhibition.

7. Fungal conidia viability staining assay

The fungicidal activity of 1 was determined by a fungal conidia viability staining assay using fluorescein diacetate (FDA) and propidium iodide (PI) (Wako Pure Chemical Industries, Osaka, Japan) according to Tang et al. (2020)²³⁾ with some modifications. A fungal conidial suspension adjusted to 5×10⁵ conidia/mL was added with the test compound dissolved in DMSO to achieve the following compound concentrations: 1, 3, 10, 30, 100, 300 and 1000 ppm, with a final concentration of DMSO at 1%. As a control, DMSO was added to the fungal suspension at 1%. The mixture was vortexed and incubated for 24 hr at 25°C. The conidia were then washed twice with sterile distilled water by microcentrifugation at 1200 rpm for 3 min, and 100 µL of conidial suspension was added with 20 µL FDA and 20 µL PI solutions. The viability of fungal conidia was observed under a fluorescence microscope (Keyence BZ-X810, Osaka, Japan) within 10 min. Dead conidia exhibited red fluorescence while living conidia appeared in green when stained with PI and FDA, respectively.²⁴⁾

8. ECD Calculation

Chem3D (version 20.1, Revvity, Waltham, MA, U.S.A.) was used to generate pdb file of 1. Conformational searches were performed using GROMACS program (version 2018.6) with the general AMBER force field (GAFF2) in the Amber Tools 21 package. Possible rotamers of 1 in terms of the methoxy group at C-6 and hydroxy group at C-3a were built using Avogadro (version 12.0). Optimization and frequency analysis were performed using Gaussian16 software²⁵⁾ at the B3LYP/631G(d) level in the gas phase. The three most stable conformers (Supplemental Fig. S14, Table S1–S4) were subjected to re-optimization and frequency analysis using M06-2X/6-311G+(2d,p) in methanol solution with IEFPCM. The ECD calculations were performed using TDDFT method with the M062X/aug-cc-pVTZ level in methanol solution with IEFPCM. The final ECD spectra of the conformers were weighed according to the Boltzmann distribution theory. The generated ECD spectra for the two enantiomers of 1 were compared with the experimental CD.

9. Detection of compound 1 from the mycelia of Cop. comatus

Mycelia obtained from the liquid culture of Cop. comatus were dried for 72 hr in a fume hood. Dried mycelia were soaked in MeOH 3 times every 24 hr. The MeOH extracts were collected every 24 hr and extracted 3 times with EtOAc and water. The EtOAc layer was dried over Na₂SO₄ overnight and evaporated to dryness by a rotary evaporator. The resulting extract was subjected to two rounds of silica gel column chromatography and ODS column chromatography under the same conditions used for the fractionation of the culture filtrate. The MeOH fraction obtained from the ODS column chromatography was subjected to HPLC analysis under the following conditions: column: Cosmosil 5C₁₈ AR-II 4.6 ID×150 mm; temperature: 40°C; flow rate: 0.8 mL/min; solvent: MilliQ water (A), ACN (B), gradient: B/(A+B)=5–100%, 30 min; detection: 210 nm. The peaks corresponding to orsellinaldehyde was detected at ODS–MeOH 20% fraction, while peak corresponding to 1 was detected at ODS–MeOH 40% fraction. 4,6-dimethoxyphthalide was detected at both ODS–MeOH 20% and 40% fractions.

Results

1. Identification of a novel antifungal compound from Cop. comatus

We extracted the culture filtrate of Cop. comatus using ethyl acetate and performed bioassay-guided fractionation of the extract to identify a new antifungal compound. Compound 1 was purified as a pale-yellow oil by two rounds of silica gel column chromatography, ODS column chromatography and preparative high performance liquid chromatography (HPLC). It has a molecular formula of C₁₅H₁₈O₅ and molecular weight of 278 as determined by the exact masses of positive (m/z 279.1223 [M+H]⁺ and m/z 301.1042 [M+Na]⁺) and negative ions (m/z 277.1083 [M−H]⁻, m/z 313.0853 [M+³⁵Cl]⁻ and m/z 315.0824 [M+³⁷Cl]⁻) in HR ESI-MS. Thus, the hydrogen deficiency index of 1 was seven. The ¹H NMR analysis revealed two doublet signals (δ_H 6.43 and 6.32 ppm; J=2.4 Hz) corresponding to a tetra-substituted benzene ring, as well as the signals derived from one methine (δ_H 4.65 ppm), one methylene (δ_H 2.82 and 2.76 ppm), and four methyl groups (δ_H 3.74, 2.21, 1.49 and 1.23 ppm). On the other hand, ¹³C NMR spectra showed signals corresponding to carbons in a benzene ring (δ_C 159.1, 152.6, 137.9, 110.9, 110.4 and 100.1 ppm), a carbonyl (δ_C 177.4 ppm), a methoxy (δ_C 55.3 ppm), an aliphatic methylene (δ_C 30.3 ppm), a hemiketal (δ_C 102.2 ppm), an oxymethine group (δ_C 80.0 ppm), a quaternary carbon (δ_C 44.9 ppm), and three methyl groups (δ_C 19.3, 16.0 and 12.8 ppm) (Table 1, Supplemental Fig. S2). The proton–carbon bonds were determined via HMQC analysis (Supplemental Fig. S3). ¹H–¹H COSY analysis showed a signal connecting H-3 and H-10, indicating a substructure of –CH–CH₃ (Fig. 1B, Supplemental Fig. S4). Furthermore, the presence of OH group was confirmed by ¹H NMR analysis measured using DMSO-d₆ as the solvent (Supplemental Fig. S6). The proton signal at δ_H 7.38 ppm corresponded to a hydroxyl group, shifted low field due to its attachment in a hemiacetal carbon at C-3a (δ_C 102.2 ppm). These partial structures were connected based on the HMBC correlations (Fig. 1B, Supplemental Fig. S5). The IUPAC name of 1 is 3a-hydroxy-6-methoxy-3,8,9a-trimethyl-3,3a,9,9a-tetrahydro-1H-furo[3,4-b]chromen-1-one (Fig. 1A).

Fig. 1. The antifungal compound isolated from the culture filtrate of Cop. comatus. (A) Chemical structure of coprinolide (compound 1). (B) HMBC and ¹H–¹H COSY correlations, (C) Key NOE correlations. (D) Experimental and calculated ECD spectra of coprinolide in MeOH.

The relative configurations of 1 were determined by 2D NOESY spectrum that revealed the correlations from H-10 (δ_H 1.32 ppm) to H-13 (δ_H 1.1 ppm) and OH-3a (δ_H 7.38 ppm). This indicated that the methyl groups attached to C-3 (δ_C 80.0 ppm) and C-9a (δ_C 44.9), and the OH group at C-3a (δ_C 102.2) were on the same spatial directions (Fig. 1C, Supplemental Fig. S6). The absolute configurations were determined by TDDFT calculations of the ECD spectra of the two possible enantiomers of 1. The circular dichroism (CD) spectrum of 1 showed a negative cotton effect at 234 nm (−2.1Δε) (Fig. 1D). One of the calculated ECD of two possible enantiomers was in accordance with the experimental spectrum leading to the assignment of the absolute configurations of 1 as 3R, 3aS, 9aR (Fig. 1D). These data established the overall identification of 1. Because 1 has not yet been reported up to date, we named it as “coprinolide.”

The mycelial extracts of Cop. comatus was also examined for the presence of compounds isolated from the culture filtrate. The HPLC analysis showed peaks corresponding to coprinolide as well as peaks corresponding to orsellinaldehyde²⁰⁾ and 4,6-dimethoxyphthalide.²¹⁾ The ODS–MeOH 20% fraction of the mycelial extracts showed peaks corresponding to orsellinaldehyde and 4,6-dimethoxyphthalide, while ODS–MeOH 40% fraction showed peaks corresponding to 4,6-dimethoxyphthalide and coprinolide. In comparison with the culture filtrate extracts, orsellinaldehyde was isolated from ODS–MeOH 40% fraction, while 4,6-dimethoxyphthalide and coprinolide were isolated from ODS–MeOH 60% fractions (Fig. 2A and 2B). The difference of the fraction containing these compounds between the culture filtrate and mycelia extracts may be because the differences in other substances present in the culture filtrate and mycelial extracts affected the timing of elution of the compounds. The presence of these compounds in both culture filtrate and mycelial extracts of Cop. comatus suggest intracellular production and eventually extracellular release of the compounds as well as their possible biosynthetic relationship.

Fig. 2. HPLC analyses of the fractions showing the peaks corresponding to the isolated compounds from Cop. comatus. Fractions obtained from the culture filtrate (A) and mycelia (B) were analyzed by gradient HPLC.

2. Coprinolide inhibits conidial germination and germ tube elongation of plant pathogenic fungi

The antifungal activity of coprinolide was determined through the inhibition of the conidial germination and germ tube elongation assay against Col. gloeosporioides, Col. orbiculare, F. oxyporum f. sp. cubense R1, F. oxyporum f. sp. cubense TR4, and P. oryzae. All tested fungi showed sensitivity to coprinolide in a dose-dependent manner starting from 1 ppm. Complete inhibition of conidial germination and germ tube elongation of Col. gloeosporioides, F. oxyporum f. sp. cubense R1 and F. oxyporum f. sp. cubense TR4 was observed at 1000 ppm of coprinolide (Figs. 3 and 4, Supplemental Figs. S7 and S8) whereas the complete inhibition of P. oryzae was observed starting at 300 ppm. Among the tested fungi, Col. orbiculare was the most sensitive in conidial germination with IC₅₀ value 7.1 ppm and completely inhibited at concentration of 100 ppm or higher. For germ tube elongation, P. oryzae, Col. orbiculare and Col. gloeosporioides were the most sensitive with IC₅₀ values of 7.7 ppm, 8.2 ppm and 8.6 ppm, respectively (Table 2). Furthermore, judging from the IC₅₀ values, coprinolide showed a relatively stronger effect on the germ tube elongation of all the tested pathogenic fungi than on their conidial germination.

Fig. 3. Inhibitory activity of coprinolide on conidial germination of different plant pathogenic fungi. DMSO solutions of compounds were added to the fungal conidial suspension (5×10⁵ conidia/mL). As control, DMSO was added to the conidial suspension at 1%. After 24 hr, the germinated conidia were counted. Data presented are mean (n=3)±SD.

Fig. 4. Inhibitory activity of coprinolide on germ tube elongation of different plant pathogenic fungi. DMSO solutions of compounds were added to the fungal conidial suspension (5×10⁵ conidia/mL). As control, DMSO was added to the conidial suspension at 1%. After 24 hr, the lengths of germ tubes were measured using ImageJ software. Data presented are mean (n=3)±SD.

　

Table 2. IC₅₀ values of coprinolide on conidial germination and germ tube elongation of different plant pathogenic fungi

Plant pathogenic fungi	Conidial germination	Germ tube elongation
	*IC50 (ppm)	*IC50 (ppm)
Colletotrichum gloeosporioides	123.5b	8.6a
Colletotrichum orbiculare	7.1a	8.2a
Fusarium oxysporum f. sp. cubense R1	207.7c	35.6b
Fusarium oxysporum f. sp. cubense TR4	157.6bc	25.8ab
Pyricularia oryzae	152.2bc	7.7a

*The IC₅₀ values of coprinolide were calculated from the data for Figs. 3 and 4, and Supplemental Figs. S7 and S8. Different letters indicate significant differences among fungi in the same column (p<0.05; Tukey–Kramer test).

3. Coprinolide exhibits fungicidal activity against various plant pathogenic fungi

The fungicidal activity of coprinolide was assessed by viability staining using FDA and PI. Conidia of the Col. gloeosporioides, F. oxyporum f. sp. cubense R1 and F. oxyporum f. sp. cubense TR4 treated with 1–300 ppm of coprinolide showed bright green fluorescence, indicative of live conidia. However, treatment of the compound at 1000 ppm resulted in the emission of red fluorescence indicating the death of the conidia (Fig. 5A, C–E, Supplemental Figs. S9, and S11–S13). On the other hand, coprinolide at 300 ppm showed fungicidal activity against P. oryzae. In agreement with the inhibition of conidial germination, where in Col. orbiculare was completely inhibited by coprinolide at 100 ppm or higher, fluorescence microscopy confirmed that coprinolide at these concentrations was fungicidal against Col. orbiculare (Fig. 5B and Supplemental Fig. S10).

Fig. 5. Effect of coprinolide on conidial viability of different plant pathogenic fungi. (A) Col. gloeosporioides, (B) Col. orbiculare, (C) F. oxysporum f. sp. cubense R1, (D) F. oxysporum f. sp. cubense TR4, (E) P. oryzae. DMSO solutions of compounds were added to the fungal conidial suspension (5×10⁵ conidia/mL). As control, DMSO was added to the conidial suspension at 1%. After 24 hr, conidia were washed with sterile distilled water, added with equal volume of FDA and PI, and immediately observed in fluorescence microscope (scale bar=10 µm).

Discussion

Mushrooms are essential producers of compounds for agricultural applications. For instance, the first natural strobilurin, strobilurin A was isolated from the mushroom Strobilurus tenacellus.²⁶⁾ Strobilurins have been practically used as fungicides globally to control different plant diseases caused by fungi. They are considered as quinone outside inhibitors (QoI) fungicides that inhibit mitochondrial respiration by targeting the quinol oxidation site of cytochrome b.²⁷⁾ The unique mechanism of action of strobilurins of targeting one specific site of fungal pathogens made them susceptible to resistance. There are studies that reported the resistance of some fungi to strobilurin fungicides.^28,29) Due to these, the discovery of novel compounds for agricultural applications specifically as antifungals against plant pathogenic fungi is highly desirable as it will lead to the development of new fungicide classes against plant pathogenic fungi.

In our previous study, we successfully isolated and identified orsellinaldehyde from the culture filtrate of Cop. comatus which can suppress anthracnose disease.²⁰⁾ In the present study, we found that another fraction of Cop. comatus culture filtrate showed strong antifungal activity against Col. gloeosporioides. We then subjected the fraction to further fractionation, leading to the identification of a novel antifungal compound together with a known compound, 4,6-dimethoxyphthalide. The structure of the novel compound was elucidated and identified as 3a-hydroxy-6-methoxy-3,8,9a-trimethyl-3,3a,9,9a-tetrahydro-1H-furo[3,4-b]chromen-1-one. To the best of our knowledge, this compound has not been reported. Hence, we named it as coprinolide.

Coprinolide is a tricyclic polyketide characterized by the presence of furanone-fused chromene units. Similar furan-fused chromene units are present in some natural products isolated from fungi such as alboatrin from the fungus Verticillium albo-atrum,³⁰⁾ xyloketal B from the marine-derived fungus Xylaria sp.,³¹⁾ and recently isolated simpliketal A, B, C and D from the fungus Simplicillium sp. AHK071-01.³²⁾ Xyloketal B and its derivatives have been extensively studied in various disease models, and have been shown to possess medicinal potentials.³³⁾ Notably, coprinolide harbors a 3,3a,9,9a-tetrahydro-1H-furo[3,4-b]chromen-1-one skeleton. Natural compounds containing this skeleton are very rare. A literature search showed three compounds having the closest or same skeleton with coprinolide. These include penilactones A and B, isolated from the Antarctic deep-sea derived fungus Penicillium crustosum PRB-2³⁴⁾ and pallenic acid isolated from the plant Calyptranthes pallens.³⁵⁾ Hence, reports on the biological activities of compounds bearing such skeleton are also rare. Penilactones A and B were evaluated for their cytotoxic activities against five human cancer cell lines as well as for NF-κB inhibitory activity through transient transfection and reporter gene expression assay, but both compounds showed no cytotoxic activity against the tested human cancer cell lines with IC₅₀>50 µM (>23 ppm for Penilactone A, >25 ppm for Penilactone B), and only penilactone A showed weak NF-κB inhibitory activity with 40% inhibition rate at 10 µM (4.7 ppm).³⁴⁾ Pallenic acid had no cytotoxic effects on different human cancer cell lines.³⁵⁾ Thus, the isolation of coprinolide from Cop. comatus is a significant finding to expand the structural diversity and biological activities of compounds with a rare 3,3a,9,9a-tetrahydro-1H-furo[3,4-b]chromen-1-one skeleton.

In this study, the inhibitory activity on conidial germination and fungicidal activity of coprinolide were evaluated against five plant fungal pathogens. Coprinolide had the most pronounced inhibitory activity based on low IC₅₀ values against the conidial germination and germ tube elongation of Col. orbiculare, a Colletotrichum species responsible for anthracnose disease in cucurbits, with complete inhibition of the conidia at 100 ppm and higher. Other fungal pathogens, P. oryzae, the causative agent of rice blast disease was completely inhibited at 300 ppm of coprinolide, while Col. gloeosporioides, and the two strains of F. oxysporum f. sp. cubense (R1 and TR4), were completely inhibited at a higher concentration (1000 ppm). These findings are important as development of resistance to fungicides have been reported in P. oryzae³⁶⁾ and some Colletotrichum species.^37,38) On the other hand, F. oxysporum f. sp. cubense cannot be easily eradicated despite several practices and strategies to eliminate this pathogen.¹¹⁾ Inhibiting these plant pathogenic fungi would be a huge contribution to the agricultural industry, as these plant pathogenic fungi cause detrimental effects on different crops worldwide.

Coprinolide showed significant stronger activity against Col. orbiculare than Col. gloeosporioides in the germination inhibition assay, indicating species-specific inhibition in the genus. This variation may be attributed to differences in gene sequences encoding proteins involved in the expression of coprinolide’s toxicity, such as the target protein, transporters, or enzymes responsible for detoxification or activation of the compound. This seems likely because Col. orbiculare belongs to orbiculare clade that is outside of Col. gloeosporioides species complex, which is a diverse clade including a number of important plant pathogens.^39,40) In the absence of the data that identify the factors underlying the differential sensitivity of the two Colletotrichum species towards coprinolide, it is important to further examine the coprinolide sensitivity of other Colletotrichum species under the same and different clades.

The adhesion of fungal spores to the plant surface is a preliminary stage towards spore germination until successful invasion and establishment of disease on host plant.⁶⁾ Hence, the inhibitory effect of coprinolide on conidial germination led to the prevention of the possible pathogenic mechanisms of fungi. Further, the fungicidal activity of coprinolide was confirmed by fluorescence microscopy using FDA and PI. The treatment of fungal conidia with coprinolide at specific concentrations resulted in red fluorescence, indication of dead conidia. FDA and PI are used to determine the viability and non-viability of cells, respectively. Viable cells fluoresce in bright green due to the conversion of FDA to fluorescein by the cellular esterases present in the cells.^41,42) On the other hand, PI can only traverse into the membranes of dead cells or cells with a damaged membrane, leaving the cells in red fluorescence.⁴¹⁾ Although the higher concentrations of coprinolide induced conidial death, signs of damage or malformation on conidia were not observed. This may indicate the possible specific mechanism of coprinolide on fungal conidia of inhibiting only the conidial germination and germ tube elongation. Further investigation is needed to determine its specific mechanism on membrane integrity of fungal conidia.

The activity of coprinolide can be deduced based on its chemical structure. The presence of a furanone skeleton in coprinolide which is a characteristic structure of phthalide compounds, may be responsible for its possible antifungal mechanisms. Phthalide and its derivatives exhibit a wide range of biological activities, including antifungal activity against various plant pathogenic fungi.^21,43,44) With regards to phytotoxicity, an antifungal phthalide derivative 3-butylidene phthalide was found to be non-toxic against the plant Lemna minor at the same concentrations that inhibited the fungus Macrophomina phaseolina. However, it posed toxicity on the same plant at 1000 µg/mL (5.3 mmol/L).⁴⁵⁾ On the other hand, a phthalide compound, 5,7-dihydroxy-3(R)-methylphthalide isolated from the endophytic fungus Xylaria brevipes was found to be phytotoxic at 4.6 µg/mL against Brassica chinensis.⁴⁶⁾ Coprinolide at concentrations ranging from 100 to 1000 ppm induced conidial death to plant pathogenic fungi. Given the shared pharmacophore with phthalide compounds, these concentrations of coprinolide should be subjected for phytotoxicity testing for further development as antifungal agent against plant pathogens.

The isolation of orsellinaldehyde from the same mushroom prompted us to hypothesize a plausible biosynthetic pathway of coprinolide (Fig. 6). Orsellinaldehyde is the aldehyde analogue of orsellinic acid, a structural backbone of many secondary metabolites.⁴⁷⁾ Reyes-Fernández et al. (2021)⁴⁸⁾ proposed the biosynthesis of melanin in the fungus Ustilago maydis, which involves the production of orsellinic acid from 1 acetyl-CoA and 3 malonyl-CoA by polyketide synthases (Pks3 and Pks4) and the subsequent reduction of orsellinic acid to orsellinaldehyde by another polyketide synthase (Pks5). In our hypothetical pathway, orsellinaldehyde is further reduced and dehydrated to a key intermediate, 3-hydroxy-5-methyl-6-methylenecyclohexa-2,4-dien-1-one (i in Fig. 6). The other key intermediate, 3,5-dimethylfuran-2,4(3H,5H)-dione (ii), is likely derived from malic acid. Malic acid is widely produced by fungi.⁴⁹⁾ Wu et al. (2012)³⁴⁾ suggested that the key intermediate (5-methylfuran-2,4(3H,5H)-dione) in the biosynthesis of penilactone A may be derived from L-malic acid. An analogous reaction with propionyl-CoA instead of acetyl-CoA in the biosynthesis of penilactone A would provide 3,5-dimethylfuran-2,4(3H,5H)-dione (ii). Interestingly, a structurally related compound with intermediate ii named coprinuslactone ((3R,4S)-2-methylene-3,4-dihydroxypentanoic acid 1,4-lactone) was previously isolated from Cop. comatus.⁵⁰⁾ A Michael-addition reaction between intermediates (i) and (ii) may possibly be resulted in the formation of intermediate (iii). Finally, the dehydration and O-methylation of (iii) generate coprinolide. Orsellinaldehyde and coprinolide were simultaneously detected in the mycelial extracts of Cop. comatus suggesting that these compounds are biosynthetically related to each other. In addition, the phthalide derivative 4,6-dimethoxyphthalide, isolated from the culture filtrate and detected in the mycelia of Cop. comatus which shares a common furanone structure with coprinolide, may also be derived from orsellinic acid. In relation to this, Regueira et al. (2011)⁵¹⁾ proposed a biosynthesis of a phthalide intermediate which was originated from 5-methylorsellinic acid, a compound structurally like orsellinic acid. Both orsellinaldehyde and 4,6-dimethoxyphthalide have been reported in our previous studies as antifungals against multiple plant pathogenic fungi.^20,21)

Fig. 6. Hypothetical biosynthetic pathway of coprinolide.

Comparing the antifungal activity of coprinolide with orsellinaldehyde and 4,6-dimethoxyphthalide in our previous studies,^20,21) coprinolide showed weaker activity than orsellinaldehyde and 4,6-dimethoxyphthalide against Col. gloeosporioides and P. oryzae, respectively, but had stronger activity than 4,6-dimethoxyphthalide against Col. gloeosporioides Cop. comatus likely survives in environments inhabited by diverse microorganisms by utilizing multiple compounds with varying antifungal spectra.

The present study identified a novel compound coprinolide which possessed a rare tricyclic skeleton, isolated from the mushroom Cop. comatus through a bioassay-guided fractionation. Coprinolide exhibited antifungal activity against five economically important plant pathogenic fungi. The inhibitory and fungicidal activities of coprinolide on fungal conidia indicated its potential as an antifungal agent for agricultural crops. This study further supports mushrooms as promising sources of novel natural products for agricultural applications. Studying the mode of action of coprinolide and in-vivo experiment is needed to fully establish its promising and underlying antifungal mechanisms.

Acknowledgements

The mushroom strain used in this study, Cop. comatus TUFC 30838, was provided by the Fungus/Mushroom Resource and Research Center, Tottori University, with partial support from the National BioResource Project (NBRP), MEXT, Japan.

Funding

Enrico M. Cabutaje is indebted to the Department of Science and Technology-Science Education Institute (Foreign Graduate Scholarship) (DOST-SEI FGS) of the Republic of the Philippines for the scholarship grant and support on this research.

Competing interests

There are no financial and non-financial competing interests.

Author Contributions

E.M.C.: conceptualization, performed experiment, data analysis, writing of original draft, K.S.: data analysis, review of manuscript, M.K. and T.A. prepared the fungal strains, review of manuscript. K.U.: data curation, review and editing of the manuscript, T.E.E.D.C. supervision, review and editing of the manuscript, and A.I.: conceptualization, supervision, review and editing of the manuscript.

Electronic supplementary materials

The online version of this article contains supplementary materials, which are available at https://www.jstage.jst.go.jp/browse/jpestics/.

References

• 1) L. Zakaria: Diversity of Colletotrichum species associated with anthracnose disease in tropical fruit crops—A review. Agriculture 11, 297 (2021).
• 2) N. J. Talbot: On the trail of a cereal killer: Exploring the biology of Magnaporthe grisea. Annu. Rev. Microbiol. 57, 177–202 (2003).
• 3) F. Magdama, L. Monserrate-Maggi, L. Serrano, J. García Onofre and M. del M. Jiménez-Gasco: Genetic diversity of Fusarium oxysporum f. sp. cubense, the Fusarium wilt pathogen of banana, in ecuador. Plants 9, 1133 (2020).
• 4) K. W. T. Chethana, R. S. Jayawardena, Y.-J. Chen, S. Konta, S. Tibpromma, P. D. Abeywickrama, D. Gomdola, A. Balasuriya, J. Xu, S. Lumyong and K. D. Hyde: Diversity and function of appressoria. Pathogens 10, 746 (2021).
• 5) M.-H. Lee and R. M. Bostock: Induction, regulation, and role in pathogenesis of appressoria in Monilinia fructicola. Phytopathology 96, 1072–1080 (2006).
• 6) S. L. Tucker and N. J. Talbot: Surface attachment and pre-penetration stage development by plant pathogenic fungi. Annu. Rev. Phytopathol. 39, 385–417 (2001).
• 7) L. S. Ryder and N. J. Talbot: Regulation of appressorium development in pathogenic fungi. Curr. Opin. Plant Biol. 26, 8–13 (2015).
• 8) C. Li, S. Chen, C. Zuo, Q. Sun, Q. Ye, G. Yi and B. Z. Huang: The use of GFP-transformed isolates to study infection of banana with Fusarium oxysporum f. sp. cubense race 4. Eur. J. Plant Pathol. 131, 327–340 (2011).
• 9) C. Li, J. Yang, W. Li, J. Sun and M. Peng: Direct root penetration and rhizome vascular colonization by Fusarium oxysporum f. sp. cubense are the key steps in the successful infection of Brazil Cavendish. Plant Dis. 101, 2073–2078 (2017).
• 10) N. Maryani, L. Lombard, Y. S. Poerba, S. Subandiyah, P. W. Crous and G. H. J. Kema: Phylogeny and genetic diversity of the banana Fusarium wilt pathogen Fusarium oxysporum f. sp. cubense in the Indonesian centre of origin. Stud. Mycol. 92, 155–194 (2019).
• 11) M. Dita, M. Barquero, D. Heck, E. S. G. Mizubuti and C. P. Staver: Fusarium wilt of banana: Current knowledge on epidemiology and research needs toward sustainable disease management. Front. Plant Sci. 9, 1468 (2018).
• 12) S. Bind, S. Bind and D. Chandra: Beneficial microbes for sustainable agroecosystem. In “Advanced Microbial Techniques in Agriculture, Environment, and Health Management,” ed. by. S. Pandey, V. Pande, D. Sati and M. Samant, Academic Press Inc, pp. 1–19, 2023.
• 13) L. Yao, J.-H. Lv, M.-C. Pan, L. Xing, L.-P. Wang, C.-T. Li, S.-Y. Liu and Y. Li: Two new compounds from edible mushroom Sarcomyxa edulis. Nat. Prod. Res. 37, 1491–1497 (2022).
• 14) Q. Dai, F.-L. Zhang, Z.-H. Li, J. He and T. Feng: Immunosuppressive sesquiterpenoids from the edible mushroom Craterellus odoratus. J. Fungi (Basel) 7, 1052 (2021).
• 15) J. Lv, L. Yao, D. Li, C. Jia, J. Zhang, L. Wang, C. Li and Y. Li: Novel hypoglycemic compounds from wild mushroom Paxillus involutus. Bioorg. Chem. 112, 104984 (2021).
• 16) Y. Kobayashi, Y. Akagi, K. Tsubaki, E. Shimoda, T. Kikuchi, N. Endo, T. Ichiyanagi, A. Nakagiri, T. Nishida and A. Ishihara: Identification of Cyclocybe erebia metabolites that affect the circadian rhythm of Eluc expression under control of Bmal1 promoter in mouse fibroblast cells. J. Biosci. Bioeng. 136, 278–286 (2023).
• 17) A. Kawamura, A. Mizuno, M. Kurakake, A. Yamada, H. Makabe and A. Inaoside: New antioxidant phenolic compound from the edible mushroom Laetiporus cremeiporus. Heliyon 10, e24651 (2024).
• 18) N. Stilinović, I. Čapo, S. Vukmirović, A. Rašković, A. Tomas, M. Popović and A. Sabo: Chemical composition, nutritional profile and in vivo antioxidant properties of the cultivated mushroom Coprinus comatus. R. Soc. Open Sci. 7, 200900 (2020).
• 19) P. Nowakowski, S. K. Naliwajko, R. Markiewicz-Żukowska, M. H. Borawska and K. Socha: The two faces of Coprinus comatus—Functional properties and potential hazards. Phytother. Res. 34, 2932–2944 (2020).
• 20) E. M. Cabutaje, K. Ueno, T. E. E. dela Cruz and A. Ishihara: Suppression of anthracnose disease by orsellinaldehyde isolated from the mushroom Coprinus comatus. J. Appl. Microbiol. 135, lxae128 (2024).
• 21) E. M. Cabutaje, K. Ueno, K. Osaki-Oka, K. Kido, T. E. E. dela Cruz and A. Ishihara: Identification of two phthalide derivatives and an oxindole compound isolated from the edible mushroom Pleurotus ostreatus and their inhibitory activities against plant pathogenic microorganisms. J. Pestic. Sci. 48, 156–167 (2023).
• 22) N. Morimoto, K. Ueno, M. Teraishi, Y. Okumoto, N. Mori and A. Ishihara: Induced phenylamide accumulation in response to pathogen infection and hormone treatment in rice (Oryza sativa). Biosci. Biotechnol. Biochem. 82, 407–416 (2018).
• 23) L. Tang, J. Mo, T. Guo, S. Huang, Q. Li, P. Ning and T. Hsiang: In vitro antifungal activity of dimethyl trisulfide against Colletotrichum gloeosporioides from mango. World J. Microbiol. Biotechnol. 36, 4 (2020).
• 24) K. Xing, Y. Xing, Y. Liu, Y. Zhang, X. Shen, X. Li, X. Miao, Z. Feng, X. Peng and S. Qin: Fungicidal effect of chitosan via inducing membrane disturbance against Ceratocystis fimbriata. Carbohydr. Polym. 192, 95–103 (2018).
• 25) Gaussian 16, Revision C.02: M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, G. A. Petersson, H. Nakatsuji, X. Li, M. Caricato, A. V. Marenich, J. Bloino, B. G. Janesko, R. Gomperts, B. Mennucci, H. P. Hratchian, J. V. Ortiz, A. F. Izmaylov, J. L. Sonnenberg, D. Williams-Young, F. Ding, F. Lipparini, F. Egidi, J. Goings, B. Peng, A. Petrone, T. Henderson, D. Ranasinghe, V. G. Zakrzewski, J. Gao, N. Rega, G. Zheng, W. Liang, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, K. Throssell, J. A. Montgomery Jr., J. E. Peralta, F. Ogliaro, M. J. Bearpark, J. J. Heyd, E. N. Brothers, K. N. Kudin, V. N. Staroverov, T. A. Keith, R. Kobayashi, J. Normand, K. Raghavachari, A. P. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi, J. Millam, M. Klene, C. Adamo, R. Cammi, J. W. Ochterski, R. L. Martin, K. Morokuma, O. Farkas, J. B. Foresman and D. J. Fox: Gaussian, Inc., Wallingford CT, 2019.
• 26) T. Anke, F. Oberwinkler, W. Steglich and G. Schramm: The strobilurins-new antifungal antibiotics from the basidiomycete Strobilurus tenacellus (Pers. ex Fr.) Sing. J. Antibiot. (Tokyo) 30, 806–810 (1977).
• 27) Y. Feng, Y. Huang, H. Zhan, P. Bhatt and S. Chen: An overview of strobilurin fungicide degradation: Current status and future perspective. Front. Microbiol. 11, 389 (2020).
• 28) L. S. D’Ávila, M. C. Corsi De Filippi and A. C. Café-Filho: Sensitivity of Pyricularia oryzae populations to fungicides over a 26-year time frame in Brazil. Plant Dis. 105, 1771–1780 (2021).
• 29) S.-C. Chu, K.-H. Lin, T.-C. Lin, C. Thanarut and W.-H. Chung: Sensitivity of Colletotrichum gloeosporioides species complex (CGSC) isolated from strawberry in Taiwan to benzimidazole and strobilurin. J. Pestic. Sci. 47, 172–183 (2022).
• 30) A. Ichihara, M. Nonaka, S. Sakamura, R. Sato and A. Tajimi: Structure and synthesis of alboatrin, a novel Phytotoxic metabolite from Verticillium albo-atrum. Chem. Lett. 17, 27–30 (1988).
• 31) W.-L. Chen, Y. Qian, W.-F. Meng, J.-Y. Pang, Y.-C. Lin, Y.-Y. Guan, S.-P. Chen, J. Liu, Z. Pei and G.-L. Wang: A novel marine compound xyloketal B protects against oxidized LDL-induced cell injury in vitro. Biochem. Pharmacol. 78, 941–950 (2009).
• 32) J.-H. Huang, J.-M. Lv, Y.-F. Xie, H. Zhao, L.-Y. Xiao, P. Dai, S.-Y. Qin, D. Hu, H. Gao and X.-S. Yao: Isolation of new compounds related to xyloketals biosynthesis implies an alternative pathway for furan-fused-chromene formation. Org. Biomol. Chem. 21, 4309–4318 (2023).
• 33) H. Gong, J. Bandura, G.-L. Wang, Z.-P. Feng, H.-S. Sun and B. Xyloketal: A marine compound with medicinal potential. Pharmacol. Ther. 230, 107963 (2022).
• 34) G. Wu, H. Ma, T. Zhu, J. Li, Q. Gu and D. Li: Penilactones A and B, two novel polyketides from Antarctic deep-sea derived fungus Penicillium crustosum PRB-2. Tetrahedron 68, 9745–9749 (2012).
• 35) T. Lobo-Echeverri, J. F. Rivero-Cruz, B.-N. Su, H.-B. Chai, G. A. Cordell, J. M. Pezzuto, S. M. Swanson, D. D. Soejarto and A. D. Kinghorn: Constituents of the leaves and twigs of Calyptranthes pallens collected from an experimental plot in Southern Florida. J. Nat. Prod. 68, 577–580 (2005).
• 36) L. S. D’Ávila, M. C. C. De Filippi and A. C. Café-Filho: Fungicide resistance in Pyricularia oryzae populations from southern and northern Brazil and evidence of fitness costs for QoI-resistant isolates. Crop Prot. 153, 105887 (2022).
• 37) P. Kongtragoul, K. Imamoto and H. Ishii: Resistance to quinone-outside inhibitor (QoI) fungicides in Colletotrichum Species isolated from anthracnose disease occurring in Thailand. Curr. Appl. Sci. Technol. 20, 79–89 (2020).
• 38) C. Q. Cortaga, B. W. P. Cordez, L. S. Dacones, M. A. O. Balendres and F. M. Dela Cueva: Mutations associated with fungicide resistance in Colletotrichum species: A review. Phytoparasitica 51, 569–592 (2023).
• 39) P. F. Cannon, U. Damm, P. R. Johnston and B. S. Weir: Colletotrichum—current status and future directions. Stud. Mycol. 73, 181–213 (2012).
• 40) B. S. Weir, P. R. Johnston and U. Damm: The Colletotrichum gloeosporioides species complex. Stud. Mycol. 73, 115–180 (2012).
• 41) H. Firstencel, T. M. Butt and R. I. Carruthers: A fluorescence microscopy method for determining the viability of entomophthoralean fungal spores. J. Invertebr. Pathol. 55, 258–264 (1990).
• 42) A. M. Svircev, R. J. Smith, T. Zhou, M. Hernadez, W. Liu and C. L. Chu: Effects of thymol fumigation on survival and ultrastracture of Monilinia fructicola. Postharvest Biol. Technol. 45, 228–233 (2007).
• 43) Y.-Y. Ding, Y. Sun, X.-F. Luo, S.-Y. Zhang, R. Wang, Z.-G. Yang, J.-R. Wang, B.-Q. Zhang, Z.-J. Zhang, Y. Ma, J.-X. An, H. Zhou and Y.-Q. Liu: Anti-phytopathogenic activity and the mechanisms of phthalides from Angelica sinensis (Oliv.) Diels. Pest Manag. Sci. 79, 2135–2146 (2023).
• 44) R. E. Sánchez-Fernández, R. Sánchez-Fuentes, H. Rangel-Sánchez, S. Hernández-Ortega, J. G. López-Cortés and M. L. Macías-Rubalcava: Antifungal and antioomycete activities and modes of action of isobenzofuranones isolated from the endophytic fungus Hypoxylon anthochroum strain Gseg1. Pestic. Biochem. Physiol. 169, 104670 (2020).
• 45) M. Abbod, N. Safaie, K. Gholivand, M. Mehrabadi and M. Bonsaii: Mode of action of 3-butylidene phthalide as a competent natural pesticide. Pestic. Biochem. Physiol. 164, 228–236 (2020).
• 46) T. Oktriawan, N. Ariefta, T. Raharjo, E. Astuti, T. Koseki and Y. Shiono: Phytotoxic and cytotoxic polyketides produced by fungal endophytes isolated from Psidium guajava. Hayati J. Biosci. 30, 473–479 (2023).
• 47) H. Han, C. Yu, J. Qi, P. Wang, P. Zhao, W. Gong, C. Xie, X. Xia and C. Liu: High-efficient production of mushroom polyketide compounds in a platform host Aspergillus oryzae. Microb. Cell Fact. 22, 60 (2023).
• 48) E. Z. Reyes-Fernández, Y. M. Shi, P. Grün, H. B. Bode and M. Bölker: An unconventional melanin biosynthesis pathway in Ustilago maydis. Appl. Environ. Microbiol. 87, e01510–e01520 (2021).
• 49) Z. Wei, Y. Xu, Q. Xu, W. Cao, H. Huang and H. Liu: Microbial biosynthesis of L-malic Acid and related metabolic engineering strategies: Advances and prospects. Front. Bioeng. Biotechnol. 9, 765685 (2021).
• 50) M. P. de Carvalho, G. Gulotta, M. W. do Amaral, H. Lünsdorf, F. Sasse and W. R. Abraham: Coprinuslactone protects the edible mushroom Coprinus comatus against biofilm infections by blocking both quorum-sensing and MurA. Environ. Microbiol. 18, 4254–4264 (2016).
• 51) T. B. Regueira, K. R. Kildegaard, B. G. Hansen, U. H. Mortensen, C. Hertweck and J. Nielsen: Molecular basis for mycophenolic acid biosynthesis in Penicillium brevicompactum. Appl. Environ. Microbiol. 77, 3035–3043 (2011).