¹H and ¹³C NMR spectra were recorded using a Bruker AVANCE-400 spectrometer (400.13 MHz for ¹H, 100.61 MHz for ¹³C; Bruker Biospin K.K., Kanagawa, Japan). Chemical shifts (δ) were measured in ppm using tetramethylsilane as an internal standard. Samples were analyzed with PDA-HPLC using a PU-980 and PU-1580 pump (JASCO Co., Ltd., Tokyo, Japan) and Inertsil ODS-3 column (5 µm, 4.6 mm × 250 mm; GL Science, Inc., Tokyo, Japan) maintained at a temperature of 30˚C using a CO-2065 Plus column oven (JASCO Co.) and equipped with a MD-2010 Plus photodiode array (JASCO Co.). Compounds were isolated using a reverse-phase MPLC system with an SSC-3160 pump (Senshu Scientific Co., Ltd., Tokyo, Japan) and ULTRA PACK ODS-SM-50A column (50 µm, 300 × 11 mm; Yamazen Corp., Osaka, Japan) equipped with a YRU-8883 RI-UV detector (Shimamuratach, Tokyo, Japan), and reverse-phase HPLC system with a LC-20AT prominence pump (Shimadzu Corp., Kyoto, Japan) and a Inertsil ODS-3 column (5 µm, 250 × 4.6 mm, GL Science Inc., Tokyo, Japan) in a CO-965 column oven (JASCO Co., Ltd., Tokyo, Japan) equipped with a SPD-20A UV detector (Shimadzu Corp., Kyoto, Japan).

Bamboo vermicompost was produced as described previously [11]. The initial substrate materials of bamboo vermicompost contained moso-bamboo shoots powder and kudzu vines (Pueraria lobata (Willd) Ohwi, <10 cm in length). Moso-bamboo shoots were cut from an abandoned bamboo forest in Eboshi-yama, Kawachinagano City, Japan. Kudzu vines were collected from the Nakamozu Campus of Osaka Prefecture University. Earthworms used were Eisenia fetida Savigny (commercial name: “Kumatarofutomushi”) and purchased from Fishing Azumino Company, Azumino, Japan. Rhizoctonia solani AG1-IB isolate SLS1 (MAFF244980) used in the study was provided by Dr. Misawa Tomoo (Hokkaido Research Organization, Japan). This isolate was recovered from diseased spinach in Hokkaido, Japan [12].

The water extracts of bamboo vermicompost were prepared by mixing the samples with distilled water at a ratio of 2:3 (v/v) followed by active aeration with a small air pump at 20˚C for 24 h according to a previous study [13] with some modifications. The supernatant of the water was filtered through a filter paper (Whatman No.1, 9 cm in diameter, GE Healthcare UK Ltd., Little Chalfont, UK) to obtain water extract of bamboo vermicompost, followed by filter-sterilization through a 0.22 µm cellulose acetate filter (Corning Inc., Corning, NY, USA) to obtain sterilized water extracts of bamboo vermicompost (SWE).

One milliliter of SWE was pipetted onto a PDA plate and expanded over the entire surface with a sterilized glass rod according to a previous study [13] with some modifications. A 5 mm-diameter agar plug with 7-day-old mycelium of R. solani AG1-IB was placed on the center of the PDA plate and incubated in the dark at 25˚C. Sterilized distilled water was used as a control. Mycelium growth was evaluated by visual measurement of the colony radius at 24 h intervals for 2 days. The experiments were repeated five times.

The separation scheme of the antifungal compounds from bamboo vermicompost is shown in Figure 1. Ten liters of bamboo vermicompost were extracted with acetone, and the organic layer was evaporated. The resulting extracts were suspended in H₂O and extracted with EtOAc, and then the organic layer was evaporated in vacuo for further analysis. The EtOAc extracts (1.67 g) were used to determine their antifungal activities against R. solani AG1-IB by the paper disc diffusion method [14]. The EtOAc extracts were then chromatographed on Sephadex LH-20 (GE Healthcare Bio-Sciences, Uppsala, Sweden; solvent system: hexane-CHCl₃ (1:4; 200 mL), CHCl₃-acetone (3:2; 200 mL), (1:4; 200 mL), acetone (200 mL) and MeOH (1000 mL) to collect nine fractions: 2.6 mg for Fr. A, 233.3 mg for Fr. B, 57 mg for Fr. C, 136.4 mg for Fr. D 355.1 mg for Fr. E, 105 mg for Fr. F, 169 mg for Fr. G, 113 mg for Fr. H, 495.8 mg for Fr. I. The nine fractions were used to determinate the antifungal activities of these samples against R. solani AG1-IB by paper disc diffusion method [14].

The fraction (C) showing two major peaks was further isolated by normal-phase-HPLC with CHCl₃: acetone to produce five fractions (Fr. C.1 - C.5). The fraction (C.4, 8 mg) which contained the two major peaks was further purified by HPLC on octadecylsilyl (ODS) and eluted with 100% CH₃CN to obtain compounds 1 (5.0 mg) and 2 (1.2 mg). Their chemical structures and mass spectra were determined by NMR and mass spectrometry as described previously [15].

Antifungal activity of ergosterol peroxide (1) against R. solani AG1-IB was tested as described previously [16] with some modifications. Briefly, pathogen plugs (5-mm diameter) from the growing edges of colonies were placed at the center of Petri dishes containing PDA. Samples of 10 μL of the compound at concentrations of 150, 300, 600, and 900 μg∙plug⁻¹ were applied on the top of each plug. The negative controls were obtained by applying 10 μL of solvent (ethyl acetate) alone. The solvent was evaporated in a laminar flow cabinet, and the plates were incubated at 25˚C for 48 h. Pathogen growth was measured daily as the colony diameter (mm). Each treatment consisted of three replicates. The percentage of inhibition was calculated based on the following formula: Relative efficacy of inhibition (%) = (mycelial growth of the control-mycelial growth of the treatment)/mycelial growth of the control × 100.

The initial substrate materials of bamboo vermicompost contained bamboo shoots powder and kudzu vine pieces. We examined whether the antifungal compounds in bamboo vermicompost were originally in the initial substrate materials. The bamboo shoots powder or kudzu vine pieces were extracted in 100% methanol at a ratio of 1:2 (v/v, dry plant material/solvent), and then the organic layer was evaporated. The resultant extracts were suspended in H₂O and extracted with EtOAc, and then the organic layer was evaporated in vacuo. Bamboo vermicompost was used as a positive control. The EtOAc extracts were used to determinate their antifungal activities against R. solani AG1-IB by paper disc diffusion method [14].

Data are represented in terms of the mean ± standard error. Comparison between the effect of treatment with SWE and sterilized distilled water on R. solani AG1-IB mycelium growth was carried out according to Student’s t-test (P < 0.05). The other data were evaluated by Tukey’s HSD test (P < 0.05) using IBM SPSS Statistics 25 (IBM Corp., Armonk, NY, USA).

SWE significantly (P < 0.05) inhibited the mycelium growth of R. solani AG1-IB on PDA medium as compared to the control (Figure 2(a)). The presence of SWE in PDA medium decreased the fungal growth rate from 23.8 mm/24 h to 20.3 mm/24 h (Figure 2(b)).

The fractions (C, D, E, F and G) showed antifungal activities, whereas the other fractions did not show these effects (Figure 3). The fraction C was chosen for further fractioned as it showed two major peaks (Figure 4). However, no major peaks were observed on the fractions (D, E, F and G) (data not shown). Two compounds, 1 and 2, were obtained from the fraction C. For compound 1, appearing as the major peak (tR 45.78 min) shown in Figure 4 was determined as ergosterol peroxide by detailed analysis of the ¹H and ¹³C NMR spectra (Table 1). Compound 2, the minor peak (tR 44.48 min) in Figure 4 was determined a

(22E, 24R)-5α,8α-epidioxyergosta-6,9(11),22-trien-3β-ol by detailed analysis of the ¹H and ¹³C NMR spectra (Table 2). The physical and chemical properties of the two isolated compounds were as follows:

Compound 1 was obtained as a colorless amorphous powder and the molecular formula was determined as C₂₈H₄₄O₃ by electrospray ionization-mass spectrometry. The ¹H-NMR spectrum of compound 1 showed four olefinic protons, an oxygenated proton, four doublet methyl protons, and two singlet methyl protons. The number of these methyl groups and coupling patterns suggested that the carbon flame of 1 was ergostane. The ¹³C-NMR spectrum showed four sp² carbons, three oxygenated carbons, and 21 aliphatic carbons, for a total of 28 carbons. From these data compound 1 was assumed to be ergosterol peroxide. Therefore, compound 1 was confirmed to be ergosterol peroxide, as the ¹H- and ¹³C-NMR data of compound 1 matched previous data [17].

Compound 2 was obtained as a colorless amorphous powder and the molecular formula was confirmed to be C₂₈H₄₂O₃ by electrospray ionization-mass spectrometry. ¹H- and ¹³C-NMR spectra of compound 2 were very similar to those of compound 1 except for the appearance of an olefinic proton in the ¹H-NMR-spectrum and appearance of two sp² carbons and disappearance of two aliphatic carbons. Therefore, compound 2 was considered to be an ergosterol

derivative with one additional double bond. The compound (22E, 24R)-5α,8α-epidioxyergosta-6,9(11),22-trien-3β-ol satisfied the above data. Therefore, the ¹H- and ¹³C-NMR data of compound 2 were compared to previous data [18]. Compound 2 was identified as (22E, 24R)-5α,8α-epidioxyergosta-6,9(11),22-trien-3β-ol, based on these data showing very good agreement.

The structures of the compounds 1 and 2 are shown in Figure 5. Compound 1 was then assayed for antifungal activity against R. solani AG1-IB, but compound 2 was not tested because of its insufficient amount.

Mycelial growth by R. solani AG1-IB was found to decrease gradually in response to increasing doses of ergosterol peroxide from 150 to 900 μg (Figure 6(a)). Ergosterol peroxide at 150, 300, 600, and 900 μg showed 13%, 22%, 34%, and 53% mycelial growth inhibition of R. solani AG1-IB, respectively, as compared to control (Figure 6(b)).

The EtOAc extracts from bamboo vermicompost inhibited the mycelium growth of R. solani AG1-IB, whereas those from bamboo shoots powder and kudzu

vines did not show these effects (Figure 7).