Antifungal Activity of Compounds Isolated from Bamboo Vermicompost against Rhizoctonia solani AG1-IB

Vermicompost has become a promising alternative to chemical pesticide because of its disease suppression effect during these decades. However, the mechanisms by which vermicompost suppress plant disease are not well-understood. Antifungal compounds, which are released by beneficial organisms in the vermicompost, may play an important role in inhibiting plant pathogens; however, these mechanisms have not been widely examined. In this study, Sterilized Water Extracts of bamboo vermicompost (SWE), using a 0.22-μm cellulose acetate filter, significantly (P < 0.05) inhibited the mycelium growth of Rhizoctonia solani AG1-IB on a Potato Dextrose Agar (PDA) plate. This suggests that antifungal compounds are present in bamboo vermicompost. The ethanol acetate (EtOAc) crude extracts of bamboo vermicompost showing antifungal activity were further separated. Two compounds were isolated from the EtOAc fraction of bamboo vermicompost and characterized as ergosterol peroxide (1) and (22E, 24R)-5α,8α-epidioxyergosta6,9(11),22-trien-3β-ol (2). Their chemical structures and mass spectra were determined by nuclear magnetic resonance and mass spectrometry analyses. Ergosterol peroxide tested at 150, 300, 600, and 900 μg showed 13%, 22%, 34%, and 53% mycelial growth inhibition against R. solani AG1-IB, respectively. Because EtOAc crude extracts of the initial substrate materials of bamboo vermicompost did not inhibit mycelium growth of R. solani AG1-IB, antifungal compounds in the vermicompost may be released by microbes but not from the original substrates during vermicomposting.


Introduction
Seedling damping-off diseases caused by Rhizoctonia solani are economically important in a broad range of crops worldwide [1] [2]. This pathogen is difficult to control because of its ability to persist under adverse soil conditions, as it contains structures such as sclerotia that survives for several years in the absence of host crops and exhibits saprophytic activity, a wide host range, and versatility [3]. Although fungicide application is effective for suppressing such diseases, frequent use of fungicides can result in the emergence of fungicide-resistance strains, leading to undesirable effects on human health and environmental safety [4] [5]. Therefore, alternative strategies for controlling this disease are urgently needed.
Vermicompost is a composting product of accelerated biodegradation of organic matter by earthworms and their associated microbes through non-thermophilic decomposition. Recently, several studies have focused on the potential of vermicompost to suppress soil-borne plant pathogens including R. solani [6] [7].
The activities of microbial antagonists present in vermicompost are considered as an important mechanism for disease suppression. Several antagonistic bacteria and fungi, such as Bacillus subtilis, Streptomyces spp., Trichoderma sp., and Aspergillus sp., were isolated from vermicompost and shown to suppress soil-borne plant pathogens [8] [9] [10]. These microbes are well-known to produce diversity bioactive compounds including antifungal compounds. Release of antifungal compounds by these microbes into the vermicompost may play an important role in inhibiting plant pathogens, which has not been widely examined. Understanding the chemical factors of how vermicompost suppresses soil-borne plant pathogens may promote effective utilization of the vermicompost. In our previous studies, a vermicompost produced from moso-bamboo (Phyllostachys edulis (Carrière) J. Houz.) showed the ability to suppress cucumber damping-off caused by R. solani AG1-IB [11].
In the present study, we examined the effects of water extracts of bamboo vermicompost, sterilized with a Corning 0.22-µm cellulose acetate filter (SWE), on R. solani AG1-IB mycelium growth on potato dextrose agar (PDA). Although the SWE did not contain microbes in the bamboo vermicompost, it significantly (P < 0.05) inhibited the mycelium growth of R. solani AG1-IB. This suggests that the SWE contained antifungal compound(s). This study was conducted to isolate the antifungal compound(s) from the bamboo vermicompost, determine the chemical structures and mass spectra by nuclear magnetic resonance (NMR) and mass spectrometry analyses, and evaluate the suppressive effect on R. solani AG1-IB.

Materials
Bamboo vermicompost was produced as described previously [11].

Preparation of SWE
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

Effect of SWE Treatment on Mycelium Growth Rate of R. solani AG1-IB
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.

Isolation of Antifungal Compounds from Bamboo Vermicompost
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 H 2 O 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]. fractions were used to determinate the antifungal activities of these samples against R. solani AG1-IB by paper disc diffusion method [14].

Inhibitory Effect of Ergosterol Peroxide (1) Isolated from Bamboo Vermicompost on Mycelium Growth of R. solani AG1-IB
Antifungal activity of ergosterol peroxide (1) against R. solani AG1-IB was tested as described previously [16] with some modifications. Briefly, pathogen plugs

Inhibitory Effect of EtOAc Extracts from Initial Substrate Materials of Bamboo Vermicompost on Mycelium Growth of R. solani AG1-IB
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 H 2 O 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].

Treatment Effect of SWE on Mycelium Growth Rate of R. solani AG1-IB
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)).

Isolation of Antifungal Compounds from Bamboo Vermicompost
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 1 H and 13 C 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 1 H and 13 C 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 C 28 H 44 O 3 by electrospray ionization-mass spectrometry. The 1 H-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 13 C-NMR spectrum showed four sp 2 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.
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.

Inhibitory Effect of EtOAc Extracts from Initial Substrate Materials of Bamboo Vermicompost on Mycelium Growth of R. solani AG1-IB
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).

Discussion
Rhizoctonia solani, one of the most common soil-borne plant pathogenic fungi worldwide, causes serious economic losses of different crops and its control strategies rely mainly on chemical fungicide applications. An eco-friendly disease management strategy is urgently needed. Vermicompost has become a promising alternative to chemical pesticide because of its disease suppression effects. However, the mechanisms of how vermicompost activity in plant disease suppression are not well-understood [19]. Antifungal compounds, which are released by beneficial organisms in the vermicompost, may play an important role in inhibiting plant pathogens; however, these mechanisms have not been widely examined. Although Mu et al. (2017) showed that some volatile organic compounds, such as 3-methyl-3-hexanol, released by Bacillus subtilis isolated from a cow dung based vermicompost showed significant inhibitory activity against Botrytis cinerea [8], no antifungal compounds have been isolated directly from vermicompost. In this study, five EtOAc fractions (C, D, E, F and G), obtained by silica gel column chromatography of the acetone layer from the bamboo vermicompost showed antifungal activities against R. solani AG1-IB. Two known compounds were obtained from the fraction (C) showing two major peaks and characterized as ergosterol peroxide (1) and (22E, 24R)-5α,8α-epidioxyergosta-6,9(11), 22-trien-3β-ol (2). This is the first study to isolate these two compounds from vermicompost. The mycelium growth of R. solani AG1-IB was significantly suppressed by ergosterol peroxide. The antifungal capacity of ergosterol peroxide against R. solani was demonstrated in this study. The EtOAc extracts from bamboo vermicompost inhibited R. solani AG1-IB mycelium growth, whereas those from the original substrates did not, including bamboo shoots powder and kudzu vines. This result suggests that the antifungal compounds in vermicompost were released by microbes in the vermicompost during vermicomposting, but not from the original substrates. This agrees with a previous study demonstrating that filter sterilized aqueous extract of vermicompost prepared from paper sludge and dairy sludge significantly inhibited the spore germination of Fu-  [20].
Ergosterol peroxide is a non-volatile compound that has been isolated from various species of mushrooms, microscopic fungi such as Trichoderma longibrachiatum, and yeasts, and has been reported to exhibit antitumor, anti-inflammatory and antimicrobial activities in vitro [21] [22]. Some of the producers of ergosterol peroxide likely actively grow in the vermicompost. However, the antifungal compounds in the other EtOAc fractions, including D, E, F, and G, have not yet been identified. Further studies are required to identify these antifungal compounds and determine their suppressive effects on seedling damping-off diseases caused by R. solani. In this study, we demonstrated that vermicompost inhibited the development of fungal pathogens through the antifungal compounds present in it, which may promote effective utilization of vermicompost.

Conclusion
In conclusion, several antifungal compounds were present in the bamboo vermicompost, which were released by microbes, and played an important role in inhibiting plant pathogens. Ergosterol peroxide isolated from the vermicompost, showed significant in vitro inhibitory effect on mycelium growth of R. solani AG1-IB.