Introduction

Sclerotinia sclerotiorum is an ascomycete fungal pathogen that causes stem rot in crucifer crops (rapeseed, canola and mustard) and several other plant families such as grain legumes (soybean, bean and pea) and other oilseeds (sunflower), resulting in enormous yield losses all over the world (Bardin and Huang 2001; Pedras and Hossain 2007). This necrotrophic pathogen exhibits little host specificity and has a host range that includes >400 primarily dicotyledonous plant species (Boland and Hall 1994). Populations of S. sclerotiorum are predominantly clonal in temperate regions of North America, Europe and New Zealand (Hambleton et al. 2002). There is now evidence of an extensive epidemic of this pathogen in South America, Australia and Asia (Sexton et al. 2006; Wang et al. 2003). It is very crucial to find some efficient inhibitors to control the spread of this disease.

The mechanism of S. sclerotiorum pathogenicity is well understood after many molecular biological and proteomic studies (Hegedus and Rimmer 2005; Pedras and Hossain 2007; Yajima and Kav 2006). The highly effective inhibitors against S. sclerotiorum have been studied (Chrysayi-Tokousbalides and Kastanias 2003; Rodriguez et al. 2006) but an effective application has not been developed to date. At present, cultivation of resistant cultivars and biological control have become the two traditional ways to battle against S. sclerotiorum (Bardin and Huang 2001; del Rio et al. 2002; Heritage 2003). The third way is perhaps to find bioactive compounds or biological reagents to inhibit the growth and spread of the pathogen.

In our search for biologically potential natural products from marine sponges and associated microorganisms (Jiang et al. 2007), we observed that the EtOAc extract of a fungal isolate JS9 from the marine sponge Axinella sp., identified as Myrothecium sp., strongly inhibited the growth of S. sclerotiorum. The extract also inhibited the other two fungi, Saccharomyces cerevisiae and Magnaporthe grisea. Two active fractions of the EtOAc extract of the JS9 culture were purified by passing them through an open silica gel column, followed with Sephadex LH-20 column fractionation and preparative high-performance liquid chromatography to obtain two antifungal trichothecenes, roridin A and roridin D (see Scheme 1). Both compounds were strongly bioactive against S. sclerotiorum.

Scheme 1
scheme 1

Structure of roridin A and roridin D

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Materials and methods

General experimental procedures

Melting points were determined on a XRS-1 digital-melting point apparatus, and the values were uncorrected. The 1H and 13C NMR data were collected on a Bruker AVANCE-500 (Bruker, Switzerland) spectrometer at 500 MHz, and the chemical shifts were recorded in δ (ppm) relative to Si(Me)4 with coupling constants J in Hz. Electron ionization-mass spectrometry (EI-MS) was performed using a MAT95XP mass spectrometer (Thermo, USA). Silica gel (100–200 mesh) for open column chromatography and prepared silica gel (300 mesh) plates for TLC were produced by the Qingdao Marine Chemical Factory (Qingdao, China). Sephadex™ LH-20 was from GE Healthcare (GE, USA, made in Sweden). Ultimate purification of compounds was performed on a HITCHI L-2000 preparative high-performance liquid chromatography (HITCHI, Japan) with one YMC semi-preparative ODS column. All other chemicals used in this study were of analytical grade.

Isolation of strain

The fungal isolates JS1 to JS9 were obtained by a serial dilution method from sponge samples collected from the South China Sea (18° 13′ N; 109° 29′ E) near Sanya, Hainan, China in July, 2005. The sponge sample was later identified as Axinella sp. by Dr. K. J. Lee (Department of Biology, Hannam University, 133 Ojungdong, Daedukgu, Daejeon, Korea). The samples were kept in fresh seawater on ice and stored at −20°C until further analysis.

Culture media

The strain isolation was performed on the starch casein KNO3 agar (SCKA) medium composed of 20 g starch, 2 g KNO3, 2 g K2HPO4, 2 g NaCl, 0.3 g casein, 0.05 g MgSO4 7H2O, 0.02 g CaCO3, 0.01 g FeSO4 7H2O, and 18.0 g agar in 500 ml sterile distilled water (SDW) premixed with 500 ml filtered sea water. The germination and growth of the marine fungal isolates were undertaken on the potato dextrose agar sea water (PDAS) medium, which was composed of 300 g diced potato, 20 g dextrose, 15 g agar in 500 ml SDW premixed with 500 ml filtered sea water.

Indicator strains

The initial bioactivity screening procedure involved fungal indicator organisms, as well as some Gram-positive (G+) and Gram-negative (G−) bacteria. The indicator strains included two plant pathogenic fungi S. sclerotiorum (ACCC 30046) and Magnaporthe grisea (Pyricularia oryzae) (ACCC 30320) from the Agricultural Culture Collection of China (ACCC) through professor Zanmin Hu of the Institute of Genetics and Developmental Biology, CAS. The two G− strains, Escherichia coli (GIM 1.115) and Pseudomonas aeruginosa (GIM 1.46), two G+ strains, Bacillus subtilis (GIM 1.181) and Staphylococcus aureus subsp. aureus (GIM 1.178), and a common yeast, Saccharomyces cerevisiae (GIM 2.86) were obtained from the Microbial Culture Collection Centre, Guangdong Institute of Microbiology.

Antagonistic test

For the fungal inhibition test, an inoculum plug (5 mm diam) of each isolate, prepared by punching the growth agar gel of a 72-h Petri dish culture, was transferred into the four corners of a 9 cm diam Petri dish containing PDAS medium. A plug of the fungal indicator organism (5 mm diam) was placed in the middle of the same Petri dish. The inoculated Petri dish was cultured at 28°C for 48 h. The antagonistic action was evaluated by measuring the diameter of the inhibition zones with fluconazole as a positive control. For the bacterial inhibition test, an indicator organism was spread on top of the Petri dish containing LB agar. An inoculum plug (5 mm diam) of each isolate, prepared by punching the growth agar gel of a 72-h Petri dish culture, was transferred into the four corners of the Petri dish with the indicator bacteria. The inoculated Petri dish was cultured at 28°C for 48 h. The antagonistic action was evaluated by measuring the diameter of the inhibition zones with fluconazole as a positive control. Each antagonistic test was performed in triplicate.

Fungal fermentation

The fresh mycelium of each of nine fungal strains grown on PDAS medium at 28°C for 5 days was inoculated respectively into 500 ml Erlenmeyer flasks containing 150 ml PDAS (without agar) medium. After 2 days of incubation at 28°C on a rotary shaker at 200 rpm, a 40 ml seed culture liquid was transferred into each 1000 ml Erlenmeyer flask containing 300 ml PDAS medium. The flasks were incubated for 10 days at 28°C and 200 rpm on a rotary shaker.

Antifungal tests of fungal fermentation extract

Each culture filtrate (5 l) was extracted four times with the same volume of EtOAc. After evaporation of the solvent from the combined extract in vacuo, the residue was used in an in vitro antifungal assay against the indicator fungi S. sclerotiorum, M. grisea and S. cerevisiae according to the methods described above. Each EtOAc extract of the isolated fungus was tested in triplicate with fluconazole as a positive control.

Identification of the isolated strain

The fungal strain JS9 was identified as Myrothecium sp. by comparing its morphological features with the reference description (Nag Raj 1995). The strain JS9 was further identified to Myrothecium sp. by comparison of its 18S rDNA sequence with those submitted sequences on GenBank (http://www.ncbi.nlm.nih.gov/) via online BLAST analysis.

Thin plate chromatography (TLC) bioautography

The EtOAc extracts of each isolated fungus were spotted onto GF254 silica gel plates (5 cm × 2 cm) preloaded with fluorescent indicator, and developed by placing them in a glass chromatography tank containing 10 ml evenly mixed solution of CHCl3/MeOH (v/v, 10:1). Plates were removed from the tank as soon as the observable solvent front reached 4.8 cm in height. The dark spots on the dried plates visualised under UV radiation (254 nm) showed the presence of the UV-absorbing compounds. After the observed spots were marked with pencil, each of the dried plates (silicon gel side) was placed onto the surface of PDAS medium which was smeared smoothly with the test pathogenic microbe in advance. Preparation of the test fungi Petri dishes and drying of plates was done using gloves in a super-clean hood. Petri dishes containing plates were sealed with parafilm and kept at 28°C in a light incubator with a 12 h photoperiod. The inhibition zones were usually visible within 6 days, appearing as semi-transparent circular areas of decreased density. This TLC bioautography, combined with the activity tracking method, was used in this study to develop the purification procedure of the metabolites.

Separation and purification of metabolites

Myrothecium sp. was cultured in PDAS medium at 28°C and 200 rpm on a rotary shaker for 10 days. The culture broth (50 l) was filtered, and the filtrate was adjusted to pH 2.0 with 2.0 mol l−1 HCl, before being extracted with EtOAc four times. The combined filtrates were concentrated in vacuo, and the obtained residue (15.2 g) was first fractionated by open column chromatography on silica gel (petroleum ether-EtOAc gradient). The activity tracking method was applied throughout the separation and purification procedure. One active fraction (2.4 g), obtained by elution with 70% petroleum ether-EtOAc, was chromatographed again on a silica gel column (CHCl3–MeOH gradient). The active fraction (255 mg) eluted with 99% CHCl3–MeOH, was further chromatographed on a PHPLC equipped with a 10 mm × 250 mm ODS column, and was eluted with 64% CH3OH–H2O. This fraction yielded 78 mg of compound 2 as colourless needle crystals from n-hexane. Another active fraction (3.2 g), obtained by elution with 60% petroleum ether-EtOAc, was chromatographed again on a silica gel column (CHCl3–MeOH gradient). The active fraction (347 mg), eluted with 95% CHCl3–MeOH was further purified on a Sephadex LH-20 column (CHCl3–MeOH, 1:1) and recrystallized from CHCl3 to yield 109 mg of compound 1 as a colourless plate.

Bioassays and minimal inhibitory concentration detections of the purified compounds

Antifungal activities and minimal inhibitory concentration (MIC) values of compounds 1 and 2 (dissolved in dimethyl sulphoxide, DMSO) were assessed in vitro using test fungi S. sclerotiorum, M. grisea and S. cerevisiae by the agar serial dilution method described in the literature (Ter Laak et al. 1991), with DMSO as a negative control and fluconazole as a positive control. Each isolated compound of different concentration was performed in triplicate.

Characterizations and structure elucidations of metabolites

Roridin A (compound 1, see Scheme 1)

Colourless plate (CHCl3), mp. 197–203°C. EI–MS: 533 [M+H]+; 1H-NMR data (CDCl3, 500 MHz): 3.85 (1H, d, J = 5.0 Hz, H-2), 2.22(1H, ddd, J = 4.7,4.7,15.3 Hz, H-3a), 2.46 (1H, dd, J = 8.3, 15.4 Hz, H-3b), 5.78 (1H, m, H-4), 1.90 (2H, m, H-7), 2.03 (2H, m, H-8), 5.43 (1H, d, J = 4.5 Hz, H-10), 3.59 (1H, m, H-11), 2.80 (1H, d, J = 3.95 Hz, H-13a), 3.12 (1H, d, J = 3.95 Hz, H-13b), 0.81 (3H, s, H-14), 4.44 (2H, s, H-15), 1.74 (3H, br.s, H-16), 4.10 (1H, s, H-2′), 2.01 (1H, m, H-3′), 1.78 (2H, m, H-4′), 3.54 (2H, m, H-5′), 3.65 (1H, m, H-6′), 6.00 (1H, dd, J = 3.0, 15.5 Hz, H-7′), 7.66 (1H, dd, J = 11.8, 15.4 Hz, H-8′), 6.66 (1H, dd, J = 11.3, 11.3 Hz, H-9′), 5.80 (1H, d, J = 11.5 Hz, H-10′), 1.09 (3H, d, J = 6.85 Hz, H-12′), 3.62 (1H, m, H-13′), 1.19 (3H, d, J = 6.1 Hz, H-14′); 13C-NMR data (CDCl3, 500 MHz): 79.07 (C-2), 34.85 (C-3), 74.31 (C-4), 49.36 (C-5), 43.77 (C-6), 20.26 (C-7), 27.70 (C-8), 140.95 (C-9), 118.23 (C-10), 67.18 (C-11), 65.24 (C-12), 47.79 (C-13), 7.51 (C-14), 64.58 (C-15), 23.31 (C-16), 174.88 (C-1′) 75.58 (C-2′), 37.17 (C-3′), 33.06 (C-4′), 69.87 (C-5′), 84.05 (C-6′), 139.19 (C-7′), 126.10 (C-8′), 143.92 (C-9′), 117.55 (C-10′), 166.53 (C-11′), 14.77 (C-12′), 70.84 (C-13′), 18.25 (C-14′). These data are identical to those described by Breitenstein and Tamm (1975).

Roridin D (compound 2, see Scheme 1)

Colourless needle crystals (CHCl3), mp. 223–225°C. EI–MS: 531 [M+H]+; 1H-NMR data (500 MHz): 3.85(1H, d, J = 5.1 Hz, H-2), 2.18(1H, ddd, J = 4.7, 4.7, 15.3 Hz, H-3a), 2.46 (1H, dd, J = 8.3, 15.4 Hz, H-3b), 5.80 (1H, m, H-4), 1.95(2H, m, H-7), 2.05(2H, m, H-8), 5.44(1H, d, J = 4.45 Hz, H-10), 3.85(1H, m, H-11), 2.81(1H, d, J = 3.95 Hz, H-13a), 3.12(1H, d, J = 4.0 Hz, H-13b), 0.85(3H, s, H-14), 4.27(1H, d, J = 12.4 Hz, H-15a), 4.38(1H, d, J = 12.4 Hz, H-15b), 1.74(3H, s, H-16), 3.31(1H, s, H-2′), 1.48(1H, m, H-4′α), 1.81(1H, m, H-4′β), 3.63(2H, m, H-5′), 3.67(1H, m, H-6′), 5.97(1H, dd, J = 3.2, 15.7 Hz, H-7′), 7.53(1H, dd, J = 11.6, 15.6 Hz, H-8′), 6.60(1H, t, J = 11.3Hz, H-9′), 5.84(1H, d, J = 10.8 Hz, H-10′), 1.62(3H, s, H-12′), 3.81(1H, m, H-13′), 1.20(3H, d, J = 6.1 Hz, H-14′); 13C-NMR data (CDCl3, 500 MHz): 79.10 (C-2), 35.17 (C-3), 74.49 (C-4), 49.21 (C-5), 43.26 (C-6), 20.62 (C-7), 27.61 (C-8), 140.69 (C-9), 118.49 (C-10), 67.10 (C-11), 65.38 (C-12), 47.83 (C-13), 7.05 (C-14), 64.60 (C-15), 23.30 (C-16), 168.15 (C-1′), 63.26 (C-2′), 58.21 (C-3′), 39.65 (C-4′), 67.46 (C-5′), 85.75 (C-6′), 138.19 (C-7′), 126.37 (C-8′), 143.11 (C-9′), 118.19 (C-10′), 166.39 (C-11′), 17.44 (C-12′), 70.91 (C-13′), 18.22 (C-14′). These data are identical to those described by Breitenstein and Tamm (1975).

Results and discussion

Several culture media including LB, SCKA, 2216E, PDA, YPD and PDAS were selected for germination and growth of the fungi isolated from the marine sponge Axinella sp. collected in the South China Sea near Sanya. As a result, the PDAS medium in 50% natural filtered sea water was the most suitable for marine fungal cultures.

Nine fungal strains numbered from JS1 to JS9 were isolated from Axinella sp. The antagonistic actions of these fungi are shown in Table 1, although nearly no antibacterial activity was detected when G+ strains B. subtilis and S. aureus, G− strains E. coli and P. aeruginosa were used as test strains, but their antifungal actions as indicated by the antagonistic tests were fairly similar to those of EtOAc extracts derived from their corresponding cultures. The results of antimicrobial activity tests show that the fungus numbered JS9 produced the most effective antifungal metabolites. Moreover, the antifungal activity of the EtOAc extract of the JS9 culture was much stronger than that of fluconazole, which was used as a positive control under the same mass concentration (Table 1). The inhibition of the JS9 EtOAc extract was especially more effective against S. sclerotiorum than the other two test fungi M. grisea and S. cerevisiae. Subsequently, a scaled-up culture of JS9 was prepared to purify and characterise the antifungal metabolites.

Table 1 The antimicrobial activity of isolated fungi from the marine sponge Axinella sp
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Using the antifungal activity tracking method, two known metabolites (compound 1 and 2) were purified from the EtOAc extract of Myrothecium sp. JS9. Compound 1 was identified as roridin A (Böhner and Tamm 1966b; Breitenstein and Tamm 1975) and compound 2 was identified as roridin D (Böhner and Tamm 1966a; Breitenstein and Tamm 1975), by spectral analyses including MS, 1H-NMR, 13C-NMR and DEPT.

The two purified fungal metabolites were bioassayed against test fungi S. sclerotiorum, M. grisea and S. cerevisiae by the serial dilution method (DMSO as solvent) and the MIC values are shown in Table 2. The results based on activity tracking and MICs test suggested that roridin A and roridin D are the major antifungal components in the fungal culture. Both compounds were strongly bioactive against the three test fungi, and especially against S. sclerotiorum with the MICs relatively lower than those of fluconazole used as a positive control in this study.

Table 2 The MIC values of compounds 1 and 2 (in μg ml−1)
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Because of its strong antifungal metabolites, strain JS9 was subjected to biological identification. The morphological features were recorded as follows. The fungus grew fast on both PDA and PDAS media, the colony attained 8–9 cm in diam after incubation in light at 28°C for 4 days. Aerial mycelia were white, floccose and concentrically scattered with large blackish wet drops, which were collected in a mass of mucous dark green conidia. The conidiomata were sporodochial, flat, and formed by densely branched and brush-like conidiophores. These features of strain JS9 were consistent with the description of the genus Myrothecium (Nag Raj 1995). This identification was further confirmed by comparison of the 18S rDNA sequence of strain JS9 (Access number: EU159531) with the data available in GenBank using online BLAST, and the homology to the 18S rDNA sequences of Myrothecium verrucaria, Myrothecium atroviride, Myrothecium leucotrichum and Myrothecium roridum was 99%.

As we know, roridin A and roridin D belong to the macrocyclic trichothecenes class. Trichothecenes comprise a group of sesquiterpenes characterised structurally by the 12, 13-epoxy-trichothec-9-ene moiety. Very interestingly, it is reported that this sesquiterpene nucleus has been isolated both from fungal cultures like those of Fusarium spp. (Ishii and Ueno 1981; Kim et al. 1990), Myrothecium spp. (Abbas et al. 2002; Amagata et al. 2003) and Stachyobotrys spp. (Sudakin 2003), and from some higher plants such as Bacchairis artemisioides and Baccharis coridifolia (Habermehl et al. 1985; Rizzo et al. 1997), Ficus fistulosa and Rhaphidophora decursiva (Zhang et al. 2002). Biologically, macrocyclic trichothecenes were reported to be antileukemic (Jarvis et al. 1980, 1984), antimalarial (Isaka et al. 1999; Zhang et al. 2002), antimicrobial (Liu et al. 2006; Wagenaar and Clardy 2001), antiviral (Tani et al. 1995), phytotoxic and cytotoxic (Abbas et al. 2002; Masuda et al. 2007), and some were reported to possess animal toxicity (Hughes et al. 1989). In the present study, the two trichothecenes (compounds 1 and 2), obtained from the EtOAc extract of the fungus JS9 isolated from the marine sponge Axinella sp., were ascertained to be antifungal compounds with the MICs comparable with those of fluconazole.

In conclusion, this study demonstrated that the fungus Myrothecium sp. JS9, a symbiotic fungus in the marine sponge Axinella sp., was an efficient producer of antifungal trichothecenes including roridin A and roridin D. Both roridin A and roridin D are potentially useful inhibitors of S. sclerotiorum.