Two new polyhydroxysterols produced by Fusarium solani, an endophytic fungus from Chloranthus multistachys

Abstract A highly antagonistic endophytic fungus, designated strain CL39, was originated from the leaves of Chloranthus multistachys collected in Wulong of Chongqing municipality of China in November 2015. The strain was identified as Fusarium solani based on morphological characteristics, 5.8S gene and internal transcribed spacer sequence analysis. Two new compounds, 2β, 9α-dihydroxy-5α-methoxyergosta-7, 22-diene (1), 2β, 6β-dihydroxy-5α-methoxyergosta-7, 22-diene (2) have been isolated from the culture broth of the strain. Structures of the new compounds were elucidated by detailed analysis of their spectroscopic data aided by the comparison with reported data of related derivatives, and found to belong to the polyhydroxylated steroids with a hydroxyl at C-2 instead of C-3, a rare structure among the steroids. The extract of this strain and all isolated compounds were evaluated for their antagonistic activities.


Introduction
Plant fungal endophytes are a hyperdiverse and abundant group, which spends the whole or part of their life cycle colonising inter-and/or intra-cellularly inside the healthy tissue of the host plant, typically causing no apparent symptoms of disease (De Bary 1866;Carroll 1986). They have proved to be a rich and important source of secondary metabolites with ABSTRACT A highly antagonistic endophytic fungus, designated strain CL39, was originated from the leaves of Chloranthus multistachys collected in Wulong of Chongqing municipality of China in November 2015. The strain was identified as Fusarium solani based on morphological characteristics, 5.8S gene and internal transcribed spacer sequence analysis. Two new compounds, 2β, 9α-dihydroxy-5α-methoxyergosta-7, 22-diene (1), 2β, 6β-dihydroxy-5α-methoxyergosta-7, 22-diene (2) have been isolated from the culture broth of the strain. Structures of the new compounds were elucidated by detailed analysis of their spectroscopic data aided by the comparison with reported data of related derivatives, and found to belong to the polyhydroxylated steroids with a hydroxyl at C-2 instead of C-3, a rare structure among the steroids. The extract of this strain and all isolated compounds were evaluated for their antagonistic activities. a large variety of novel chemical structures and diverse biological activities (Saikkonen et al. 2004;Strobel et al. 2004).
Chloranthus multistachys (Chloranthaceae), is a perennial herb that is mainly distributed in the wet areas of eastern Asia (Kawabata et al. 1990;Zhou 1993). It is applied in Chinese folk medicine to treat bone fractures, lumbocrural pain and pruritus (Jiangsu New Medical Jiangsu New Medical College 1977). This plant produces a class of dimeric lindenanes, diterpenoids and sesquiterpenoids (Wang et al. 2015). However, little is known about secondary metabolites of endophytes harbored inside the healthy tissues of C. multistachys.

Isolation and identification of CL39
Comparison of the ITS1-5.8S-ITS2 gene of isolated strain CL39 with those available in the GenBank database (http://www.ncbi.nlm.nih.gov/blast) by the phylogenetic tree ( Figure 2) indicated that it belongs to the genus Fusarium, which was most similar to F. solani (accession  No. KF918582). The strain was maintained as cryopreserved glycerol stocks deposited as Fusarium sp. CL39 at the key laboratory of eco-environments in the Three Gorges Reservoir Region, Ministry of education, School of Life Science, Southwest university, Chongqing, China.
Compound 2, a white solid, possessed the same molecular formula as 1 from eI-MS (C 29 H 48 o 3 ). It exhibited uV and IR absorption bands similar to those of 1. The proton and carbon signals in the 1 H and 13 C NMR spectra of 2 (Table S1) demonstrated a close resemblance with those of 1, and the only difference was that 2 had one hydroxyl group at C-9 instead of at C-6 in 1. The 1 H-1 H CoSY and HMBC correlations of 2 were shown in Figure  S1. The location of the hydroxy group at C-9 (δ C 74.5) was confirmed by HMBC correlations between H 3 -19 (δ H 0.98) and C-9 as well as the chemical shift values of C-9, C-10 (δ C 41.6), C-11 (δ C 27.8) and respective proton signals at C-11 (δ H 1.94 and 1.40). The NoeSY correlations of H 3 -18 (δ H 0.66) with H a -1 (δ H 1.45), H b -1 (δ H 2.38) with H-2 (δ H 3.94) in the NoeSY spectrum of 2 ( Figure S2), allowed us to establish the stereochemistry of the hydroxy group at C-2 in 2 as the same β-configuration as that in 1. The stereochemistry for the hydroxyl group at C-9 was not determined due to the low quantity. We presumed to R according to the previous reports (Moss 1989;Mansoor et al. 2006), because all natural sterols have a trans B/C ring fusion (Dewick 2002). on the basis of the above evidence, the structure of 2 was established as 2β, 9α-dihydroxy-5α-methoxyergosta-7, 22-diene.

Antagonistic properties of compounds against plant pathogenic fungi
All isolated compounds were subjected to a panel of bioassays to evaluate their potential activities against these seven plant pathogenic fungi. unfortunately, the isolated natural products only showed very weak antagonistic activity (zone of inhibition ＜1 mm) in any of the bioassays performed.

Fungal isolation and identification of morphology
The endophytic strain, designated CL39, was isolated from the leaves of C. multistachys which were collected in Wulong of Chongqing municipality of China in November 2014. The C. multistachys samples were thoroughly washed in running tap water to remove debris, and then air-dried naturally. The dry samples were individually surface sterilised with 75% ethanol for 5 min and rinsed with sterile distilled water 3 times, followed by immersion in 0.1% mercuric chloride (HgCl) (v/v) for 3 min. Afterwards, the samples were rinsed 3 times in sterile distilled water and transferred to PDA (potato 200 g, glucose 20 g, agar 18 g and water 1000 mL) medium supplemented with 60 mg/mL of streptomycin and 100 mg/mL of ampicillin using an aseptic technique. The inoculated plates were incubated at 28 °C darkness for 3-15 days to allow the growth of endophytic fungal hyphae, and checked regularly. Pure isolates were checked for purity and transferred to another PDA plate by the hyphal tip method (Strobel et al. 1996). After isolation, the strain CL39 was purified and maintained at 4 °C in PDA medium (Shirling & Gottlieb 1966).
The strain was preliminarily identified based on morphological characterisations. The macroscopic characteristics of the colonies (color, aspect and presence of pigmentation) and microscopic characteristics (morphology of vegetative spores'structures) were observed with an Axio Lab.A1 microscope (Carl Zeiss, Germany) equipped with an Axiocam ICc 5 digital camera (Carl Zeiss Vision, Germany) (Booth 1971;ellis 1976;Barnett & Hunter 1977;Hanlin 2000).

Phylogenetic analysis of the strain
The fungal isolates were inoculated individually into 250 mL erlenmeyer flasks containing 100 mL potato dextrose broth (PDB) medium. Cultures were incubated at 180 rpm at 28 °C for two days and harvested by centrifugation at 12,000 rpm for 10 min. The genomic DNA of the strain was extracted from 0.5 to 1 g chilled in liquid nitrogen mycelia using CTAB method (Zhang et al. 2006). Primers ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) and ITS5 (5′-GGAAGTAAAAGTCGTAAC AAGG-3′) were used to amplify the 5.8S gene and flanking ITS1 and ITS2 regions (White et al. 1990). The thermal cycle programme was as follows: 94 °C for 3 min, 32 cycles at 94 °C for 30 s, 56 °C for 30 s, 72 °C for 90 s and a final extension at 72 °C for 10 min. The PCR products were analysed by agarose gel electrophoresis and purified using a DNA gel extraction kit (omega Biotechnology Ltd China). The purified PCR products were sequenced with the same primers by BGI-Beijing (Beijing, China). The ITS sequence was compared with the data available in genomic database banks, using the NCBI BLAST program.
The resulting sequences were aligned with the Clustal X 2.0 software (Larkin et al. 2007), with gaps treated as missing data. Phylogenetic analysis was carried out by the neighbour-joining method using Mega 5.2 software. The bootstrap was 1000 replications to assess the reliability of the inferred tree (Tamura et al. 2011).

Antagonistic properties of fermentation broth against plant pathogenic fungi
The antagonistic properties of the strain CL39 fermentation broth against seven pathogens (G. cingnlata, A. brassicae, P. digitatum, B. cinerea, V. alboatrum, P. capsici and E. turcicum)were determined. (The test pathogens were provided from the key laboratory of eco environments in the Three Gorges Reservoir Region, Ministry of education, School of Life Science, Southwest university, Chongqing, China.) The fungus was incubated in three 250 mL erlenmeyer flasks, each containing 70 mL of PDB medium on a rotary shaker (180 rev/min) at 28 °C for 15 days. one flask of PDB medium was kept for control. After incubation, cultures were centrifuged. every 3 mL supernatant was mixed to 27 mL PDA. The mixed media was poured in three plates (10 mL each). The mixed media were used as crude antagonistic materials. each test pathogen was precultivated on the surface of PDA in Petri dishes at 28 °C for 7 days. Then, three 5 mm disks of PDA with pathogens' mycelium were inoculated independently at the centre of three mixed media plates and average radial growth were measured until the check plate attained full growth. Substrate controls consisted of same amount of substrate (1 mL sterile PDB medium mixed to 9 mL PDA each plate) and were inoculated and measured under the same conditions. Growth inhibition (GI) was calculated as per the following formula, where A = radial diameter of plant pathogenic fungi in PDB mixed plates; B = radial diameter of plant pathogenic fungi in fermentation broth mixed plates. All experiments were conducted in triplicate. The average of the GI for each pathogenic fungus was calculated.

Production, isolation and purification of the secondary metabolites of the strain
The fungus strain CL39 grew well and produced antagonistic active substance in potato extract medium in our preliminary work. So the fermentations were carried out in the same medium. The culture was subcultured in PDA at 25 °C for a period of 15 days. The mycelium containing agar was then cut into pieces (1 × 1 cm) and inoculated into 20 × 250 mL erlenmeyer flasks, each containing 50 mL of PDB (potato 200 g, glucose 20 g and water 1000 mL) (five pieces for each flask). After incubation at 25 °C for 15 days on a rotary shaker (180 rpm), the culture was transferred into 100 × 500 mL erlenmeyer flasks containing 150 mL of liquid (10 mL for each flask) and final fermentation was carried out at 25 °C for 15 days on a rotary shaker (180 rpm).
The culture broths were filtered through cotton yarn and concentrated under vacuum to 3 L below 60 °C and extracted successively three times with an equal volume of etoAc at room temperature (3 × 3 L, 72 h each). The combined organic layers were dried in vacuo to yield a brown gum residue (3.5 g). The extract was fractionated on a silica gel column chromatography (100-200 mesh; 4 cm 80 cm; with 175 g of silica gel), eluted with petroleum ether (b.p. 60-90 °C)-acetone (from 10:1 to 6:4, 5% gradient, 350 mL each eluent) to deliver five main fractions (Fr. 1-Fr. 5) based on thin-layer chromatography (TLC) behaviour. Fr. 3 was purified on Sephadex LH-20 (acetone) to afford compound 2 (10 mg). Fr. 5 was purified on Sephadex LH-20 (MeoH) to afford compound 1 (7 mg). Column chromatography was performed on silica gel G (Qingdao Marine Chemical Factory) and/or Sephadex LH-20 (Amersham Pharmacia Biotech). Precoated silica gel plates GF254 (Qingdao Marine Chemical Factory) were used for TLC. Spots were visualised under uV light and/or detected by spraying with 5% H 2 So 4 in 95% etoH followed by heating. All solvents used in our experiments were analytical grade.

Antagonistic properties of compounds against plant pathogenic fungi
Antimicrobial bioassays were conducted according to the agar diffusion method (Wicklow et al. 1998). The fungi were grown on potato dextrose agar. Test compounds were absorbed onto individual paper disks (5 mm diameter) at 50 μg/disk and placed on the surface of agar. The assay plates were incubated at 28 °C for 48 h for testing their antifungal activities. The plates were examined for the presence of a zone of inhibition.

Conclusion
endophytic fungi particularly from medicinal plants as a new source of natural products, which can sometimes produce compounds with unique chemical structure and biological activity analogous to their hosts, have recently received more attention (Maione et al. 2015). In previous articles, various secondary metabolites produced by endophytic Fusarium sp. were reported (Burmeister & Vesonder 1990;Yang et al. 2011;Yang, Wang, et al. 2012;Yang, Xiao, et al. 2012). In this study, we sampled Chinese folk medicinal plants, C. multistachys, and isolated several endophytic fungi from it. Among them, the strain CL39 identified as a highly antagonistic endophyte, belongs to Fusarium genus according to its phylogenetic analysis. In this study's programme of isolation and purification, two metabolites of the strain CL39 were obtained, both of them are new polyhydroxylated steroids according to its comprehensively spectroscopic analyses. They possess a rare structure, a hydroxyl at C-2 instead of C-3, which is different from the steroids in previous reports (Mansoor et al. 2006;Liu et al. 2013). The publications and our experimental result prove the genus Fusarium fungi is a rich source of novel and bioactive secondary metabolites of great importance.

Disclosure statement
No potential conflict of interest was reported by the authors.