A new sesquiterpene from the entomogenous fungus Phomopsis amygdali

A new sesquiterpene, (+)-S-1-methyl-abscisic-6-acid (1), together with five known compounds, (+)-S-abscisic acid (2), fusicoccin J (3), 3α-hydroxyfusicoccin J (4), (R)-5-hydroxymethylmellein (5) and 4-hydroxyphenethyl acetate (6) was isolated from the fermentation extract of Phomopsis amygdali, an entomogenous fungus isolated from Call midge. Their structures were determined mainly by analysis of MS and NMR spectroscopic data. Compounds 1–6 were tested for antimicrobial activity against three plant pathogenic fungi: Gibberella zeae, Verticillium albo-atrum, and Fusarium nivale, and two bacteria: Escherichia coli and Pseudomonas aeruginosa 2033E. As a result, compounds 1–4 displayed antibacterial activity against Gram-negative P. aeruginosa 2033E, and the minimum inhibition concentration (MIC value) of 1–4 is 30 μg/mL, 58 μg/mL, 26 μg/mL, and 26 μg/mL, respectively.


General
Optical rotations were measured on an Anton Paar MCP 200 Analytical Automatic Polarimeter (Anton Paar GmbH, Austria). 1 H, 13 C NMR, and 2D NMR data were acquired with Bruker Avance III 500 (Bruker Corporation, Germany). ESIMS and HR ESIMS data were obtained using an Agilent HPLC -QTOF/MS 6520 System (Agilent Technologies Inc., USA) instrument equipped with electrospray ionisation source. HPLC separations were performed on an Agilent 1100 instrument (Agilent, Santa Clara, CA, USA) equipped with a variable wavelength UV detector. IR data were recorded using a Nicolet IS5 FT-IR spectrophotometer (Thermo Scientific, USA). Column chromatography (CC) was performed using ODS (Qingdao Marine Chemical Factory, Qingdao, China) and Sephadex LH-20 (Pharmacia Biotech Ltd., Uppsala, Sweden).

Fungal material
The culture of P. amygdali (No. RAXL Y-6) was isolated from a Call midge collected from Ai Sha forest farm, Guang Xi Region, People's Republic of China, in August 2012. The isolate was identified by Wang Lin based on morphology. The isolate was identified by one of the authors (Yunfei Pei) based on morphology and sequence (Genbank Accession No. AF102998) analysis of the ITS region of the rDNA. The strain has been preserved at the State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, China. The fungal strain was cultured on slants of potato dextrose agar (PDA) at 258C for five days. Agar plugs were cut into small pieces (about 0.5 £ 0.5 £ 0.5 cm 3 ) under aseptic conditions, and 15 pieces were used to inoculate three Erlenmeyer flasks (250 mL), each containing 50 mL of media (0.4% glucose, 1% malt extract, and 0.4% yeast extract; the final pH of the media was adjusted to 6.5 and sterilised by autoclave). Five flasks of the inoculated media were incubated at 258C on a rotary shaker at 200 rpm for five days to prepare the seed culture. Fermentation was carried out in 20 Fernbach flasks (500 mL), each containing 80 g of rice. Distilled H 2 O (120 mL) was added to each flask, and the contents were soaked overnight before autoclaving at 15 psi for 30 min. After cooling to room temperature, each flask was inoculated with 30 mL of the spore inoculum and incubated at 258C for 40 days.

Antimicrobial activity assay
The antimicrobial activity assays were carried out in the 96-well sterilised microplates using a micro-dilution method (Zhou et al. 2014). All experiments were repeated three times. MICs were determined as the lowest concentrations that produce complete growth inhibition of the tested microorganisms.

Supplementary material
Supplemental data for this article can be accessed at http://dx.doi.org/10.1080/14786419.2015.1055742

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