Chemical constituents of the mangrove-associated fungus Capnodium sp. SZ-F22. A new eremophilane sesquiterpene

Abstract A new eremophilane sesquiterpene, capnodiumone (1), along with five known eremophilane sesquiterpenes (2–6) and eight other compounds (7–14), have been isolated from a mangrove-associated fungus Capnodium sp. SZ-F22. The chemical structures were elucidated on the basis of extensive spectroscopic analysis. The broth extract of the fungus exhibited a good inhibitory effect on the mycelium growth against Fusarium graminearum at 100 μg/mL, however, all the 14 compounds showed no expected antifungal activity. The probable reasons were discussed.


Introduction
Mangrove forests are complex ecosystems and the special conditions result in an active microbial community, which can adapt to the changing environment via generation of unique functional metabolites of pharmaceutical importance (Xu 2015). The study of mangrove-associated microbial natural products is a hotspot (Deng et al. 2013;Klaiklay et al. 2013;Yang et al. 2013;Zhu et al. 2013;. 348 biomolecules were discovered until 2011 (Xu 2011), and 464 new notable bioactivities of secondary metabolites were isolated during 2011-2013 (Xu 2015).
Fusarium head blight (FHB) is the most diffuse wheat ear disease throughout the world and Fusarium graminearum is one of the most important causal agents of the fungal disease (Crespo-Sempere et al. 2015). FHB of wheat and other small-grain cereals not only reduces yields but also contaminates grains with mycotoxins, which pose a threat to animals and ABSTRACT A new eremophilane sesquiterpene, capnodiumone (1), along with five known eremophilane sesquiterpenes (2-6) and eight other compounds (7-14), have been isolated from a mangrove-associated fungus Capnodium sp. SZ-F22. The chemical structures were elucidated on the basis of extensive spectroscopic analysis. The broth extract of the fungus exhibited a good inhibitory effect on the mycelium growth against Fusarium graminearum at 100 μg/mL, however, all the 14 compounds showed no expected antifungal activity. The probable reasons were discussed. human. Since FHB-resistant varieties are still not available (Hue et al. 2009), fungicides containing triazole, imidazole, or triazolinthione active ingredients, are the most effective pesticides against FHB infection and DoN contamination in wheat grain (Haidukowski et al. 2005). Nevertheless, the problems of fungicide-resistance and environmental pollution have arisen, there is a clear need to discover new biomolecules to replace currently available fungicides.
In this study, we selected Capnodium sp. SZ-F22 from more than 30 mangrove-associated fungi according to their inhibitory activity against in vitro assay. We further purified and identified 14 compounds, including a new eremophilane sesquiterpene, from the broth extract of the fungus Capnodium sp. SZ-F22. The antifungal activity of the isolated metabolites was tested against F. graminearum.

Results and discussion
The fungus Capnodium sp. SZ-F22 showed a significant antagonistic effect in qualitative antifungal assays against F. graminearum ( Figure S1a), and the broth extract also exhibited a good inhibitory effect on the radial growth of F. graminearum at 100 μg/mL with the inhibition rate of 70.7% ( Figure S1b).
The relative configuration of 1 was determined by the NoeSY experiments, according to the literature, the relative configuration of methyl group at C-4 and C-5 were both β-orientation in the eremophilane sesquiterpenes (Tabata et al. 1997;McDonald et al. 2004;Yamada et al. 2004Yamada et al. , 2005Sørensen et al. 2007), and the relative stereochemistry of 3-ooCCH 3 at C-3 and epoxide group at C-1 and C-2 could be deduced as α-orientations based on the NoeSY correlation between H 3 -14 and H-1,H-3, H 3 -15, and H-2. Therefore, 1 was identified as a new naturally occurring eremophilane sesquiterpene.
The antifungal activity of the isolated metabolites was tested against F. graminearum. None of the tested compounds exhibited inhibitory effects toward tested fungus ( Figure S1c).

Antagonistic test of the Capnodium sp. strain against F. graminearum
A 5-mm plug taken from the edge of a 3-day-old colony of F. graminearum was inoculated in the centre of a 90-mm flat plate with 15 mL of PDA medium, and three fungus cakes containing mangrove-associated fungus Capnodium sp. SZ-F22 were placed symmetrically at 25 mm around the centre of the culture plate, then incubated at 28 °C. The experiment was repeated three times independently. The method was modified by a method reported before (Zhao et al. 2014).

Fermentation and extraction
The strain SZ-F22 was inoculated in 500 mL erlenmeyer flasks each containing 250 mL YGP broth and fermented at 28 °C, 180 rpm for 10 days. The flask culture (200 L) was filtered to separate into the filtrate and wet mycelia, then the two parts were extracted separately, afforded 13 g of dark brown gum finally (The procedure of fermentation and extraction was seen on supplementary material 1.3).

Inhibitory activity of all compounds against F. graminearum
The DMSo solution (60 μL) of the each compound (1-14) and carbendazim was added to 6 mL of PDA held at 50 °C. The agar medium was poured onto a 60-mm plate. each compound was tested at a concentration of 100 μg/mL. A PDA disc (5 mm in diameter) with fresh mycelia of F. graminearum was placed on each plate containing a compound. The pathogen was incubated at 28 °C for 3 days，then the diameter of the colony on each plate was measured. The pathogen growing on PDA (6 mL) containing 60 μL of DMSo was used as control, and the carbendazim treatment group was used as the positive control. All isolates were tested in triplicate.

Conclusion
In summary, we isolated a fungus Capnodium sp. SZ-F22 from mangrove sample, and the fungus colony showed distinct antagonistic effect on the Hyphae dispersal of F. graminearum. Moreover, the broth extract of the fungus exhibited good inhibition effect on mycelia growth of F. graminearum at a concentration of 100 μg/mL. Additionally, the borth extract of fungus Capnodium sp. SZ-F22 afforded 14 metabolites, a new eremophilane sesquiterpene, capnodiumone, and five known eremophilane sesquiterpenes together with eight other compounds. Nevertheless, F. graminearum was not inhibited at all by any of the isolated compounds at concentration of 100 μg/mL, we speculated the probable reasons were that the effective component was not isolated, or was volatile easily so that we could not get it by the conventional purification methods.

Supplementary material
NMr and LC-MS data relating to this article was available on line, alongside Table S1 and Figures S2-S9.