The antifungal metabolites from coculture of Aspergillus fumigatus and Alternaria alternata associated with Coffea arabica

Abstract One new cyclohexenone derivative, asperfumtone A (1) along with six known compounds were obtained from the coculture of Aspergillus fumigatus and Alternaria alternata associated with Coffea arabica. The configuration of 2 was first reported in the research. The structures were determined by extensive spectroscopic analyses, and ECD calculation. Compounds 3, 4 and 7 showed significant antifungal activities against coffee phytopathogens A. alternata and Fusarium incarnatum with MICs of 1 μg/mL. Compounds 1 and 2 showed weak antifungal activities against A. alternata and F. incarnatum with MICs of 32–64 μg/mL. Graphical abstract


Introduction
Plant-fungal interactions crucially impacted crop productions and can range from extremely beneficial to harmful (Redkar et al. 2022).The beneficial endophytes facilitated the growth of host plants by producing anti-pathogenic metabolites (Wen et al. 2022).The endophyte Aspergillus fumigatus produces metabolites with great interest to the scientifc research community (Vadlapudi et al. 2017).
Pathogen Alternaria alternata can produce toxins and affect food commodities including green coffee (Mujahid et al. 2020).Endophytes communicate with invading pathogens but their language remains largely unknown (Barenstrauch et al. 2020).The biotic stress existed in coculture of endophyte and phytopathogen can induce the productions of anti-phytopathogen metabolites from endophyte (Barthelemy et al. 2021).In this study, the coculture of endophyte A. fumigatus and phytopathogen A. alternata was investigated to find antifungal compounds against phytopathogens (Figure 1) and understand the biological control mechanism of endophyte on plant pathogen.

Results and discussion
Compound 1 was obtained as a yellow oil.The molecular formula was determined as C 8 H 10 O 5 with four degrees of unsaturation by HR-ESI-MS m/z: 209.0420 [M + Na] + , 185.0440 [M − H] − .The 1 H and 13 C NMR spectral data of 1 (Table S1) showed a total of 8 carbon signals, including two methyls (δ C 26.4, 58.7), one methylene (δ C/H 49.3/2.74,2.85).The data analysis revealed the structure of compound 1 to be similar to that of fumigatin (Frisvad et al. 2009).The cyclohexene-1,4-dione fraction of compound 1 was determined by HMBC correlations from H-3 to C-1, C-2, C-4 and C-5, and the CH 3 at C-7 was confirmed by the HMBC correlations from H-7 to the C-1, C-2 and C-3.The HMBC correlation from H-8 to δ C 144.0 showed the methoxy connected at C-5 or C-6 (Figure S25).Asperstone, like 1 with methoxy at C-5 was found from A. fumigatus in previous work, but the obvious NMR differences (Table S1) existed between those of 1 and asperstone in the same solvent (Chen et al. 2022).So the methoxy of 1 only connected to C-6.The absolute configuration of 1 was determined as 2R by the similarity between the calculated electronic circular dichroism (ECD) and the experimental one (Figure S26).So the structure of 1 was identified and named as asperfumtone A.
Compound 2 was isolated as a colorless oil with the molecular formula of C 8 H 8 O 5 deduced from its HRESI-MS ion at m/z 183.0315 [M − H] − .The similarities between the NMR data (Table S1) of 1 and 2 indicated that the structure of 2 was similar to that of 1.The deduction was further confirmed by extensive analysis of the 2D NMR (Figure S25).The differences of the chemical shifts at C-2 (δ C 73.5 for 1, δ C 58.0 for 2), and H-3 (δ H 2.74, 2.85 for 1, δ H 3.66 for 2) showed an epoxy bond substituted at C 2 -C 3 in 2 (Figure S25).In addition, the calculated ECD of 2 (Figure S26) demonstrated that the absolute configurations of 2 were 2R, 3S.So the structure of 2 was identified and named as asperfumtone B.

General experimental procedures
Silica gel (100-200, 200-300 mesh Qingdao Marine Chemical Group Company), Lichroprep RP-18 (40-63 mm Merck), and Sephadex LH-20 (GE Healthcare Company) were used for chromatographic technology.1D and 2D NMR spectra were performed on an AVANCE 400 NMR instrument from Bruker, MS spectra were recorded with Agilent G3250AA.AP-1020 polarimeter from JASCO and a Chirascan spectrometer from Applied Photophysics were used to determine optical rotations and circular dichroism.HPLC analyses were performed on an Agilent 1260 instrument.

Fungus material and fermentation
The fungus A. alternata KFZ-32 and A. fumigatus KFQG-2 were isolated from Coffea arabica collected at Baoshan in Yunnan Province and were identified by morphological and genetic (ITS) characteristics (GenBank accession No. of A. fumigatus: MG674662, GenBank accession No. of A. alternata: MN790783).The fungi were maintained in the School of Chemical Science and Technology, Yunnan University.
A. alternata KFZ-32 and A. fumigatus KFQG-2 were maintained on modified PDA medium prepared with 200 g/L fresh potato, glucose (20 g/L), and agar (19 g/L).The A. alternata KFZ-32 and A. fumigatus KFQG-2 were cocultured 0.5 L Erlenmeyer flasks containing 120 mL of potato dextrose broth (PDB) prepared by infusion of potato (200 g), dextrose (20 g), and 1.0 L of water at 160 rpm and 28 °C for 1 day for the seed culture.Each 10-15 mL seed culture was incubated in a 0.5 L Erlenmeyer flask containing 150 mL of PDB at 160 rpm and 28 °C for 7 days.

Bioactive assay
In the in vitro antifungal test, the minimal inhibitory concentrations (MICs) of the metabolites against phytopathogens A. alternata and F. incarnatum isolated from Coffea arabica, were measured with the double dilution method.Test compounds were dissolved in dimethyl sulfoxide (DMSO).The final concentrations were 512,256,128,64,32,16,8,4, 2 and 1 µg/mL in medium.The 96-well microplates containing nystatin (Taicheng Pharmaceutical Company, purity >99%) were used as positive controls.The wells containing samples were cultured for 18 h at 28 °C.The tests were repeated three times (Wang et al. 2021).

ECD calculation
To establish the absolute configurations for the isolated compounds, the theoretical calculations of 1 and 2 were compared with the corresponding experimental data.Conformational searches for 1 and 2 were performed via molecular mechanics using the MM + method implemented in CONFLEX software.The geometries were further optimized at the B3LYP/6-31G level using GaussiView 5.0 software to give the energy-minimized conformers.Then, the optimized conformers were subjected to the calculations of ECD spectra using TDDFT at the B3LYP/6-31G (d, p) level, solvent effects of the methanol solution were evaluated at the same DFT level using the polarizable continuum model (PCM) method.The final calculated ECD spectra were obtained according to the Boltzmann-calculated contribution of each conformer (Wu et al. 2022).