Since the early 1990s, European ash (Fraxinus excelsior) has been facing an onslaught of severe dieback in most of its natural distribution area in Europe. The causal agent of this epidemic disease is the invasive ascomycete Hymenoscyphus fraxineus (=Hymenoscyphus pseudoalbidus).1, 2, 3 This fungal pathogen most probably originates from East Asia and has almost replaced the European sister species Hymenoscyphus albidus.4, 5 The latter species, H. albidus, is known in Europe since 18516 as a widespread, non-pathogenic decomposer of ash litter and produces numerous white-stalked apothecia on fallen, previous year’s rachises of F. excelsior.5, 7 Apparently, H. albidus is threatened with local extinction in regions colonized by H. fraxineus due to competitive exclusion from its ecological niche (fallen petioles of F. excelsior).4

Over the past years, H. fraxineus has been studied intensively for virulence factors ultimately responsible for the pathogenicity. The role of previously described secondary metabolites, like the tetramic acid antibiotic hymenosetin,8 during infection and necrosis formation has yet to be defined. Actually, the only known phytotoxins of H. fraxineus, viridiol and 3,4-dimethylpentan-4-olide, are, however, also produced by the non-pathogenic H. albidus.9, 10

In the current study, we report the isolation and structure elucidation of two new polyketides, albiducins A and B (1 and 2, Figure 1), from liquid culture extracts of H. albidus. Further, these small salicylaldehyde derivatives were evaluated for cytotoxic and antimicrobial activities (Table 1).

Figure 1
figure 1

Structures of albiducins A (1) and B (2).

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Table 1 Antimicrobial and cytotoxic activities of albiducins A (1) and B (2)
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The examined strain H. albidus 2009-111/1/4 was isolated from apothecia collected in 2009 in Bergen, Norway outside H. fraxineus-infected areas11 and unambiguously determined by morphological characterization and sequencing of its ITS rDNA region (details see Supplementary Information). A 50 l scale fermentation of H. albidus 2009-111/1/4 was carried out for 20 days at 23 °C in ZM/2 medium. This medium was chosen because previous investigations had revealed its optimal composition for secondary metabolite production of H. fraxineus.8 After cultivation had been stopped, the biomass was separated by centrifugation and extracted with acetone. The resulting mycelial extract was fractionated using normal-phase and reversed-phase column chromatography, yielding the two new bioactive compounds 1 and 2 (details see Supplementary Information).

Albiducin A (1) was isolated as yellow oil. Its molecular formula C16H20O3 was determined by HRESIMS, indicative of seven units of unsaturation. The UV/Vis spectrum with absorption maxima of 226, 262, 298 and 398 nm indicated an extensive conjugated π system. The 1H and 1H,13C HSQC spectra showed signals of an aldehyde, six aromatic/olefinic methines, four methylenes along one methyl group. In addition, the 13C spectrum revealed the presence of four additional sp2 hybridized quaternary carbons; two of which were bound to oxygen as indicated by their deep field chemical shifts. The nona-1,3-dienyl side chain was deduced from 1H,1H COSY and TOCSY correlations between protons H-8 to H3-16 and confirmed by a multitude of intraresidue 1H,13C HMBC correlations (Supplementary Figure S1). HMBC correlations of H-8 to C-2/C-6, H-6 to C-2/C-4/C-8, H-5 to C-3/C-7 and H-1 to C-2/C-3/C-4/C-7 revealed the backbone of 1 (Supplementary Figure S1). Finishing the structure elucidation, the all-trans-geometry of the Δ8, 9 and Δ10, 11 double bonds was assigned based on the vicinal coupling constants (J8,9=15.4 Hz; J10,11=15.1 Hz). The IUPAC nomenclature of 1 is 2,3-dihydroxy-6-[(1E,3E)-nona-1,3-dien-1-yl]benzaldehyde.

Albiducin B (2) was analyzed having the molecular formula C16H20O2, which indicated the formal loss of an oxygen atom compared to 1. The proton and carbon spectra of 2 were very similar to those of 1, with the key difference of the appearance of an additional aromatic methine instead of a quaternary carbon. This methine was part of the H-4/H-5/H-6 spin system. Again, JH8,H9=15.6 Hz and JH10,H11=14.9 Hz indicated all-trans-geometry of the Δ8, 9 and Δ10, 11 double bonds. The IUPAC nomenclature of 2 is 2-hydroxy-6-[(1E,3E)-nona-1,3-dien-1-yl]benzaldehyde.

Albiducins A (1) and B (2) are members of a group of structurally related small polyketides formally derived from salicylaldehyde (Supplementary Figure S2). Despite their relative simplicity, these small molecules have been found to possess a variety of interesting bioactivities. Their closest relative aurocitrin was isolated from Hypocrea citrina (currently valid name Trichoderma citrinum) and showed strong antibiotic activity against Staphylococcus aureus.12 Furthermore, aurocitrin and its derivatives exhibited moderate antiplasmodial activities and cytotoxicity against three cancer cell lines.13 Pyriculariol, produced by the rice blast fungus Pyricularia oryzae, was found to be phytotoxic due to dark necrotic spots caused on rice leaves and growth inhibition of rice seedlings.14 2,4-dihydroxy-6-((1E,3E)-penta-1,3-dien-1-yl)benzaldehyde from the endophytic fungus Periconia atropurpurea was able to induce slight increase in cell proliferation of HeLa and CHO cell lines and showed potent antifungal activity.15 Another similar compound family, auroglaucin and its derivatives dihydroauroglaucin, isodihydroauroglaucin, flavoglaucin were isolated from ‘Eurotium herbariorum’ (currently valid name Aspergillus glaucus) and displayed strong antioxidative activity.16

In our study, the antimicrobial activities of albiducins A (1) and B (2) were evaluated in a serial dilution assay against a broad panel of bacteria and fungi in a similar manner as previously reported17 (Table 1, details see Supporting Information). Both compounds exhibited weak inhibitory activity against Gram-positive bacteria, including the pathogen Staphylococcus aureus. In addition, 1 displayed slight antifungal activity against all tested fungi. The cytotoxicity of 1 and 2 were assayed in vitro against the mouse fibroblast cell line L929 and the human cervical carcinoma cell line KB3-1. Both compounds showed moderate IC50 values with stronger effect on human cancer cells. In general, 1 was found to be more active than 2. These results are in accordance with above described activities of previously reported structural related compounds.

Albiducin A ( 1): Yellow oil; UV (MeOH) λmax (log ɛ): 226 nm (3.84), 260 nm (3.81), 296 nm (3.68), 398 (3.26); 1H NMR (700 MHz, DMSO-d6): δH 10.41 (s, H-1), 7.18 (d, J=15.5 Hz, H-8), 7.03 (d, J=8.4 Hz, H-5), 7.00 (d, J=8.4 Hz, H-6), 6.66 (dd, J=15.5 Hz, 10.4 Hz, H-9), 6.22 (dd, J=15.1 Hz, 10.4 Hz, H-10), 5.86 (dt, J=15.1 Hz, 7.1 Hz, H-11), 2.12 (q, J=7.1 Hz, 7.1 Hz, H2-12), 1.39 (dt, J=14.0 Hz, 7.3 Hz, H2-13), 1.33—1.25 (m, H2-14, H2-15), 0.87 (t, J=7.0 Hz H3-16); 13C NMR (175 MHz, DMSO-d6): δC 195.3 (CH, C-1), 150.6 (qC, C-3), 145.0 (qC, C-4), 135.7 (CH, C-11), 131.5 (CH, C-9), 131.3 (qC, C-7), 130.8 (CH, C-10), 125.6 (CH, C-8), 121.7 (CH, C-5), 118.2 (qC, C-2), 117.0 (CH, C-6), 32.2 (CH2, C-12), 30.8 (CH2, C-14), 28.4 (CH2, C-13), 21.9 (CH2, C-15), 13.9 (CH3, C-16); ESIMS m/z 261.24 [M+H]+, 259.10 [M-H]-; HRESIMS m/z 261.1493 [M+H]+ (calcd. for C16H21O3, 261.1485); Rt=14.7 min.

Albiducin B ( 2): Yellow oil; UV (MeOH) λmax (log ɛ): 254 nm (3.58), 288 nm (3.34), 372 (2.94); 1H NMR (500 MHz, DMSO-d6): δH 11.27 (br s, 3-OH), 10.46 (s, H-1), 7.45 (t, J=8.0 Hz, H-5), 7.30 (d, J=15.6 Hz, H-8), 7.14 (d, J=8.0 Hz, H-6), 6.85 (m, H-4), 6.83 (m, H-9), 6.26 (dd, J=15.1 Hz, 10.4 Hz, H-10), 5.95 (dt, J=15.1 Hz, 7.2 Hz, H-11), 2.14 (m, H2-12), 1.40 (m, H2-13), 1.33—1.25 (m, H2-14, H2-15), 0.87 (t, J=7.0 Hz, H3-16); 13C NMR (125 MHz, DMSO-d6): δC 194.4 (CH, C-1), 162.0 (qC, C-3), 140.8 (qC, C-7), 137.6 (CH, C-11), 136.1 (CH, C-5), 133.9 (CH, C-9), 130.7 (CH, C-10), 125.9 (CH, C-8), 118.1 (qC, C-2), 117.1 (CH, C-6), 115.6 (CH, C-4), 32.2 (CH2, C-12), 30.9 (CH2, C-14), 28.3 (CH2, C-13), 21.9 (CH2, C-15), 13.9 (CH3, C-16); ESIMS m/z 245.20 [M+H]+, 243.10 [M-H]-; HRESIMS m/z 245.1535 [M+H]+ (calcd. for C16H21O2, 245.1536); Rt=16.0 min.