Discovery of Novel Phenolic Compounds from Eutypa lata Through OSMAC Approach: Structural Elucidation and Antibiotic Potential

Abstract

Among grapevine trunk diseases, Eutypa dieback, caused by the fungus Eutypa lata, is one of the most critical ones, due to its widespread infection in vineyards and the lack of effective treatments. This fungus is a vascular pathogen that enters grapevines through pruning wounds. The infection process is associated with phytotoxic metabolites produced by the fungus, and as such, the identification of new metabolites from different culture conditions and broths could provide valuable insights into the fungus's enzymatic system and help its control. For the purposes of this study, the OSMAC (one strain, many compounds) approach was applied to investigate the secondary metabolism of E. lata strain 311 isolated from Vitis vinifera plants in Spain. A total of twenty metabolites were isolated, including five reported for the first time from E. lata and four that are newly identified compounds in the literature: eulatagalactoside A, (R)-2-(4'-hydroxy-3'-methylbut-1'-yn-1'-yl)-4-(hydroxymethyl)phenol, (S)-7-hydroxymethyl-3-methyl-2,3-dihydro-1-benzoxepin-3-ol, and (3aR,4S,5R,7aS)-4,5-dihydroxy-6-((R)-3'-methylbuta-1',3'-dien-1'-ylidene)hexahydrobenzo[d][1,3]dioxol-2-one. These compounds were extracted from fermentation broths using silica gel column chromatography and high-performance liquid chromatography (HPLC). Their structures were elucidated through extensive 1D and 2D NMR spectroscopy, along with high-resolution electrospray ionization mass spectrometry (HRESIMS). Compounds were evaluated for phytotoxicity against Phaseolus vulgaris, with only eulatagalactoside A producing white spots after 48 h. Additionally, the antibacterial activity against Escherichia coli, Staphylococcus aureus, and Klebsiella pneumoniae of selected compounds was tested. The compounds (R)-2-(4'-hydroxy-3'-methylbut-1'-yn-1'-yl)-4-(hydroxymethyl)phenol and (S)-7-(hydroxymethyl)-3-methyl-2,3-dihydrobenzo[b]oxepin-3-ol showed the most significant antimicrobial activity against Gram-positive bacteria, inhibiting S. aureus by over 75%, with IC₅₀ values of 511.4 µg/mL and 617.9 µg/mL, respectively.

Keywords: Eutypa; GTDs; phytopathogen; secondary metabolites; toxins.

Figure 1

Isolated compounds from E. lata 311.

Figure 2

HMBC spectra correlations for 5.

Figure 3

NOESY spectra correlations for 5.

Figure 4

Experimental and calculated ECD spectra for compounds 7, 8, 11, 12, 14, and 16.

Figure 5

HMBC and COSY spectra correlations for 7.

Figure 6

HMBC and COSY spectra correlations for 11.

Figure 7

HMBC and COSY spectra correlations for 16.

Figure 8

Selected NOE interactions and correlations exhibited by 16.

Figure 9

Phytotoxicity assay of 5 after (a) 0 h; (b) 24 h and (c) 72 h at 1000 ppm.

Figure 10

Heatmap plot of the percentage inhibition (PI) of the isolated compounds against different microbial strains. All determinations were performed in triplicate and values are expressed as the means of replicates.