Investigation of Secondary Metabolites from the Co-cultivation of the Mangrove Endophytic Fungi Phomopsis asparagi DHS-48 and Pestalotiopsis sp. HHL-101

【Objective】To investigate the secondary metabolites produced by the co-culture system of two mangrove endophytic fungi: Phomopsis asparagi DHS-48 (isolated from Rhizophora mangle) and Pestalotiopsis sp. HHL-101 (isolated from Rhizophora stylosa). 【Methods】Interspecific competitive interactions and micromorphological changes were observed using plate confrontation assays and scanning electron microscopy (SEM). Metabolic differences between the crude extracts of mono-cultures and the co-culture were analyzed using HPLC profiling. The ethyl acetate extract from the co-culture fermentation was isolated and purified using various chromatographic techniques, including normal-phase silica gel column chromatography, reversed-phase ODS column chromatography, Sephadex LH-20 gel column chromatography, and semi-preparative high-performance liquid chromatography (HPLC). The structures of the isolated compounds were identified using spectroscopic methods, including NMR and MS, in combination with literature data comparison. 【Results】Plate confrontation assays and SEM results revealed the formation of a pigmented line in the contact zone, accompanied by dense mycelial coiling and cell membrane fusion at the interface, indicating intense competitive interactions within the co-culture system. Metabolic analysis demonstrated changes in the HPLC profile of the co-culture, characterized by significantly enhanced intensity of characteristic peaks and an increased number of peaks, suggesting that co-cultivation improved both the abundance and diversity of metabolic products. Twelve compounds were isolated from the co-culture system and identified as: 2-phenylethyl-β-D-glucopyranoside (1), tyrosol (2), 4-hydroxyphenethyl acetate (3), 4-hydroxybenzaldehyde (4), 4-hydroxybenzoic acid (5), 3-chlorogentisyl alcohol (6), 2'-O-methyluridine (7), 2'-deoxythymidine (8), thymine (9), cyclo(L-Hyp-L-Ala) (10), (1R,2R,4R)-trihydroxymenthane (11), and ergosterol (12). Notably, compounds 6, 9, and 11 were isolated from the genera Phomopsis and Pestalotiopsis for the first time. 【Conclusion】Twelve secondary metabolites were successfully isolated and identified from the co-culture of Phomopsis asparagi DHS-48 and Pestalotiopsis sp. HHL-101, encompassing diverse structural types such as aromatic derivatives, nucleosides, monoterpenes, cyclic dipeptides, and sterols. The discovery of three compounds reported for the first time from these two genera enriches the structural diversity of secondary metabolites known to be produced by them.