Two new sulfur-containing pyridine alkaloids from the co-culture of Diaporthe fungi

The untapped metabolome of filamentous fungi represents a profound reservoir for therapeutic lead discovery. Although genomic mining confirms an abundance of cryptic biosynthetic gene clusters (BGCs), axenic laboratory culture frequently lacks the ecological stimuli required to trigger their expression, inevitably leading to the redundant isolation of known metabolites. To circumvent this biosynthetic silencing and unlock hidden chemical diversity, strategies such as heterologous expression, the one-strain-many-compounds (OSMAC), epigenetic modulation, and microbial co-cultivation have been increasingly deployed. Herein, we harnessed an interspecies co-cultivation strategy to systematically investigate the biogenetic potential of mangrove-associated fungi belonging to the genus Diaporthe . Four fungal strains ( Phomopsis phyllanthicola ZMU-32-2-1, Diaporthe kyushuensis ZMU-48-1, Diaporthe phaseolorum ZMU-49-3, and Diaporthe sp. ZMU-11) were initially isolated and purified. Subsequent co-cultivation screening revealed that strains ZMU-48-1, ZMU-32-2-1, and ZMU-11 showed altered metabolic profiles, with the appearance of novel chromatographic peaks that were not observed in their respective monocultures. Scaled-up co-fermentation and subsequent chromatographic purification led to the isolation of five structurally diverse compounds. Notably, this included two unprecedented sulfur-containing pyridine alkaloids, designated as diaridine A (1) and B (2), alongside three previously described secondary metabolites. The isolated compounds were evaluated for their antifungal properties; however, none displayed significant inhibitory activity at a concentration of 200 μg/mL. Despite the absence of potent antifungal efficacy, these findings highlight the power of microbial crosstalk in activating the cryptic chemical space of Diaporthe species, reaffirming their potential as a robust resource for novel drug discovery.