In situ cultivation and chromogenic screening: a dual approach for rapid discovery of PDE1 inhibitors from pinus-tree associated microbes

The discovery of natural phosphodiesterase 1 (PDE1) inhibitors from Pinus-tree associated microbes faces critical bottlenecks: the inability to culture uncultured species in situ and the inefficiency of traditional activity screening. To overcome these challenges, we developed a miniaturized in situ cultivation device for direct insertion into plant tissues, enabling targeted isolation of uncultured microbes. Complementing this, a novel filter paper chromogenic (FPC) method was designed to simultaneously detect PDE1 inhibitory activity during microbial cultivation. By integrating these approaches, we rapidly screened bioactive strains from four Pinus species ( P. tabuliformis , P. armandii , P. bungeana , and P. massoniana ), isolating 88 actinomycetes and 2 fungi. Notably, the FPC method demonstrated high consistency with UV-based assays in 96-well plates, validating its reliability for real-time activity monitoring. Strain YSTR-10, identified as Penicillium sp., exhibited significant PDE1 inhibition. Subsequent chemical characterization of YSTR-10 yielded four compounds 4-(2-hydroxybutynyloxy)benzoic acid ( 1 ), p-hydroxyacetophenone ( 2 ), penicilazaphilone B ( 3 ), and graminin B ( 4 ), with graminin B (4) showing moderate inhibitory activity (61.32% at 100.0 µg/mL). This study not only pioneers a dual-method framework for efficient enzyme inhibitor discovery but also highlights the untapped potential of plant-associated microbiota in drug development.