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.