Comparative Genomics and Gene Cluster Family Analysis Reveal Conserved and Isolate-Specific Biosynthetic Potential in Medicinal Plant-Associated Endophytic Diaporthe

Endophytic fungi associated with medicinal plants represent promising reservoirs of specialized metabolites; however, the genomic diversity underlying their biosynthetic potential remains incompletely characterized. Here, we performed comparative genomic and genome-mining analyses of four medicinal plant-associated endophytic Diaporthe isolates: D. inconspicua LGMF1612, D. cf. heveae LGMF1633, D. vochysiae LGMF1583, and Diaporthe sp. HANT25. Genome sizes ranged from 52.45 to 60.11 Mb, with 16,033–17,434 predicted proteins. Comparative orthology identified 16,196 protein clusters, of which 11,230 (69.3%) were conserved across all four isolates, indicating a substantial shared proteomic repertoire accompanied by smaller variable components. Functional annotation revealed extensive representation of membrane transporters, fungal transcriptional regulators, cytochrome P450s, redox and detoxification enzymes, and protein domains associated with specialized metabolism. Genome mining using antiSMASH identified 481 biosynthetic gene clusters (BGCs), with 112–129 BGCs per isolate. Type I polyketide synthase clusters were predominant, together with substantial NRPS, NRPS-like, terpene, and hybrid/multiclass repertoires. BiG-SLiCE grouped the predicted BGCs into 348 gene cluster families (GCFs), only 15 of which were classified as core GCFs, with members represented in all four isolates, revealing extensive variation in biosynthetic repertoires despite a largely conserved proteomic background. Integration with MIBiG-associated predictions identified core GCFs showing similarity to characterized pathways associated with squalestatin S1, choline, scytalone/T3HN, cercosporin, and nectriapyrone biosynthesis. Additional characterized pathway-associated signatures showed partial or isolate-restricted distributions, including α-acorenol- and phyllostictine-associated BGCs in D. inconspicua, chaetolivacine, chrysogine-, patulin-, and AKML-associated BGCs in D. vochysiae, and enniatin-, azaphilone-, curvupallide-B-, aspyridone-, and wortmanamide-associated BGCs in D. sp. HANT25. Importantly, a substantial fraction of GCFs lacked significant correspondence to characterized MIBiG clusters, indicating considerable chemically uncharacterized biosynthetic potential. Collectively, these findings reveal a conserved genomic foundation coupled with extensive diversification of specialized-metabolite biosynthetic repertoires in medicinal plant-associated Diaporthe and identify conserved and isolate-restricted BGC families as candidates for future metabolomic and functional characterization.