Identification of Endophytic Fungi from Red Betel (Piper crocatum Ruiz & Pav.): Exploration of Anticariogenic Bioactive Against Oral Pathogens

The rapid rise of antimicrobial resistance underscores the urgent need to identify novel bioactive compounds from underexplored natural resources. This study aimed to isolate, classify, and characterize endophytic fungi associated with Piper crocatum leaves, an ethnomedicinal plant in Indonesia known for its antimicrobial potential, and to evaluate their capacity as alternative sources of oral antimicrobial agents. Endophytic fungi were isolated from P. crocatum leaves collected across 12 locations in Tasikmalaya Regency, Indonesia, yielding 66 pure isolates after repeated subculturing. Morphological characterization was conducted based on 33 phenotypic traits, converted into binary codes, and analyzed using numerical taxonomy with Jaccard similarity coefficients and UPGMA clustering. Representative isolates from 10 major clusters were subjected to antimicrobial screening. Fungal crude extracts were prepared solid-state fermentation on red rice ( Oryza sativa var. andel abang), followed by methanolic maceration. Antimicrobial activity was tested against Streptococcus mutans using the disc diffusion (Kirby–Bauer) method with 2% chlorhexidine as a positive control. Isolates displaying significant activity were further identified via ITS1–4 rDNA sequencing and phylogenetic analysis. Among the 10 representative isolates, four demonstrated notable inhibitory activity against S. mutans . Molecular characterization revealed that Isolate 059 clustered with Colletotrichum truncatum , Isolate 062 with Colletotrichum cliviae , Isolate 064 with Torula canangae , and Isolate 007 with Aspergillus rhizopodus . Phylogenetic analysis confirmed their taxonomic placement with strong bootstrap support. Notably, bioactive derived from Isolate 059 ( C. truncatum ) exhibited the highest antimicrobial potential. This study demonstrates that endophytic fungi from P. crocatum represent a promising reservoir of antimicrobial agents against oral pathogens. The combined application of numerical taxonomy, solid-state fermentation, and molecular identification provides a robust framework for bioprospecting studies. In particular, C. truncatum (Isolate 059) emerges as a compelling candidate for further fractionation, bioactive compound isolation, and development into alternative therapeutics targeting oral infectious diseases.