Characterization of anticariogenic mycosymbiotic fungi associated with the medicinal plant Piper crocatum

Abstract The escalating antibiotic resistance in oral pathogens, particularly Streptococcus mutans, necessitates the discovery of alternative bioactive scaffolds. While Piper crocatum (red betel) is an ethnobotanically significant plant, its symbiotic mycobiota remains a negligible niche compared to heavily mined species like P. nigrum or P. betle. This study explored the diversity of fungal syimbiont from P. crocatum as potential anticariogenic agents. Fungal samples were isolated from leaves collected across 13 locations in Tasikmalaya, Indonesia. Purification for each fungi was conducted using the hyphal tip transfer technique, yielding 66 axenic isolates. Phenetic characterization was employed as a dereplication strategy to select ten representative based on 33 morphotypes for screening. Similarity analysis was conducted using the Jaccard coefficient via the UPGMA, then visualized in RStudio using the ape and ggtree packages to generate a phenetic dendrogram. One representative isolate from each major cluster was selected for bioactivity screening, with priority given to isolates producing visible extracellular exudates. Results indicated a positive correlation between colony pigmentation and bioactivity, with isolate t5-059 exhibiting the strongest inhibition against S. mutans (21.5 mm). Molecular identification via ITS rDNA resolved the bioactive strains as Colletotrichum truncatum (t5-059), Colletotrichum cliviae (t-9052), Torula canangae (t10-062), and Aspergillus clavatonanicus (t1-007). The recurrence of these specific taxa across geographically heterogeneous sites supports the hypothesis of host filtering, where P. crocatum selects for a core microbiome. These findings highlight P. crocatum as a reservoir of unique fungal associates capable of producing potent metabolites for oral health applications.