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

Identification of Endophytic Fungi from Red Betel (Piper crocatum Ruiz & Pav.): Exploration of Anticariogenic Bioactive Against Oral Pathogens | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Identification of Endophytic Fungi from Red Betel (Piper crocatum Ruiz & Pav.): Exploration of Anticariogenic Bioactive Against Oral Pathogens Syifa Zahara Kultsum Azmi, Dikdik Kurnia, Rianti Nurpalah, Dewi Peti Virgianti, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8006293/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract 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. endophytic fungi anticariogenic numerical taxonomy ITS sequencing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 1 Introduction Indonesia is globally recognized as one of the world’s megadiverse country, harboring extraordinary biological wealth. The archipelago is home to more than 30,000 plant species, of which approximately 7,000 are known to possess medicinal properties (Navia et al. 2022 ). This exceptional floristic diversity is paralleled by a wide spectrum of chemotaxonomic profiles, offering an immense repository for the discovery of novel bioactive compounds with potential pharmaceutical applications (Silalahi et al. 2015 ). Such biodiversity-driven chemical diversity provides a strategic advantage for bioprospecting and drug discovery, especially in the search for functional metabolites that can address pressing global health challenges. However, direct exploitation of medicinal plants for large-scale production presents considerable challenges (Astutik et al. 2019 ). Cultivation and harvesting of these plants often require long growth cycles, are susceptible to environmental fluctuations, and demand resource-intensive agronomic adjustments (Schafer et al. 2019; Shukla et al. 2025 ). Consequently, scalable production of plant-derived bioactives remains constrained by both biological and logistical limitations. An alternative and increasingly attractive avenue lies in endophytic fungi, microorganisms that colonize the internal tissues of plants without causing apparent harm (Shukla et al. 2025 ; Nair and Padmavathy 2014 ; Yan et al. 2019 ). Endophytes are hypothesized to have co-evolved and exchanged genetic material with their host plants through long-term mutualistic associations (Mesny et al. 2023 ). This intimate relationship may involve horizontal gene transfer, metabolic pathway convergence, and co-regulation of biosynthetic gene clusters (Hiruma et al. 2023 ). As a result, endophytic fungi are capable of synthesizing secondary metabolites structurally and functionally analogous to those produced by their hosts, and in some cases, generating novel compounds with enhanced bioactivity (Hashem et al. 2023 ). Despite this promising potential, the diversity and biotechnological value of many endophytes, particularly those from medicinal plants, remain underexplored (Hashem et al. 2023 ; Hiruma et al. 2023 ). Among Indonesia’s medicinal flora, red betel leaf ( Piper crocatum Ruiz & Pav.) holds a prominent position in ethnobotanical and ethnopharmacological traditions. This species has been widely used for its antimicrobial, anti-inflammatory, and antioxidant properties (Hashem et al. 2023 ; Heliawati et al. 2022 ; Lestari et al. 2024 ). Recent studies have demonstrated that extracts and isolated compounds from red betel exhibit significant inhibitory effects against cariogenic bacteria in both in vitro and clinical settings (Kurnia et al. 2024 ; Siswina et al. 2024). Given these attributes, exploring the endophytic fungi residing in the mesophyll tissues of red betel represents a promising strategy to discover bioactive metabolites capable of modulating microbial communities, particularly those associated with oral diseases. Dental caries remains a major public health burden in Indonesia, affecting approximately 8 out of 10 adults in the productive age group, with the mean of DMF-T (Decayed, Missing, and Filled Teeth) index of 5.0 (Kementerian Kesehatan Republik Indonesia 2023 ; Lestari et al. 2025 ). The prevalence continues to rise, increasing the risk of edentulism in later age. Current preventive measures are largely inadequate (Borg-Bartolo et al. 2022 ), and conventional treatments focus predominantly on restorative interventions rather than proactive microbial modulation (Hitch et al. 2022 ). Therefore, a holistic prevention strategy that targets the ecological balance of oral microbiota, rather than solely treating the clinical manifestation of caries, is urgently required (Luo et al. 2024 ). The present study aims to characterize the diversity of endophytic fungi isolated from red betel leaves, screen their bioactivity against cariogenic microbes, and isolate active secondary metabolites with potential application as microbial colony modulators in the context of cariogenic consortia. 2 Methods 2.1 Collection of Plant Material and Selection Criteria Pathogen-free leaves of Piper crocatum Ruiz & Pav. (red betel) were selected as the primary source of observation in this study. The leaves were screened visually to ensure the absence of necrosis, chlorosis, or any other visible signs of pathogenic infection (Viret 1993 ). Sampling was conducted from twelve distinct locations across the Tasikmalaya region, West Java, Indonesia, to capture a broad representation of the plant’s endophytic fungal diversity. The distribution map of the sampling sites was generated using ArcGIS 10.8 (Esri, Redlands, CA, USA). Administrative boundary layers (province, district, and subdistrict levels) were obtained from the official Rupa Bumi Indonesia (RBI) dataset provided by the Geospatial Information Agency of Indonesia (Badan Informasi Geospasial, BIG). The geographic coordinates of the sampling plants were recorded in the field using a handheld Global Positioning System (GPS) device and compiled into a comma-separated values (CSV) file containing latitude, longitude, and sample ID. The map was constructed using the Universal Transverse Mercator (UTM) projection, WGS 1984 datum, Zone 48S. Sampling points were visualized using distinct symbols and colors to differentiate each sample code (SM1001–SM1013). The administrative boundaries were displayed as polygon layers with contrasting outlines for clarity. Map elements including a north arrow, scale bar, and legend were added to improve readability. 2.2 Surface Sterilization and Tissue Preparation All collected samples were placed in sterile petri dishes, transported under cooled conditions, and processed within 24 hours to minimize changes in the endogenous microbial community. Leaves were rinsed under running tap water to remove debris, followed by immersion in 70% (v/v) ethanol for 1 min, 1.3 mol/L sodium hypochlorite (NaOCl) for 3 min, and rinsed three times with sterile distilled water (Meng et al. 2024 ). The abaxial surface of each leaf was oriented upward (inverted position), and a longitudinal medial section was excised along the midrib (primary vein) using a sterile scalpel blade (no. 11). Sections were prepared with 2.5 mm spacing on either side of the midrib, resulting in a 5 mm-wide central strip. This strip was then transversely subdivided into three segments, each measuring 2.5 mm in length, to obtain explants from mesophyll tissue. 2.3 Isolation of Endophytic Fungi Endophytic fungal isolation was carried out using a direct planting method. Three leaf explants were aseptically placed on the periphery of solidified Sabouraud Dextrose Agar (SDA) medium supplemented with chloramphenicol (0.4 µg/mL) (Achraya and Hare 2022) to suppress bacterial growth. Explants were positioned equiangularly at an estimated center-to-center distance of 2–3 cm to avoid colony interference. Plates were incubated aerobically at 37°C for 48 h. Emergent fungal colonies from the mesophyll tissue were subcultured repeatedly onto fresh SDA plates until pure single-spore isolates were obtained. 2.4 Phenetic Characterization and Cluster Analysis Phenetic characterization was performed on each purified isolate based on 33 macromorphological traits of colony morphology (Crous et al. 2009 ), including both upper and reverse colony characteristics. Each character state was recorded in binary format (presence = 1, absence = 0) and compiled into a binary data matrix. Similarity analysis was conducted using the Jaccard coefficient via the UPGMA clustering tool available at http://genomes.urv.cat/UPGMA/ . The resulting Newick-formatted (.nwk) dendrogram file was visualized in RStudio (2025.05.1–513) using the ape and ggtree packages to generate a phenetic dendrogram (Yu et al. 2017 ). One representative isolate from each major cluster was selected for bioactivity screening, with priority given to isolates producing visible extracellular exudates. 2.5 Preliminary Bioactivity Screening Selected isolates were rejuvenated on SDA plates and subsequently inoculated into red rice ( Oryza sativa var. andel abang) as a substrate for small-scale solid-state fermentation (El-Sayed et al. 2022 ). Fermentation was conducted under aerobic conditions for 28 days at ambient temperature. The fermented substrate was extracted by maceration in methanol (1:3 v/v) for three days with occasional agitation, followed by filtration. The filtrate was concentrated under reduced pressure, and crude extracts were subjected into two preliminary assays: Kirby–Bauer disk diffusion assay against cariogenic bacteria ( Streptococcus mutans ) with 2% chlorhexidine as a positive control; chemical profiling by thin-layer chromatography (TLC) using silica gel F 254 and octadecylsilyl (ODS) stationary phases under UV light (254 and 365 nm). 2.6 Molecular Identification For molecular identification, genomic DNA was extracted using the Quick-DNA Magbead Plus Kit (Zymo Research, D4082) following the internal protocol. The internal transcribed spacer (ITS1-4) region was amplified with MyTaq HS Red Mix, 2X (Bioline, BIO-25048), and PCR products were verified by using agarose gel electrophoresis. Bidirectional Sanger sequencing was performed via capillary electrophoresis, and raw sequence data were assembled and analyzed using an in-house bioinformatics pipeline. Sequencing data were used for BLAST searches against the NCBI GenBank database to determine species-level identity and infer phylogenetic relationships (Yuan et al. 2010 ). 3 Results 3.1 Sampling Map Distribution The distribution map illustrates the spatial variation of Piper crocatum (red betel) populations sampled across Tasikmalaya Regency, West Java (Fig. 1 ). A total of 13 accessions (SM1001–SM2013) were collected, representing diverse subdistricts ranging from lowland to upland areas. The sampling sites cover heterogeneous ecological zones, which are likely to influence the phytochemical diversity and bioactive metabolite profiles of the plants. Spatial heterogeneity is particularly important in medicinal plants, as environmental conditions such as altitude, soil composition, and microclimatic factors can modulate secondary metabolite biosynthesis. This variability provides a strong foundation for bioactivity-guided selection, as distinct populations may yield differential antimicrobial or antibiofilm potentials against oral pathogens. The broad geographic coverage also ensures that the sampling design captures both ecological and chemotypic diversity, thus increasing the reliability of subsequent biological and chemical analyses (Huang et al. 2008 ; Mishra et al. 2014 ; Rana et al. 2019 ). 3.2 Phenotypic Characterization and Clustering of Endophytic Fungi A total of 66 endophytic fungal isolates obtained from Piper crocatum exhibited substantial morphological diversity, as reflected by variations in colony pigmentation, texture, margin configuration, elevation, and growth rate (Fig. 2 ). Such heterogeneity is typical of endophyte communities and underscores their underlying taxonomic and ecological diversity. Comparable morphological differentiation has been reported in endophytes associated with other medicinal plants, reinforcing the notion that P. crocatum represents a rich reservoir of unexplored microbial taxa. To resolve relationships among isolates, 33 macromorphological traits (Table 1 ) were subjected to multivariate analyses. Cluster analysis using the UPGMA method (Fig. 3 , Table 2 ) partitioned the isolates into several well-defined groups with moderate to high bootstrap support (70–128). The presence of strongly supported clades (bootstrap ≥ 100) suggests that certain colony features—particularly pigmentation, texture, and margin structure, can serve as indicators of taxonomic proximity, although other traits may be influenced by culture conditions (Grant and Kluge 2008 ). Table 1 Material Samples of Piper crocatum leaf Code Scientific Name Method of Collection Specimen Source Distribution SM1001 Piper crocatum Direct sampling and observation from living specimen Cicurug, Tasikmalaya Common throughout in Indonesia (Kendari, Bogor, Bandung, Malang, Jayapura, Samarinda, and Banda Aceh) SM1002 Manonjaya, Tasikmalaya SM1003 Padakembang, Tasikmalaya SM1004 Sukaratu, Tasikmalaya SM1005 Singaparna, Tasikmalaya SM1006 Lanud, Tasikmalaya SM2007 Singajaya, Tasikmalaya SM2008 Kubangsari, Tasikmalaya SM2009 Setiaratu, Tasikmalaya SM2010 Cibeureum, Tasikmalaya SM2011 Cisalak, Tasikmalaya SM2012 Mandalajaya, Tasikmalaya SM2013 Tamansari, Tasikmalaya Table 2 Morphological character of endophytic fungi isolated from Piper crocatum leaf Morphological Characters Colony Color (Upper and Reverse Surface) WUC-P White upper colony 1. present, 0. absent WUC-H White upper colony 1. present, 0. absent WUC-O Black upper colony 1. present, 0. absent WUC-U Orange upper colony 1. present, 0. absent WRC-P Purple upper colony 1. present, 0. absent WRC-H White reverse colony 1. present, 0. absent WRC-O Black reverse colony 1. present, 0. absent WRC-U Orange reverse colony 1. present, 0. Absent Texture (Surface Colony) TK-B Velvety texture 1. present, 0. absent TK-H Smooth texture 1. present, 0. absent TK-P Powdery texture 1. present, 0. absent TK-K Cottony (floccose) texture 1. present, 0. absent TK-S Rough texture 1. present, 0. absent Margin (Edge of Colony) MG-E Entire margin 1. present, 0. absent MG-U Undulate margin 1. present, 0. absent MG-L Lobate margin 1. present, 0. absent Elevation (Vertical Profile) ELE-D Flat elevation 1. present, 0. absent ELE-C Convex elevation 1. present, 0. absent ELE-J Raised elevation 1. present, 0. absent ELE-CK Concave elevation 1. present, 0. absent Growth Rate LJ-C Rapid growth rate 1. (2–7 days), 0. (7–14 days) LJ-L Slow growth rate 1. (7–14 days), 0. (2–7 days) Pigmentation and Exudates PEP Exudate/pigment production 1. present, 0. absent ZB Medium clearing / transparent halo 1. present, 0. absent Consistency and Texture KS-P Firm consistency 1. present, 0. absent KS-L Mucilaginous consistency 1. present, 0. absent KS-K Dry consistency 1. present, 0. absent KS-LB Moist consistency 1. present, 0. absent Odor AM-T Earthy odor 1. present, 0. absent AM-A Alcoholic odor 1. present, 0. absent AM-F Fermentative odor 1. present, 0. absent Special Characteristics MUC Multicolored 1. present, 0. absent ZNT Zonate pattern 1. present, 0. absent Principal component analysis (PCA) clarified phenotypic variation, with the first two principal components (PC1 and PC2) explaining 75% of the cumulative variance. Separation along PC1 was primarily associated with colony color and growth rate, distinguishing fast-growing, pigmented clusters (e.g., t1, t3, t4, t10) from slower-growing, less-pigmented clusters (e.g., t2, t8, t9) (Fig. 4 ). In contrast, PC2 was largely driven by differences in texture and elevation, separating clusters such as t5 and t7 that displayed unique colony surface structures. These results indicate that distinct morphological traits, particularly pigmentation and texture, can serve as reliable markers for preliminary classification prior to molecular identification. Complementary insights were obtained from a Jaccard similarity heatmap (Fig. 5 ), which summarized binary trait overlap across clusters (t1–t10). Similarity coefficients ranged from 0.40 to 1.00, revealing both highly conserved groups and divergent profiles. Cluster A (t9, t2, t8) displayed the highest within-group similarity (> 0.85), whereas Cluster B (t3, t7, t4, t1) exhibited moderate similarity (0.75–0.85). Cluster C (t6 and t10) demonstrated an especially close relationship (~ 0.90), while isolate t5 emerged as an intermediate, bridging distinct groups. This clustering pattern suggests that while subsets of isolates share conserved morphologies, others may represent ecologically or functionally divergent taxa. Isolates from geographically distant sites were distributed across multiple clusters, implying that host-associated selective pressures, rather than geography, play a dominant role in structuring P. crocatum endophytic communities. Such findings align with evidence that host plant chemistry exerts a strong influence on endophyte assemblages. Collectively, the integration of morphological characterization, UPGMA clustering, PCA, and Jaccard similarity mapping provides a rational framework for selecting representative isolates. By prioritizing morphologically distinct and phylogenetically robust clusters, the likelihood of capturing metabolite diversity is maximized, thereby enhancing the efficiency of bioactivity-guided screening for novel antimicrobial compounds. 3.3 Preliminary Metabolite Profiling and Antimicrobial Screening of Representative Endophytic Fungi One representative strain from each major cluster was selected for preliminary metabolite screening. This strategy ensured that the functional potential of phylogenetically and morphologically distinct taxa could be captured, thereby increasing the likelihood of identifying novel bioactive metabolites. Ten representative isolates (Samples 1–10) (Fig. 6 ) were fermented aerobically on a small scale using sterile red rice as substrate, providing a nutrient-rich and reproducible matrix for metabolite production. After 14 days of incubation, the fermented material was extracted by methanolic maceration, subjected to sonication-assisted lysis, and concentrated using rotary evaporation to obtain crude extracts. Thin-layer chromatography (TLC) analysis revealed marked differences in metabolite banding patterns among the isolates, highlighting the chemical heterogeneity of the endophyte community. Distinct UV-active spots were observed at both 254 nm and 365 nm, with some extracts displaying unique fluorescent bands (Fig. 7 ). The appearance of isolate-specific chromatographic fingerprints supports the notion that each cluster contributes different metabolic capacities, consistent with previous studies demonstrating that fungal endophytes from medicinal plants produce diverse classes of secondary metabolites, including alkaloids, terpenoids, and polyketides (Mousa and Raizada 2013 ). (A) Separation on silica gel 60 F₂₅₄ normal-phase plates using n-hexane:ethyl acetate (7:3, v/v) as the mobile phase. (B) Separation on silica gel reverse phase R-18 60 F₂₅₄ plates using methanol:distilled water (7:3, v/v) as the mobile phase. Spots were visualized under visible light, UV 254 nm, and UV 365 nm to highlight variations in metabolite composition and chemical diversity among isolates (lanes 1–10). In addition to TLC-based profiling, crude extracts were tested for antimicrobial activity against Streptococcus mutans , the primary etiological agent of dental caries. Several isolates exhibited growth-inhibitory activity, suggesting the presence of bioactive metabolites with potential anticariogenic properties. Interestingly, isolates originating from clusters characterized by fast-growing, pigmented colonies (e.g., Samples 3 and 9) showed stronger inhibition compared to slow-growing, less pigmented representatives (e.g., Samples 2 and 8). This observation aligns with reports that pigmentation in fungi is frequently associated with the biosynthesis of polyketide-derived metabolites, many of which possess antimicrobial or antibiofilm activity (Sukmarini et al. 2024 ). The diversity of TLC patterns and the variable inhibitory activity observed against S. mutans highlight the untapped potential of P. crocatum -associated endophytes as sources of novel bioactive compounds. To complement phenetical clustering and TLC-based metabolite profiling, the crude extracts of ten representative isolates were subjected to antimicrobial screening against Streptococcus mutans (ATCC 25175). Clear inhibition zones were observed for several isolates, with marked variability in activity levels (Fig. 8 , Table 3 ). Among the tested samples, isolates 1, 4, 7, and 8 exhibited strong growth inhibition, producing mean inhibition zones of 21.5 mm, 11.5 mm, 12.75 mm, and 15.85 mm, respectively. By contrast, the remaining isolates displayed negligible or weak inhibition, with zone diameters ≤ 6.9 mm. When these results were integrated with the TLC metabolite fingerprints, a compelling pattern emerged. Isolates demonstrating strong antimicrobial activity (Samples 1, 4, 7, and 8) also exhibited richer chromatographic profiles, characterized by multiple UV-active bands under both 254 nm and 365 nm illumination. In particular, Sample 1 displayed several unique fluorescent spots, suggesting the presence of diverse and potentially bioactive secondary metabolites. This correlation between chemical complexity and bioactivity supports the use of TLC as a rapid, cost-effective proxy for identifying metabolically prolific isolates. The most potent isolate, Sample 1 (code 059) (Fig. 9 ), originated from a cluster defined by slow-growing, pigmented colonies, further reinforcing the notion that pigmentation is linked with metabolite production in endophytic fungi. The alignment of morphological traits, metabolite diversity, and strong bioactivity highlights the robustness of the integrated selection pipeline employed in this study. Table 3 Inhibition zone of endophytic fungal extracts for anticariogenic activity against S. mutans via agar disc diffusion assay (mm). Sample Inhibition Zone (mm) x̄ (mm) ± Dev. Standard 1 21 22 21,5 ± 0 2 6,5 6 6,25 ± 1,414214 3 6 0 3 ± 0,707107 4 12 11 11,5 ± 0,707107 5 5 5 5 ± 0,707107 6 7 6,8 6,9 ± 0 7 13 12,5 12,75 ± 2,12132 s8 16 15,7 15,85 ± 0 9 0 3 1,5 ± 0,141421 10 5 6 5,5 ± 0,141421 3.4 Molecular Identification of Selected Fungal Isolate Molecular characterization of the four representative isolates (samples 1, 4, 7, and 8) using the ITS1–4 rDNA region confirmed their taxonomic affiliation with diverse fungal genera. PCR amplification produced clear amplicons within the expected size range (Fig. 10 ), which were subsequently sequenced and compared against 40 reference strains retrieved from NCBI. Phylogenetic analysis demonstrated robust clustering of the isolates with Colletotrichum , Torula , and Aspergillus lineages, supported by high bootstrap values (Fig. 11 ). Specifically, Isolate 1 (code: 059) grouped with Colletotrichum truncatum , Isolate 3 (code: 062) with Colletotrichum cliviae , Isolate 4 (code: 064) with Torula canangae , and Isolate 2 (code: 007) with Aspergillus rhizopodus . These results provide novel insight into the endophytic mycobiota of Piper crocatum , a medicinal plant that has not previously been extensively surveyed for fungal associates. While Colletotrichum species are widely recognized as phytopathogens, their role as endophytes producing bioactive metabolites has only recently gained attention. Similarly, Torula , traditionally understudied in endophyte research, has been increasingly reported as a promising source of antimicrobial and antibiofilm compounds. The recovery of Aspergillus rhizopodus , a rare species with limited documentation in endophytic contexts, further expands the taxonomic spectrum of fungi associated with P. crocatum . Comparatively, most previous reports of endophytes from medicinal Piper species have been dominated by Fusarium , Penicillium , and Aspergillus spp., whereas the detection of Torula canangae and Colletotrichum cliviae in this study represents, to our knowledge, the first report of these taxa as endophytes in P. crocatum . This finding highlights the unique ecological niche of P. crocatum that accommodates both pathogenic and rare fungal taxa, suggesting an evolutionary adaptation that may stimulate the biosynthesis of structurally diverse secondary metabolites. The novelty of this study lies not only in uncovering unexpected fungal diversity within P. crocatum but also in identifying endophytes with previously unreported associations that hold significant potential for the discovery of anticariogenic compounds. Given the established antimicrobial and quorum-sensing inhibitory properties of Colletotrichum and Aspergillus metabolites, and the emerging relevance of Torula spp., these isolates represent promising candidates for further exploration in dental and oral health applications. 4 Discussion The identification of bioactive endophytic fungi from Piper crocatum carries significant clinical implications in the context of oral health in Indonesia, where the prevalence of dental caries remains alarmingly high, affecting more than 88% of the population and ranking among the most common chronic diseases nationwide. Current therapeutic approaches, such as fluoride treatment and conventional antimicrobials, are limited by reduced efficacy against biofilm-embedded pathogens, recurrent infections, and the emerging threat of antimicrobial resistance. The strong inhibitory effects of C. truncatum , C. cliviae , T. canangae , and A. rhizopodus against S. mutans observed in this study underscore their potential as alternative sources of anticariogenic agents that could overcome these limitations. Future research should prioritize the purification and structural elucidation of the active metabolites responsible for the observed antibiofilm and antimicrobial effects, in order to determine their novelty and mechanism of action. In addition, rigorous evaluation of toxicity and safety profiles is essential to ensure suitability for human application, given that many secondary metabolites of fungal origin can exhibit cytotoxicity. To bridge the gap between in vitro screening and clinical application, further validation using ex vivo oral biofilm models and in vivo systems is warranted, which would provide more realistic insights into pharmacodynamics and therapeutic potential. Ultimately, the integration of endophyte-derived natural products into preventive or adjunctive therapies could represent a transformative approach to reducing the burden of dental caries in Indonesia and beyond. 5 Conclusion This study demonstrated that endophytic fungi isolated from P. crocatum harbor promising anticariogenic potential against S. mutans . TLC profiling revealed diverse secondary metabolite patterns across isolates, suggesting chemical heterogeneity and the presence of multiple bioactive constituents. Bioactivity-guided screening confirmed that four isolates (Samples 1, 4, 7, and 8) exhibited strong inhibitory activity, highlighting their potential as candidates for anticariogenic drug discovery. Molecular characterization using ITS1–4 sequencing identified these bioactive strains as C. truncatum , C. cliviae , T. canangae , and A. rhizopodus , thereby expanding the current knowledge of endophytic biodiversity within Piper species. Declarations Acknowledgements. The author wishes to thank the Department of Chemistry, Universitas Padjadjaran, for providing laboratory facilities and technical support. Special appreciation for Department of Pharmacy, Universitas Bakti Tunas Husada is extended for their guidance during the research process. Funding: This research was supported by PMDSU Research Grant, Ministry of Education, Culture, Research, and Technology of Indonesia (Grant no. 093/C3/DT.05.00/PL/2025). Conflict of interest/Competing interests: The authors declare no conflict of interest. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Data availability: All data generated and analyzed during this study are included in this published article and its supplementary files. Additional datasets are available from the corresponding author upon reasonable request. Materials availability: The fungal isolates obtained and characterized in this study are available from the corresponding author upon reasonable request. Code availability: Not applicable. Author contribution: S.Z.K.A. conceived and designed the study, performed laboratory work. RN provided the research facilities. D.P.V. designed the sampling plot and methods. T.S. conceived the data analysis, and manuscript writing. D.K. provided supervision, critical review, and final approval of the manuscript. All authors have read and approved the final version of the manuscript. References Acharya T, Hare J (2022) Sabouraud agar and other fungal growth media. Laboratory Protocols in Fungal Biology: Current Methods in Fungal Biology. Springer International Publishing, Cham, pp 69–86. https://doi.org/10.1007/978-3-030-83749-5_2 Astutik S, Pretzsch J, Ndzifon Kimengsi J (2019) Asian medicinal plants’ production and utilization potentials: A review. Sustainability 11(19):5483. https://doi.org/10.3390/su11195483 Borg-Bartolo R, Roccuzzo A, Molinero-Mourelle P, Schimmel M, Gambetta-Tessini K, Chaurasia A et al (2022) Global prevalence of edentulism and dental caries in middle-aged and elderly persons: A systematic review and meta-analysis. J Dent 127:104335. https://doi.org/10.1016/j.jdent.2022.104335 Crous PW, Verkley GJ, Groenewald JZ, Samson RA (2009) Fungal biodiversity El-Sayed ESR, Gach J, Olejniczak T, Boratyński F (2022) A new endophyte Monascus ruber SRZ112 as an efficient production platform of natural pigments using agro-industrial wastes. Sci Rep 12(1):12611. https://doi.org/10.1038/s41598-022-16269- Grant T, Kluge AG (2008) Clade support measures and their adequacy. Cladistics 24(6):1051–1064. https://doi.org/10.1111/j.1096-0031.2008.00231.x Hashem AH, Attia MS, Kandil EK, Fawzi MM, Abdelrahman AS, Khader MS et al (2023) Bioactive compounds and biomedical applications of endophytic fungi: a recent review. Microb Cell Fact 22(1):107. https://doi.org/10.1186/s12934-023-02118-x Heliawati L, Lestari S, Hasanah U, Ajiati D, Kurnia D (2022) Phytochemical profile of antibacterial agents from red betel leaf ( Piper crocatum Ruiz and Pav) against bacteria in dental caries. Molecules 27(9):2861. https://doi.org/10.3390/molecules27092861 Hiruma K, Aoki S, Takino J, Higa T, Utami YD, Shiina A et al (2023) A fungal sesquiterpene biosynthesis gene cluster critical for mutualist-pathogen transition in Colletotrichum tofieldiae. Nat Commun 14(1):5288. https://doi.org/10.1038/s41467-023-40867-w Hitch TC, Hall LJ, Walsh SK, Leventhal GE, Slack E, de Wouters T et al (2022) Microbiome-based interventions to modulate gut ecology and the immune system. Mucosal Immunol 15(6):1095–1113. https://doi.org/10.1038/s41385-022-00564-1 Huang WY, Cai YZ, Hyde KD, Corke H, Sun M (2008) Biodiversity of endophytic fungi associated with 29 traditional Chinese medicinal plants. Fungal Divers 33:61–75. http://www.scopus.com/inward/record.url?partnerID=x8ErFbHU&eid=2-s2.0–61849133540 Kementerian Kesehatan Republik Indonesia (2023) SKI 2023 dalam angka [Internet]. Badan Kebijakan Pembangunan Kesehatan, Jakarta. Available from: https://www.kemkes.go.id/article/view/23070300003/ski-2023-dalam-angka.html Kurnia D, Lestari S, Mayanti T, Gartika M, Nurdin D (2024) Anti-infection of oral microorganisms from herbal medicine of Piper crocatum Ruiz & Pav. Drug Des Devel Ther 2024:2531-53. https://doi.org/10.2147/DDDT.S453375 Lestari ADJ, Harokan A, Ekawati D (2025) Analisis kejadian karies gigi pada anak usia 6–11 tahun di RSUD Lubai Ulu Kabupaten Muara Enim. J Ilmu Kedokteran Kesehatan 12(2):220–229. https://doi.org/10.33024/jikk.v12i2.16594 Lestari S, Kurnia D, Mayanti T, Heliawati L (2024) Antimicrobial activities of stigmasterol from Piper crocatum in vitro and in silico. J Chem 2024(1):2935516. https://doi.org/10.1155/2024/2935516 Luo SC, Wei SM, Luo XT, Yang QQ, Wong KH, Cheung PC et al (2024) How probiotics, prebiotics, synbiotics, and postbiotics prevent dental caries: an oral microbiota perspective. NPJ Biofilms Microbiomes 10(1):14. https://doi.org/10.1038/s41522-024-00488-7 Meng X, Cai H, Luo Y, Zhao X, Fu Y, Zou L et al (2024) Biocontrol potential of endophytic Bacillus velezensis LSR7 against rubber red root rot disease. J Fungi 10(12):849. https://doi.org/10.3390/jof10120849 Mesny F, Hacquard S, Thomma BP (2023) Co-evolution within the plant holobiont drives host performance. EMBO Rep 24(9):e57455. https://doi.org/10.15252/embr.202357455 Mishra Y, Singh A, Batra A, Sharma MM (2014) Understanding the biodiversity and biological applications of endophytic fungi: a review. J Microb Biochem Technol S 8(2). http://dx.doi.org/10.4172/1948-5948.S8-004 Mousa WK, Raizada MN (2013) The diversity of anti-microbial secondary metabolites produced by fungal endophytes: an interdisciplinary perspective. Front Microbiol 4:65. https://doi.org/10.3389/fmicb.2013.00065 Nair DN, Padmavathy S (2014) Impact of endophytic microorganisms on plants, environment and humans. Sci World J 2014:250693. https://doi.org/10.1155/2014/250693 Navia ZI, Adnan A, Harmawan T, Suwardi AB (2022) Ethnobotanical study of wild medicinal plants in Serbajadi protected forest of East Aceh District, Indonesia. Biodiversitas J Biol Divers 23(10):10. https://doi.org/10.13057/biodiv/d231001 Rana KL, Kour D, Sheikh I, Yadav N, Yadav A, Kumar V et al (2019) Biodiversity of endophytic fungi from diverse niches and their biotechnological applications. Advances in Endophytic Fungal Research: Present Status and Future Challenges. Springer, Cham, pp 105–144. https://doi.org/10.1007/978-3-030-03589-1_6 Schafer P (2011) The Chinese Medicinal Herb Farm: A Cultivator's Guide to Small-scale Organic Herb Production. Chelsea Green Publishing, White River Junction Shukla S, Patra D, Sinha A, Saha R, Tripathi Y, Borah NK et al (2025) Medicinal and aromatic plant cultivation, improvement and conservation for sustainable development. Industrial Crops Improvement: Biotechnological Approaches for Sustainable Agricultural Development. Springer Nature Switzerland, Cham, pp 183–204. https://doi.org/10.1007/978-3-031-75937-6_11 Silalahi M, Walujo EB, Supriatna J, Mangunwardoyo W (2015) The local knowledge of medicinal plants trader and diversity of medicinal plants in the Kabanjahe traditional market, North Sumatra, Indonesia. J Ethnopharmacol 175:432–443. https://doi.org/10.1016/j.jep.2015.09.009 Siswina T, Rustama MM, Sumiarsa D, Apriyanti E, Dohi H, Kurnia D (2023) Antifungal constituents of Piper crocatum and their activities as ergosterol biosynthesis inhibitors discovered via in silico study using ADMET and drug-likeness analysis. Molecules 28(23):7705. https://doi.org/10.3390/molecules28237705 Sukmarini L, Atikana A, Hertiani T (2024) Antibiofilm activity of marine microbial natural products: potential peptide-and polyketide-derived molecules from marine microbes toward targeting biofilm-forming pathogens. J Nat Med 78(1):1–20. https://doi.org/10.1007/s11418-023-01754-2 Viret O (1993) Infection of beech leaves by the endophyte Discula umbrinella (Berk. & Br.) Morelet teleomorph: Apiognomonia errabunda (Rob.) Höhnel: an ultrastructural study [dissertation]. ETH Zurich, Zurich Yan L, Zhu J, Zhao X, Shi J, Jiang C, Shao D (2019) Beneficial effects of endophytic fungi colonization on plants. Appl Microbiol Biotechnol 103(8):3327–3340. https://doi.org/10.1007/s00253-019-09713-2 Yu G, Smith DK, Zhu H, Guan Y, Lam TTY (2017) ggtree: an R package for visualization and annotation of phylogenetic trees with their covariates and other associated data. Methods Ecol Evol 8(1):28–36. https://doi.org/10.1111/2041-210X.12628 Yuan ZL, Zhang CL, Lin FC, Kubicek CP (2010) Identity, diversity, and molecular phylogeny of the endophytic mycobiota in the roots of rare wild rice ( Oryza granulate ) from a nature reserve in Yunnan, China. Appl Environ Microbiol 76(5):1642–1652. https://doi.org/10.1128/AEM.01911-09 Additional Declarations No competing interests reported. Supplementary Files AdditionalFiles1.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8006293","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":539611852,"identity":"86fcce08-d534-45a2-94e7-ce63a2aa64e4","order_by":0,"name":"Syifa Zahara Kultsum Azmi","email":"","orcid":"","institution":"Padjadjaran University","correspondingAuthor":false,"prefix":"","firstName":"Syifa","middleName":"Zahara Kultsum","lastName":"Azmi","suffix":""},{"id":539611854,"identity":"5bda2b7b-24b9-4368-b124-0da2d09d42f7","order_by":1,"name":"Dikdik Kurnia","email":"","orcid":"","institution":"Padjadjaran University","correspondingAuthor":false,"prefix":"","firstName":"Dikdik","middleName":"","lastName":"Kurnia","suffix":""},{"id":539611855,"identity":"132659d7-5cad-4cc5-8daf-42c442cd11fd","order_by":2,"name":"Rianti Nurpalah","email":"","orcid":"","institution":"Univesitas Bakti Tunas Husada","correspondingAuthor":false,"prefix":"","firstName":"Rianti","middleName":"","lastName":"Nurpalah","suffix":""},{"id":539611860,"identity":"f461d395-b909-4fc1-9eeb-987272785aff","order_by":3,"name":"Dewi Peti Virgianti","email":"","orcid":"","institution":"Univesitas Bakti Tunas Husada","correspondingAuthor":false,"prefix":"","firstName":"Dewi","middleName":"Peti","lastName":"Virgianti","suffix":""},{"id":539611863,"identity":"288113ad-5b56-48eb-b5f9-8becc12fefc2","order_by":4,"name":"Toto Subroto","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIiWNgGAWjYJACZjDJ3oBgH+DBo5wHqkyCgecAyVokEhBaGPBpsWdgfyZduMemjl/yjdnjghprBv72A4wH3uC1hcdMesazNAnJ2TnmxjOOpTNInElgODgHvxY2aZ4DhyUMbueYSfOwHWZguMHAcBi/X4AOA2u5eQao5d9hBnnCWhjMIFpuAF3I23aYwYCglsM8xtYzDqRJzuxJK5Oe2ZfOY3gmsQGvX9jb2x/eLjhgw8/PfnibdME3azm544cPf8AXYrCIgLOBTmJswKcBt/ZRMApGwSgYBXAAALrwQXUqnWWlAAAAAElFTkSuQmCC","orcid":"","institution":"Padjadjaran University","correspondingAuthor":true,"prefix":"","firstName":"Toto","middleName":"","lastName":"Subroto","suffix":""}],"badges":[],"createdAt":"2025-11-01 14:23:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8006293/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8006293/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":95228327,"identity":"e047934b-954b-49fe-bb41-6ff5999826a0","added_by":"auto","created_at":"2025-11-05 16:33:37","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":8202762,"visible":true,"origin":"","legend":"","description":"","filename":"ResearchArticleSpringerWorldJournalofMicrobiologyandBiotechnology.docx","url":"https://assets-eu.researchsquare.com/files/rs-8006293/v1/e6281d9a3d0c7bd37db3e02d.docx"},{"id":95212361,"identity":"bba9efce-f557-4a9b-b40f-c59cbcf1be60","added_by":"auto","created_at":"2025-11-05 14:30:48","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":8044,"visible":true,"origin":"","legend":"","description":"","filename":"c2474a95d0c84eac90fe91aeecac4db5.json","url":"https://assets-eu.researchsquare.com/files/rs-8006293/v1/aa53b10a5c4c03a200253f81.json"},{"id":95227779,"identity":"2b70a6f5-90a8-4f6e-819d-48dc1f7215a7","added_by":"auto","created_at":"2025-11-05 16:32:56","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":15905,"visible":true,"origin":"","legend":"","description":"","filename":"AdditionalFiles1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8006293/v1/1d0670eb9a86162bcc832851.xlsx"},{"id":95228940,"identity":"f5afb1f0-fa67-4294-830d-436ca35bfd69","added_by":"auto","created_at":"2025-11-05 16:34:17","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":116254,"visible":true,"origin":"","legend":"","description":"","filename":"c2474a95d0c84eac90fe91aeecac4db51enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8006293/v1/0229c72011578ccbf750ad0c.xml"},{"id":95228354,"identity":"d6965c9d-455c-484b-8727-fe5fb2dd0e42","added_by":"auto","created_at":"2025-11-05 16:33:39","extension":"jpeg","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":296543,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8006293/v1/e6405745821189f0e3f6f895.jpeg"},{"id":95212373,"identity":"892fcea3-4e37-42e3-85e8-d99f184cc955","added_by":"auto","created_at":"2025-11-05 14:30:48","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":268625,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-8006293/v1/fcf09029897a3dc0018c5cf8.png"},{"id":95227799,"identity":"60bb9691-6398-4b5a-9d31-4bba7ba0bf8b","added_by":"auto","created_at":"2025-11-05 16:32:58","extension":"png","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":376416,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-8006293/v1/5ac15e88d1d32997f4d1dcb1.png"},{"id":95212380,"identity":"83b859f7-48e8-489d-9c90-89583acf8773","added_by":"auto","created_at":"2025-11-05 14:30:48","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2118933,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8006293/v1/25432e898afbc799d6d4f794.png"},{"id":95212377,"identity":"f7aa2f40-c6c5-494c-be6e-207403482d1d","added_by":"auto","created_at":"2025-11-05 14:30:48","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":118319,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8006293/v1/ce1a1a315abe3b3d90fbc6cf.png"},{"id":95212387,"identity":"9c3626be-95fa-499b-91eb-57c3382f646f","added_by":"auto","created_at":"2025-11-05 14:30:48","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":102665,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8006293/v1/01ebd99c104561521ed18642.png"},{"id":95228532,"identity":"17f4330f-1c6c-43ae-9f02-b9e3c18ed1fa","added_by":"auto","created_at":"2025-11-05 16:33:53","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":8203,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8006293/v1/72f16c2b86a847f6068782a4.png"},{"id":95228253,"identity":"20add8f4-9486-43cc-b53b-8eab1172362f","added_by":"auto","created_at":"2025-11-05 16:33:32","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1328465,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8006293/v1/6d34ed4f99fa572c3940b0f4.png"},{"id":95228508,"identity":"54b09b8a-3717-4270-8b6b-57893fd46b39","added_by":"auto","created_at":"2025-11-05 16:33:50","extension":"jpeg","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1084482,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8006293/v1/5f848b53f9aafece61f768c7.jpeg"},{"id":95228998,"identity":"a018e8bb-bd2d-4328-a4e0-a8c4ff39644e","added_by":"auto","created_at":"2025-11-05 16:34:20","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":975132,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8006293/v1/1c6f1596f942fb215e4a6c74.png"},{"id":95228564,"identity":"b3f0bb4e-c9d3-447b-b18d-b5e45c43ea52","added_by":"auto","created_at":"2025-11-05 16:33:55","extension":"jpeg","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1619935,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8006293/v1/70bc7ade6562133761912bec.jpeg"},{"id":95212382,"identity":"4553d299-380d-45d3-bd37-6964fbd69391","added_by":"auto","created_at":"2025-11-05 14:30:48","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":126349,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8006293/v1/c4e0b9c3a2b7a4bbd92b42b8.png"},{"id":95212379,"identity":"fee23e42-6944-4f01-aec8-e839a8be62d5","added_by":"auto","created_at":"2025-11-05 14:30:48","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":23835,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-8006293/v1/f511a61271f1ee85e845a2d8.png"},{"id":95212388,"identity":"45b16688-99fe-4de6-a55c-c423775f3fe6","added_by":"auto","created_at":"2025-11-05 14:30:48","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":71353,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-8006293/v1/c98bf28a4ea7cddcd903094f.png"},{"id":95229209,"identity":"228e6974-48eb-4b3c-bea3-a6b97091362d","added_by":"auto","created_at":"2025-11-05 16:34:36","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":355684,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8006293/v1/d2659b50149a39ad18348e50.png"},{"id":95212385,"identity":"1b67e122-858d-4528-a80e-41f14614fe6f","added_by":"auto","created_at":"2025-11-05 14:30:48","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":23151,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8006293/v1/9790a9664a66fdd31fb8331e.png"},{"id":95228368,"identity":"2c7107a4-d195-423a-b25a-1e0c43a1fc56","added_by":"auto","created_at":"2025-11-05 16:33:40","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":21429,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8006293/v1/f507d3a60a60514494540e61.png"},{"id":95212395,"identity":"cfa36ba0-9efb-43ea-9c63-0017d876d681","added_by":"auto","created_at":"2025-11-05 14:30:49","extension":"png","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":7311,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8006293/v1/2442fdaaaa048826d757a53c.png"},{"id":95212394,"identity":"e78f5a52-d2f5-474b-bb7f-b790f210c441","added_by":"auto","created_at":"2025-11-05 14:30:49","extension":"png","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":126506,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8006293/v1/17821b81a2a52dd452b76305.png"},{"id":95212389,"identity":"35da2dac-f659-412b-b4a7-fe8aba770608","added_by":"auto","created_at":"2025-11-05 14:30:48","extension":"png","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":244622,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8006293/v1/be72d779c69e7ea7595ad71f.png"},{"id":95228786,"identity":"06ae2457-b9a1-435e-a8d4-67813e9b0094","added_by":"auto","created_at":"2025-11-05 16:34:08","extension":"png","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":104655,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8006293/v1/3bb6b49053dc326863217076.png"},{"id":95212391,"identity":"175218d0-2d77-49ca-a181-61f5eee98d86","added_by":"auto","created_at":"2025-11-05 14:30:48","extension":"png","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":338173,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8006293/v1/2c74766463e0b15731fdd1fa.png"},{"id":95212392,"identity":"85181029-ae2d-4eb5-94be-d781441f4b07","added_by":"auto","created_at":"2025-11-05 14:30:48","extension":"xml","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":112947,"visible":true,"origin":"","legend":"","description":"","filename":"c2474a95d0c84eac90fe91aeecac4db51structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8006293/v1/f462aa42d1e4643f883bd34e.xml"},{"id":95212398,"identity":"b8631fd2-d759-46dc-bce1-2fb9263e0653","added_by":"auto","created_at":"2025-11-05 14:30:49","extension":"html","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":125261,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8006293/v1/ffe78dee9b5a36a08a762dd6.html"},{"id":95212358,"identity":"58148b31-74ce-471d-ba4b-d364ea80f249","added_by":"auto","created_at":"2025-11-05 14:30:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":333091,"visible":true,"origin":"","legend":"\u003cp\u003eGeographic distribution of \u003cem\u003ePiper crocatum\u003c/em\u003e Ruiz \u0026amp; Pav. (red betel) sampling sites across Tasikmalaya Regency, West Java, Indonesia.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8006293/v1/0a886fb3f15f065c9d2ee80f.png"},{"id":95212362,"identity":"b0f236f9-1642-4b4c-8679-b4cdcddda9c5","added_by":"auto","created_at":"2025-11-05 14:30:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":457402,"visible":true,"origin":"","legend":"\u003cp\u003eMacromorphological diversity of endophytic fungi isolated from \u003cem\u003ePiper crocatum\u003c/em\u003e based on colony morphology on SDA medium.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8006293/v1/a113dfb1326301b3b1739651.png"},{"id":95227776,"identity":"f123c2d2-f8fd-4b0d-bbf0-51011e9e2b98","added_by":"auto","created_at":"2025-11-05 16:32:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":48703,"visible":true,"origin":"","legend":"\u003cp\u003eUPGMA phenogram of 66 endophytic fungal isolates from\u003cem\u003ePiper crocatum\u003c/em\u003e based on 33 morphological characters, with bootstrap values indicated at major nodes.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8006293/v1/83d60bba47c3508f2699b828.png"},{"id":95229242,"identity":"19016da0-cb63-46e2-81d9-edf11caf6dc1","added_by":"auto","created_at":"2025-11-05 16:34:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":38307,"visible":true,"origin":"","legend":"\u003cp\u003ePCA biplot depicting variance among ten endophytic fungal isolates\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8006293/v1/bcb963c0b6bc85b9cffcc745.png"},{"id":95212364,"identity":"edca6629-a57a-48d6-b8ee-7cf0c3b01800","added_by":"auto","created_at":"2025-11-05 14:30:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":29345,"visible":true,"origin":"","legend":"\u003cp\u003ePairwise Jaccard similarity heatmap depicting phenotypic relationships among endophytic fungi\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8006293/v1/02329cf73b46c4e878f8a28c.png"},{"id":95212366,"identity":"3b304842-11d0-4401-8365-2834547256ed","added_by":"auto","created_at":"2025-11-05 14:30:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":283595,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative Endophytic Fungal Colonies Corresponding to Clusters in the Jaccard-Based Phenogram\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8006293/v1/5f99b0dcb9061efeb9357ce7.png"},{"id":95212369,"identity":"4bcfd1ca-9d7a-4198-9240-2b3bfc69b380","added_by":"auto","created_at":"2025-11-05 14:30:48","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":503891,"visible":true,"origin":"","legend":"\u003cp\u003eTLC fingerprints of secondary metabolites from ten endophytic fungal isolates visualized under visible and UV illumination\u003c/p\u003e\n\u003cp\u003e(A) Separation on silica gel 60 F₂₅₄ normal-phase plates using n-hexane:ethyl acetate (7:3, v/v) as the mobile phase. (B) Separation on silica gel reverse phase R-18 60 F₂₅₄ plates using methanol:distilled water (7:3, v/v) as the mobile phase. Spots were visualized under visible light, UV 254 nm, and UV 365 nm to highlight variations in metabolite composition and chemical diversity among isolates (lanes 1–10).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8006293/v1/1b4bf5373948cc618a51f254.png"},{"id":95212393,"identity":"10bc89fd-2a75-4488-849b-5e4ecdbb44e9","added_by":"auto","created_at":"2025-11-05 14:30:48","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":291386,"visible":true,"origin":"","legend":"\u003cp\u003eScreening of endophytic fungal extracts for anticariogenic activity against S. mutans via agar disc diffusion assay\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8006293/v1/5c5b36ef1db8828e39705229.png"},{"id":95212378,"identity":"f2b28b6d-d8a0-43cb-9c9b-5337df29219d","added_by":"auto","created_at":"2025-11-05 14:30:48","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":390285,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological description of 4 (four) potent fungal samples\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-8006293/v1/d924ae59c40b3625913bab96.png"},{"id":95212371,"identity":"c8e2be7d-715d-445e-920f-e728e03efbfe","added_by":"auto","created_at":"2025-11-05 14:30:48","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":95791,"visible":true,"origin":"","legend":"\u003cp\u003eAgarose gel electrophoresis of PCR products showing amplification of the ITS region in selected endophytic fungi\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-8006293/v1/feb5ecb0e9167d7c479b9810.png"},{"id":95228621,"identity":"33fb3ebf-303e-4ec9-9cf6-84aef5d0ed38","added_by":"auto","created_at":"2025-11-05 16:34:00","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":215396,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular phylogeny of endophytic fungi from \u003cem\u003ePiper crocatum\u003c/em\u003e based on ITS (1-4) rDNA sequences reveals distinct taxonomic clusters\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-8006293/v1/187baab2ff4deaf5b540aaf2.png"},{"id":95312167,"identity":"df619e71-a24c-46c1-ac19-1151204c487c","added_by":"auto","created_at":"2025-11-06 15:47:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4040400,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8006293/v1/879d7779-6f01-4ad3-bd7e-3869edc05234.pdf"},{"id":95212359,"identity":"e30c8d15-ad03-470a-8afe-b35c68754997","added_by":"auto","created_at":"2025-11-05 14:30:48","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":15905,"visible":true,"origin":"","legend":"","description":"","filename":"AdditionalFiles1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8006293/v1/169192b2ba00607125cd708b.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Identification of Endophytic Fungi from Red Betel (Piper crocatum Ruiz \u0026 Pav.): Exploration of Anticariogenic Bioactive Against Oral Pathogens","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eIndonesia is globally recognized as one of the world\u0026rsquo;s megadiverse country, harboring extraordinary biological wealth. The archipelago is home to more than 30,000 plant species, of which approximately 7,000 are known to possess medicinal properties (Navia et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This exceptional floristic diversity is paralleled by a wide spectrum of chemotaxonomic profiles, offering an immense repository for the discovery of novel bioactive compounds with potential pharmaceutical applications (Silalahi et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Such biodiversity-driven chemical diversity provides a strategic advantage for bioprospecting and drug discovery, especially in the search for functional metabolites that can address pressing global health challenges. However, direct exploitation of medicinal plants for large-scale production presents considerable challenges (Astutik et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Cultivation and harvesting of these plants often require long growth cycles, are susceptible to environmental fluctuations, and demand resource-intensive agronomic adjustments (Schafer et al. 2019; Shukla et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Consequently, scalable production of plant-derived bioactives remains constrained by both biological and logistical limitations.\u003c/p\u003e\u003cp\u003eAn alternative and increasingly attractive avenue lies in endophytic fungi, microorganisms that colonize the internal tissues of plants without causing apparent harm (Shukla et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Nair and Padmavathy \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Yan et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Endophytes are hypothesized to have co-evolved and exchanged genetic material with their host plants through long-term mutualistic associations (Mesny et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This intimate relationship may involve horizontal gene transfer, metabolic pathway convergence, and co-regulation of biosynthetic gene clusters (Hiruma et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). As a result, endophytic fungi are capable of synthesizing secondary metabolites structurally and functionally analogous to those produced by their hosts, and in some cases, generating novel compounds with enhanced bioactivity (Hashem et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Despite this promising potential, the diversity and biotechnological value of many endophytes, particularly those from medicinal plants, remain underexplored (Hashem et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Hiruma et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAmong Indonesia\u0026rsquo;s medicinal flora, red betel leaf (\u003cem\u003ePiper crocatum\u003c/em\u003e Ruiz \u0026amp; Pav.) holds a prominent position in ethnobotanical and ethnopharmacological traditions. This species has been widely used for its antimicrobial, anti-inflammatory, and antioxidant properties (Hashem et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Heliawati et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Lestari et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Recent studies have demonstrated that extracts and isolated compounds from red betel exhibit significant inhibitory effects against cariogenic bacteria in both in vitro and clinical settings (Kurnia et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Siswina et al. 2024). Given these attributes, exploring the endophytic fungi residing in the mesophyll tissues of red betel represents a promising strategy to discover bioactive metabolites capable of modulating microbial communities, particularly those associated with oral diseases.\u003c/p\u003e\u003cp\u003eDental caries remains a major public health burden in Indonesia, affecting approximately 8 out of 10 adults in the productive age group, with the mean of DMF-T (Decayed, Missing, and Filled Teeth) index of 5.0 (Kementerian Kesehatan Republik Indonesia \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Lestari et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The prevalence continues to rise, increasing the risk of edentulism in later age. Current preventive measures are largely inadequate (Borg-Bartolo et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and conventional treatments focus predominantly on restorative interventions rather than proactive microbial modulation (Hitch et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Therefore, a holistic prevention strategy that targets the ecological balance of oral microbiota, rather than solely treating the clinical manifestation of caries, is urgently required (Luo et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe present study aims to characterize the diversity of endophytic fungi isolated from red betel leaves, screen their bioactivity against cariogenic microbes, and isolate active secondary metabolites with potential application as microbial colony modulators in the context of cariogenic consortia.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"2 Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Collection of Plant Material and Selection Criteria\u003c/h2\u003e\u003cp\u003ePathogen-free leaves of \u003cem\u003ePiper crocatum\u003c/em\u003e Ruiz \u0026amp; Pav. (red betel) were selected as the primary source of observation in this study. The leaves were screened visually to ensure the absence of necrosis, chlorosis, or any other visible signs of pathogenic infection (Viret \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). Sampling was conducted from twelve distinct locations across the Tasikmalaya region, West Java, Indonesia, to capture a broad representation of the plant\u0026rsquo;s endophytic fungal diversity. The distribution map of the sampling sites was generated using ArcGIS 10.8 (Esri, Redlands, CA, USA). Administrative boundary layers (province, district, and subdistrict levels) were obtained from the official Rupa Bumi Indonesia (RBI) dataset provided by the Geospatial Information Agency of Indonesia (Badan Informasi Geospasial, BIG). The geographic coordinates of the sampling plants were recorded in the field using a handheld Global Positioning System (GPS) device and compiled into a comma-separated values (CSV) file containing latitude, longitude, and sample ID. The map was constructed using the Universal Transverse Mercator (UTM) projection, WGS 1984 datum, Zone 48S. Sampling points were visualized using distinct symbols and colors to differentiate each sample code (SM1001\u0026ndash;SM1013). The administrative boundaries were displayed as polygon layers with contrasting outlines for clarity. Map elements including a north arrow, scale bar, and legend were added to improve readability.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Surface Sterilization and Tissue Preparation\u003c/h2\u003e\u003cp\u003eAll collected samples were placed in sterile petri dishes, transported under cooled conditions, and processed within 24 hours to minimize changes in the endogenous microbial community. Leaves were rinsed under running tap water to remove debris, followed by immersion in 70% (v/v) ethanol for 1 min, 1.3 mol/L sodium hypochlorite (NaOCl) for 3 min, and rinsed three times with sterile distilled water (Meng et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The abaxial surface of each leaf was oriented upward (inverted position), and a longitudinal medial section was excised along the midrib (primary vein) using a sterile scalpel blade (no. 11). Sections were prepared with 2.5 mm spacing on either side of the midrib, resulting in a 5 mm-wide central strip. This strip was then transversely subdivided into three segments, each measuring 2.5 mm in length, to obtain explants from mesophyll tissue.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Isolation of Endophytic Fungi\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eEndophytic fungal isolation was carried out using a direct planting method. Three leaf explants were aseptically placed on the periphery of solidified Sabouraud Dextrose Agar (SDA) medium supplemented with chloramphenicol (0.4 \u0026micro;g/mL) (Achraya and Hare 2022) to suppress bacterial growth. Explants were positioned equiangularly at an estimated center-to-center distance of 2\u0026ndash;3 cm to avoid colony interference. Plates were incubated aerobically at 37\u0026deg;C for 48 h. Emergent fungal colonies from the mesophyll tissue were subcultured repeatedly onto fresh SDA plates until pure single-spore isolates were obtained.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Phenetic Characterization and Cluster Analysis\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003ePhenetic characterization was performed on each purified isolate based on 33 macromorphological traits of colony morphology (Crous et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), including both upper and reverse colony characteristics. Each character state was recorded in binary format (presence\u0026thinsp;=\u0026thinsp;1, absence\u0026thinsp;=\u0026thinsp;0) and compiled into a binary data matrix. Similarity analysis was conducted using the Jaccard coefficient via the UPGMA clustering tool available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://genomes.urv.cat/UPGMA/\u003c/span\u003e\u003cspan address=\"http://genomes.urv.cat/UPGMA/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. The resulting Newick-formatted (.nwk) dendrogram file was visualized in RStudio (2025.05.1\u0026ndash;513) using the \u003cem\u003eape\u003c/em\u003e and \u003cem\u003eggtree\u003c/em\u003e packages to generate a phenetic dendrogram (Yu et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). One representative isolate from each major cluster was selected for bioactivity screening, with priority given to isolates producing visible extracellular exudates.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Preliminary Bioactivity Screening\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eSelected isolates were rejuvenated on SDA plates and subsequently inoculated into red rice (\u003cem\u003eOryza sativa\u003c/em\u003e var. andel abang) as a substrate for small-scale solid-state fermentation (El-Sayed et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Fermentation was conducted under aerobic conditions for 28 days at ambient temperature. The fermented substrate was extracted by maceration in methanol (1:3 v/v) for three days with occasional agitation, followed by filtration. The filtrate was concentrated under reduced pressure, and crude extracts were subjected into two preliminary assays: Kirby\u0026ndash;Bauer disk diffusion assay against cariogenic bacteria (\u003cem\u003eStreptococcus mutans\u003c/em\u003e) with 2% chlorhexidine as a positive control; chemical profiling by thin-layer chromatography (TLC) using silica gel F\u003csub\u003e254\u003c/sub\u003e and octadecylsilyl (ODS) stationary phases under UV light (254 and 365 nm).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Molecular Identification\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eFor molecular identification, genomic DNA was extracted using the Quick-DNA Magbead Plus Kit (Zymo Research, D4082) following the internal protocol. The internal transcribed spacer (ITS1-4) region was amplified with MyTaq HS Red Mix, 2X (Bioline, BIO-25048), and PCR products were verified by using agarose gel electrophoresis. Bidirectional Sanger sequencing was performed via capillary electrophoresis, and raw sequence data were assembled and analyzed using an in-house bioinformatics pipeline. Sequencing data were used for BLAST searches against the NCBI GenBank database to determine species-level identity and infer phylogenetic relationships (Yuan et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Sampling Map Distribution\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe distribution map illustrates the spatial variation of \u003cem\u003ePiper crocatum\u003c/em\u003e (red betel) populations sampled across Tasikmalaya Regency, West Java (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). A total of 13 accessions (SM1001\u0026ndash;SM2013) were collected, representing diverse subdistricts ranging from lowland to upland areas. The sampling sites cover heterogeneous ecological zones, which are likely to influence the phytochemical diversity and bioactive metabolite profiles of the plants. Spatial heterogeneity is particularly important in medicinal plants, as environmental conditions such as altitude, soil composition, and microclimatic factors can modulate secondary metabolite biosynthesis. This variability provides a strong foundation for bioactivity-guided selection, as distinct populations may yield differential antimicrobial or antibiofilm potentials against oral pathogens. The broad geographic coverage also ensures that the sampling design captures both ecological and chemotypic diversity, thus increasing the reliability of subsequent biological and chemical analyses (Huang et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Mishra et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Rana et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Phenotypic Characterization and Clustering of Endophytic Fungi\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eA total of 66 endophytic fungal isolates obtained from \u003cem\u003ePiper crocatum\u003c/em\u003e exhibited substantial morphological diversity, as reflected by variations in colony pigmentation, texture, margin configuration, elevation, and growth rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Such heterogeneity is typical of endophyte communities and underscores their underlying taxonomic and ecological diversity. Comparable morphological differentiation has been reported in endophytes associated with other medicinal plants, reinforcing the notion that \u003cem\u003eP. crocatum\u003c/em\u003e represents a rich reservoir of unexplored microbial taxa.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eTo resolve relationships among isolates, 33 macromorphological traits (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) were subjected to multivariate analyses. Cluster analysis using the UPGMA method (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) partitioned the isolates into several well-defined groups with moderate to high bootstrap support (70\u0026ndash;128). The presence of strongly supported clades (bootstrap\u0026thinsp;\u0026ge;\u0026thinsp;100) suggests that certain colony features\u0026mdash;particularly pigmentation, texture, and margin structure, can serve as indicators of taxonomic proximity, although other traits may be influenced by culture conditions (Grant and Kluge \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMaterial Samples of \u003cem\u003ePiper crocatum\u003c/em\u003e leaf\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCode\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eScientific Name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMethod of Collection\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSpecimen Source\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eDistribution\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSM1001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"12\" rowspan=\"13\"\u003e\u003cp\u003e\u003cem\u003ePiper crocatum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"12\" rowspan=\"13\"\u003e\u003cp\u003eDirect sampling and observation from living specimen\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCicurug, Tasikmalaya\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"12\" rowspan=\"13\"\u003e\u003cp\u003eCommon throughout in Indonesia (Kendari, Bogor, Bandung, Malang, Jayapura, Samarinda, and Banda Aceh)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSM1002\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eManonjaya, Tasikmalaya\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSM1003\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePadakembang, Tasikmalaya\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSM1004\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSukaratu, Tasikmalaya\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSM1005\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSingaparna, Tasikmalaya\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSM1006\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLanud, Tasikmalaya\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSM2007\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSingajaya, Tasikmalaya\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSM2008\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eKubangsari, Tasikmalaya\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSM2009\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSetiaratu, Tasikmalaya\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSM2010\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCibeureum, Tasikmalaya\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSM2011\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCisalak, Tasikmalaya\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSM2012\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMandalajaya, Tasikmalaya\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSM2013\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTamansari, Tasikmalaya\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMorphological character of endophytic fungi isolated from \u003cem\u003ePiper crocatum\u003c/em\u003e leaf\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e\u003cp\u003eMorphological Characters\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e\u003cp\u003eColony Color (Upper and Reverse Surface)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWUC-P\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWhite upper colony\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1. present, 0. absent\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWUC-H\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWhite upper colony\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1. present, 0. absent\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWUC-O\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBlack upper colony\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1. present, 0. absent\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWUC-U\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOrange upper colony\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1. present, 0. absent\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWRC-P\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePurple upper colony\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1. present, 0. absent\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWRC-H\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWhite reverse colony\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1. present, 0. absent\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWRC-O\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBlack reverse colony\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1. present, 0. absent\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWRC-U\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOrange reverse colony\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1. present, 0. Absent\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTexture (Surface Colony)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTK-B\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eVelvety texture\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1. present, 0. absent\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTK-H\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSmooth texture\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1. present, 0. absent\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTK-P\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePowdery texture\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1. present, 0. absent\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTK-K\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCottony (floccose) texture\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1. present, 0. absent\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTK-S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRough texture\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1. present, 0. absent\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eMargin (Edge of Colony)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMG-E\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEntire margin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1. present, 0. absent\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMG-U\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eUndulate margin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1. present, 0. absent\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMG-L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLobate margin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1. present, 0. absent\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eElevation (Vertical Profile)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eELE-D\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFlat elevation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1. present, 0. absent\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eELE-C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eConvex elevation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1. present, 0. absent\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eELE-J\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRaised elevation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1. present, 0. absent\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eELE-CK\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eConcave elevation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1. present, 0. absent\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eGrowth Rate\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLJ-C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRapid growth rate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1. (2\u0026ndash;7 days), 0. (7\u0026ndash;14 days)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLJ-L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSlow growth rate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1. (7\u0026ndash;14 days), 0. (2\u0026ndash;7 days)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePigmentation and Exudates\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePEP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eExudate/pigment production\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1. present, 0. absent\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eZB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMedium clearing / transparent halo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1. present, 0. absent\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eConsistency and Texture\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKS-P\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFirm consistency\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1. present, 0. absent\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKS-L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMucilaginous consistency\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1. present, 0. absent\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKS-K\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDry consistency\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1. present, 0. absent\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKS-LB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMoist consistency\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1. present, 0. absent\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eOdor\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAM-T\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEarthy odor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1. present, 0. absent\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAM-A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAlcoholic odor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1. present, 0. absent\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAM-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFermentative odor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1. present, 0. absent\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSpecial Characteristics\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMUC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMulticolored\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1. present, 0. absent\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eZNT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZonate pattern\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1. present, 0. absent\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003ePrincipal component analysis (PCA) clarified phenotypic variation, with the first two principal components (PC1 and PC2) explaining 75% of the cumulative variance. Separation along PC1 was primarily associated with colony color and growth rate, distinguishing fast-growing, pigmented clusters (e.g., t1, t3, t4, t10) from slower-growing, less-pigmented clusters (e.g., t2, t8, t9) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In contrast, PC2 was largely driven by differences in texture and elevation, separating clusters such as t5 and t7 that displayed unique colony surface structures. These results indicate that distinct morphological traits, particularly pigmentation and texture, can serve as reliable markers for preliminary classification prior to molecular identification.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eComplementary insights were obtained from a Jaccard similarity heatmap (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), which summarized binary trait overlap across clusters (t1\u0026ndash;t10). Similarity coefficients ranged from 0.40 to 1.00, revealing both highly conserved groups and divergent profiles. Cluster A (t9, t2, t8) displayed the highest within-group similarity (\u0026gt;\u0026thinsp;0.85), whereas Cluster B (t3, t7, t4, t1) exhibited moderate similarity (0.75\u0026ndash;0.85). Cluster C (t6 and t10) demonstrated an especially close relationship (~\u0026thinsp;0.90), while isolate t5 emerged as an intermediate, bridging distinct groups. This clustering pattern suggests that while subsets of isolates share conserved morphologies, others may represent ecologically or functionally divergent taxa.\u003c/p\u003e\u003cp\u003eIsolates from geographically distant sites were distributed across multiple clusters, implying that host-associated selective pressures, rather than geography, play a dominant role in structuring \u003cem\u003eP. crocatum\u003c/em\u003e endophytic communities. Such findings align with evidence that host plant chemistry exerts a strong influence on endophyte assemblages. Collectively, the integration of morphological characterization, UPGMA clustering, PCA, and Jaccard similarity mapping provides a rational framework for selecting representative isolates. By prioritizing morphologically distinct and phylogenetically robust clusters, the likelihood of capturing metabolite diversity is maximized, thereby enhancing the efficiency of bioactivity-guided screening for novel antimicrobial compounds.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Preliminary Metabolite Profiling and Antimicrobial Screening of Representative Endophytic Fungi\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eOne representative strain from each major cluster was selected for preliminary metabolite screening. This strategy ensured that the functional potential of phylogenetically and morphologically distinct taxa could be captured, thereby increasing the likelihood of identifying novel bioactive metabolites. Ten representative isolates (Samples 1\u0026ndash;10) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) were fermented aerobically on a small scale using sterile red rice as substrate, providing a nutrient-rich and reproducible matrix for metabolite production.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAfter 14 days of incubation, the fermented material was extracted by methanolic maceration, subjected to sonication-assisted lysis, and concentrated using rotary evaporation to obtain crude extracts. Thin-layer chromatography (TLC) analysis revealed marked differences in metabolite banding patterns among the isolates, highlighting the chemical heterogeneity of the endophyte community. Distinct UV-active spots were observed at both 254 nm and 365 nm, with some extracts displaying unique fluorescent bands (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The appearance of isolate-specific chromatographic fingerprints supports the notion that each cluster contributes different metabolic capacities, consistent with previous studies demonstrating that fungal endophytes from medicinal plants produce diverse classes of secondary metabolites, including alkaloids, terpenoids, and polyketides (Mousa and Raizada \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e(A) Separation on silica gel 60 F₂₅₄ normal-phase plates using n-hexane:ethyl acetate (7:3, v/v) as the mobile phase. (B) Separation on silica gel reverse phase R-18 60 F₂₅₄ plates using methanol:distilled water (7:3, v/v) as the mobile phase. Spots were visualized under visible light, UV 254 nm, and UV 365 nm to highlight variations in metabolite composition and chemical diversity among isolates (lanes 1\u0026ndash;10).\u003c/p\u003e\u003cp\u003eIn addition to TLC-based profiling, crude extracts were tested for antimicrobial activity against \u003cem\u003eStreptococcus mutans\u003c/em\u003e, the primary etiological agent of dental caries. Several isolates exhibited growth-inhibitory activity, suggesting the presence of bioactive metabolites with potential anticariogenic properties. Interestingly, isolates originating from clusters characterized by fast-growing, pigmented colonies (e.g., Samples 3 and 9) showed stronger inhibition compared to slow-growing, less pigmented representatives (e.g., Samples 2 and 8). This observation aligns with reports that pigmentation in fungi is frequently associated with the biosynthesis of polyketide-derived metabolites, many of which possess antimicrobial or antibiofilm activity (Sukmarini et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The diversity of TLC patterns and the variable inhibitory activity observed against \u003cem\u003eS. mutans\u003c/em\u003e highlight the untapped potential of \u003cem\u003eP. crocatum\u003c/em\u003e-associated endophytes as sources of novel bioactive compounds.\u003c/p\u003e\u003cp\u003eTo complement phenetical clustering and TLC-based metabolite profiling, the crude extracts of ten representative isolates were subjected to antimicrobial screening against \u003cem\u003eStreptococcus mutans\u003c/em\u003e (ATCC 25175). Clear inhibition zones were observed for several isolates, with marked variability in activity levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Among the tested samples, isolates 1, 4, 7, and 8 exhibited strong growth inhibition, producing mean inhibition zones of 21.5 mm, 11.5 mm, 12.75 mm, and 15.85 mm, respectively. By contrast, the remaining isolates displayed negligible or weak inhibition, with zone diameters\u0026thinsp;\u0026le;\u0026thinsp;6.9 mm.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eWhen these results were integrated with the TLC metabolite fingerprints, a compelling pattern emerged. Isolates demonstrating strong antimicrobial activity (Samples 1, 4, 7, and 8) also exhibited richer chromatographic profiles, characterized by multiple UV-active bands under both 254 nm and 365 nm illumination. In particular, Sample 1 displayed several unique fluorescent spots, suggesting the presence of diverse and potentially bioactive secondary metabolites. This correlation between chemical complexity and bioactivity supports the use of TLC as a rapid, cost-effective proxy for identifying metabolically prolific isolates. The most potent isolate, Sample 1 (code 059) (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e), originated from a cluster defined by slow-growing, pigmented colonies, further reinforcing the notion that pigmentation is linked with metabolite production in endophytic fungi. The alignment of morphological traits, metabolite diversity, and strong bioactivity highlights the robustness of the integrated selection pipeline employed in this study.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eInhibition zone of endophytic fungal extracts for anticariogenic activity against \u003cem\u003eS. mutans\u003c/em\u003e via agar disc diffusion assay (mm).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSample\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eInhibition Zone (mm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ex̄ (mm)\u0026thinsp;\u0026plusmn;\u0026thinsp;Dev. Standard\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e21,5\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e6,5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e6,25\u0026thinsp;\u0026plusmn;\u0026thinsp;1,414214\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e3\u0026thinsp;\u0026plusmn;\u0026thinsp;0,707107\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e11,5\u0026thinsp;\u0026plusmn;\u0026thinsp;0,707107\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e5\u0026thinsp;\u0026plusmn;\u0026thinsp;0,707107\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6,8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e6,9\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e12,5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e12,75\u0026thinsp;\u0026plusmn;\u0026thinsp;2,12132\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003es8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e15,7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e15,85\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e1,5\u0026thinsp;\u0026plusmn;\u0026thinsp;0,141421\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e5,5\u0026thinsp;\u0026plusmn;\u0026thinsp;0,141421\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Molecular Identification of Selected Fungal Isolate\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eMolecular characterization of the four representative isolates (samples 1, 4, 7, and 8) using the ITS1\u0026ndash;4 rDNA region confirmed their taxonomic affiliation with diverse fungal genera. PCR amplification produced clear amplicons within the expected size range (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e), which were subsequently sequenced and compared against 40 reference strains retrieved from NCBI. Phylogenetic analysis demonstrated robust clustering of the isolates with \u003cem\u003eColletotrichum\u003c/em\u003e, \u003cem\u003eTorula\u003c/em\u003e, and \u003cem\u003eAspergillus\u003c/em\u003e lineages, supported by high bootstrap values (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e). Specifically, Isolate 1 (code: 059) grouped with \u003cem\u003eColletotrichum truncatum\u003c/em\u003e, Isolate 3 (code: 062) with \u003cem\u003eColletotrichum cliviae\u003c/em\u003e, Isolate 4 (code: 064) with \u003cem\u003eTorula canangae\u003c/em\u003e, and Isolate 2 (code: 007) with \u003cem\u003eAspergillus rhizopodus\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eThese results provide novel insight into the endophytic mycobiota of \u003cem\u003ePiper crocatum\u003c/em\u003e, a medicinal plant that has not previously been extensively surveyed for fungal associates. While \u003cem\u003eColletotrichum\u003c/em\u003e species are widely recognized as phytopathogens, their role as endophytes producing bioactive metabolites has only recently gained attention. Similarly, \u003cem\u003eTorula\u003c/em\u003e, traditionally understudied in endophyte research, has been increasingly reported as a promising source of antimicrobial and antibiofilm compounds. The recovery of \u003cem\u003eAspergillus rhizopodus\u003c/em\u003e, a rare species with limited documentation in endophytic contexts, further expands the taxonomic spectrum of fungi associated with \u003cem\u003eP. crocatum\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eComparatively, most previous reports of endophytes from medicinal \u003cem\u003ePiper\u003c/em\u003e species have been dominated by \u003cem\u003eFusarium\u003c/em\u003e, \u003cem\u003ePenicillium\u003c/em\u003e, and \u003cem\u003eAspergillus\u003c/em\u003e spp., whereas the detection of \u003cem\u003eTorula canangae\u003c/em\u003e and \u003cem\u003eColletotrichum cliviae\u003c/em\u003e in this study represents, to our knowledge, the first report of these taxa as endophytes in \u003cem\u003eP. crocatum\u003c/em\u003e. This finding highlights the unique ecological niche of \u003cem\u003eP. crocatum\u003c/em\u003e that accommodates both pathogenic and rare fungal taxa, suggesting an evolutionary adaptation that may stimulate the biosynthesis of structurally diverse secondary metabolites. The novelty of this study lies not only in uncovering unexpected fungal diversity within \u003cem\u003eP. crocatum\u003c/em\u003e but also in identifying endophytes with previously unreported associations that hold significant potential for the discovery of anticariogenic compounds. Given the established antimicrobial and quorum-sensing inhibitory properties of \u003cem\u003eColletotrichum\u003c/em\u003e and \u003cem\u003eAspergillus\u003c/em\u003e metabolites, and the emerging relevance of \u003cem\u003eTorula\u003c/em\u003e spp., these isolates represent promising candidates for further exploration in dental and oral health applications.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe identification of bioactive endophytic fungi from \u003cem\u003ePiper crocatum\u003c/em\u003e carries significant clinical implications in the context of oral health in Indonesia, where the prevalence of dental caries remains alarmingly high, affecting more than 88% of the population and ranking among the most common chronic diseases nationwide. Current therapeutic approaches, such as fluoride treatment and conventional antimicrobials, are limited by reduced efficacy against biofilm-embedded pathogens, recurrent infections, and the emerging threat of antimicrobial resistance. The strong inhibitory effects of \u003cem\u003eC. truncatum\u003c/em\u003e, \u003cem\u003eC. cliviae\u003c/em\u003e, \u003cem\u003eT. canangae\u003c/em\u003e, and \u003cem\u003eA. rhizopodus\u003c/em\u003e against \u003cem\u003eS. mutans\u003c/em\u003e observed in this study underscore their potential as alternative sources of anticariogenic agents that could overcome these limitations.\u003c/p\u003e\u003cp\u003eFuture research should prioritize the purification and structural elucidation of the active metabolites responsible for the observed antibiofilm and antimicrobial effects, in order to determine their novelty and mechanism of action. In addition, rigorous evaluation of toxicity and safety profiles is essential to ensure suitability for human application, given that many secondary metabolites of fungal origin can exhibit cytotoxicity. To bridge the gap between in vitro screening and clinical application, further validation using ex vivo oral biofilm models and in vivo systems is warranted, which would provide more realistic insights into pharmacodynamics and therapeutic potential. Ultimately, the integration of endophyte-derived natural products into preventive or adjunctive therapies could represent a transformative approach to reducing the burden of dental caries in Indonesia and beyond.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThis study demonstrated that endophytic fungi isolated from \u003cem\u003eP. crocatum\u003c/em\u003e harbor promising anticariogenic potential against \u003cem\u003eS. mutans\u003c/em\u003e. TLC profiling revealed diverse secondary metabolite patterns across isolates, suggesting chemical heterogeneity and the presence of multiple bioactive constituents. Bioactivity-guided screening confirmed that four isolates (Samples 1, 4, 7, and 8) exhibited strong inhibitory activity, highlighting their potential as candidates for anticariogenic drug discovery. Molecular characterization using ITS1\u0026ndash;4 sequencing identified these bioactive strains as \u003cem\u003eC. truncatum\u003c/em\u003e, \u003cem\u003eC. cliviae\u003c/em\u003e, \u003cem\u003eT. canangae\u003c/em\u003e, and \u003cem\u003eA. rhizopodus\u003c/em\u003e, thereby expanding the current knowledge of endophytic biodiversity within \u003cem\u003ePiper\u003c/em\u003e species.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements.\u0026nbsp;\u003c/strong\u003eThe author wishes to thank the Department of Chemistry, Universitas Padjadjaran, for providing laboratory facilities and technical support. Special appreciation for Department of Pharmacy, Universitas Bakti Tunas Husada is extended for their guidance during the research process.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This research was supported by PMDSU Research Grant, Ministry of Education, Culture, Research, and Technology of Indonesia (Grant no. 093/C3/DT.05.00/PL/2025).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest/Competing interests:\u003c/strong\u003e The authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability:\u003c/strong\u003e All data generated and analyzed during this study are included in this published article and its supplementary files. Additional datasets are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials availability:\u003c/strong\u003e The fungal isolates obtained and characterized in this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability:\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution:\u003c/strong\u003e S.Z.K.A. conceived and designed the study, performed laboratory work. RN provided the research facilities. D.P.V. designed the sampling plot and methods. T.S. conceived the data analysis, and manuscript writing. D.K. provided supervision, critical review, and final approval of the manuscript. All authors have read and approved the final version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAcharya T, Hare J (2022) Sabouraud agar and other fungal growth media. Laboratory Protocols in Fungal Biology: Current Methods in Fungal Biology. Springer International Publishing, Cham, pp 69\u0026ndash;86. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/978-3-030-83749-5_2\u003c/span\u003e\u003cspan address=\"10.1007/978-3-030-83749-5_2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAstutik S, Pretzsch J, Ndzifon Kimengsi J (2019) Asian medicinal plants\u0026rsquo; production and utilization potentials: A review. Sustainability 11(19):5483. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/su11195483\u003c/span\u003e\u003cspan address=\"10.3390/su11195483\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBorg-Bartolo R, Roccuzzo A, Molinero-Mourelle P, Schimmel M, Gambetta-Tessini K, Chaurasia A et al (2022) Global prevalence of edentulism and dental caries in middle-aged and elderly persons: A systematic review and meta-analysis. J Dent 127:104335. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jdent.2022.104335\u003c/span\u003e\u003cspan address=\"10.1016/j.jdent.2022.104335\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCrous PW, Verkley GJ, Groenewald JZ, Samson RA (2009) Fungal biodiversity\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEl-Sayed ESR, Gach J, Olejniczak T, Boratyński F (2022) A new endophyte Monascus ruber SRZ112 as an efficient production platform of natural pigments using agro-industrial wastes. Sci Rep 12(1):12611. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-022-16269-\u003c/span\u003e\u003cspan address=\"10.1038/s41598-022-16269-\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGrant T, Kluge AG (2008) Clade support measures and their adequacy. Cladistics 24(6):1051\u0026ndash;1064. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1096-0031.2008.00231.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1096-0031.2008.00231.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHashem AH, Attia MS, Kandil EK, Fawzi MM, Abdelrahman AS, Khader MS et al (2023) Bioactive compounds and biomedical applications of endophytic fungi: a recent review. Microb Cell Fact 22(1):107. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12934-023-02118-x\u003c/span\u003e\u003cspan address=\"10.1186/s12934-023-02118-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHeliawati L, Lestari S, Hasanah U, Ajiati D, Kurnia D (2022) Phytochemical profile of antibacterial agents from red betel leaf (\u003cem\u003ePiper crocatum\u003c/em\u003e Ruiz and Pav) against bacteria in dental caries. Molecules 27(9):2861. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/molecules27092861\u003c/span\u003e\u003cspan address=\"10.3390/molecules27092861\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHiruma K, Aoki S, Takino J, Higa T, Utami YD, Shiina A et al (2023) A fungal sesquiterpene biosynthesis gene cluster critical for mutualist-pathogen transition in Colletotrichum tofieldiae. Nat Commun 14(1):5288. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41467-023-40867-w\u003c/span\u003e\u003cspan address=\"10.1038/s41467-023-40867-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHitch TC, Hall LJ, Walsh SK, Leventhal GE, Slack E, de Wouters T et al (2022) Microbiome-based interventions to modulate gut ecology and the immune system. Mucosal Immunol 15(6):1095\u0026ndash;1113. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41385-022-00564-1\u003c/span\u003e\u003cspan address=\"10.1038/s41385-022-00564-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHuang WY, Cai YZ, Hyde KD, Corke H, Sun M (2008) Biodiversity of endophytic fungi associated with 29 traditional Chinese medicinal plants. Fungal Divers 33:61\u0026ndash;75. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.scopus.com/inward/record.url?partnerID=x8ErFbHU\u0026amp;eid=2-s2.0\u0026ndash;61849133540\u003c/span\u003e\u003cspan address=\"http://www.scopus.com/inward/record.url?partnerID=x8ErFbHU\u0026amp;eid=2-s2.0\u0026ndash;61849133540\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKementerian Kesehatan Republik Indonesia (2023) SKI 2023 dalam angka [Internet]. Badan Kebijakan Pembangunan Kesehatan, Jakarta. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.kemkes.go.id/article/view/23070300003/ski-2023-dalam-angka.html\u003c/span\u003e\u003cspan address=\"https://www.kemkes.go.id/article/view/23070300003/ski-2023-dalam-angka.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKurnia D, Lestari S, Mayanti T, Gartika M, Nurdin D (2024) Anti-infection of oral microorganisms from herbal medicine of \u003cem\u003ePiper crocatum\u003c/em\u003e Ruiz \u0026amp; Pav. Drug Des Devel Ther 2024:2531-53. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2147/DDDT.S453375\u003c/span\u003e\u003cspan address=\"10.2147/DDDT.S453375\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLestari ADJ, Harokan A, Ekawati D (2025) Analisis kejadian karies gigi pada anak usia 6\u0026ndash;11 tahun di RSUD Lubai Ulu Kabupaten Muara Enim. J Ilmu Kedokteran Kesehatan 12(2):220\u0026ndash;229. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.33024/jikk.v12i2.16594\u003c/span\u003e\u003cspan address=\"10.33024/jikk.v12i2.16594\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLestari S, Kurnia D, Mayanti T, Heliawati L (2024) Antimicrobial activities of stigmasterol from \u003cem\u003ePiper crocatum\u003c/em\u003e in vitro and in silico. J Chem 2024(1):2935516. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1155/2024/2935516\u003c/span\u003e\u003cspan address=\"10.1155/2024/2935516\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLuo SC, Wei SM, Luo XT, Yang QQ, Wong KH, Cheung PC et al (2024) How probiotics, prebiotics, synbiotics, and postbiotics prevent dental caries: an oral microbiota perspective. NPJ Biofilms Microbiomes 10(1):14. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41522-024-00488-7\u003c/span\u003e\u003cspan address=\"10.1038/s41522-024-00488-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMeng X, Cai H, Luo Y, Zhao X, Fu Y, Zou L et al (2024) Biocontrol potential of endophytic Bacillus velezensis LSR7 against rubber red root rot disease. J Fungi 10(12):849. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/jof10120849\u003c/span\u003e\u003cspan address=\"10.3390/jof10120849\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMesny F, Hacquard S, Thomma BP (2023) Co-evolution within the plant holobiont drives host performance. EMBO Rep 24(9):e57455. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.15252/embr.202357455\u003c/span\u003e\u003cspan address=\"10.15252/embr.202357455\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMishra Y, Singh A, Batra A, Sharma MM (2014) Understanding the biodiversity and biological applications of endophytic fungi: a review. J Microb Biochem Technol S 8(2). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.4172/1948-5948.S8-004\u003c/span\u003e\u003cspan address=\"10.4172/1948-5948.S8-004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMousa WK, Raizada MN (2013) The diversity of anti-microbial secondary metabolites produced by fungal endophytes: an interdisciplinary perspective. Front Microbiol 4:65. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fmicb.2013.00065\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2013.00065\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNair DN, Padmavathy S (2014) Impact of endophytic microorganisms on plants, environment and humans. Sci World J 2014:250693. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1155/2014/250693\u003c/span\u003e\u003cspan address=\"10.1155/2014/250693\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNavia ZI, Adnan A, Harmawan T, Suwardi AB (2022) Ethnobotanical study of wild medicinal plants in Serbajadi protected forest of East Aceh District, Indonesia. Biodiversitas J Biol Divers 23(10):10. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.13057/biodiv/d231001\u003c/span\u003e\u003cspan address=\"10.13057/biodiv/d231001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRana KL, Kour D, Sheikh I, Yadav N, Yadav A, Kumar V et al (2019) Biodiversity of endophytic fungi from diverse niches and their biotechnological applications. Advances in Endophytic Fungal Research: Present Status and Future Challenges. Springer, Cham, pp 105\u0026ndash;144. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/978-3-030-03589-1_6\u003c/span\u003e\u003cspan address=\"10.1007/978-3-030-03589-1_6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSchafer P (2011) The Chinese Medicinal Herb Farm: A Cultivator's Guide to Small-scale Organic Herb Production. Chelsea Green Publishing, White River Junction\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShukla S, Patra D, Sinha A, Saha R, Tripathi Y, Borah NK et al (2025) Medicinal and aromatic plant cultivation, improvement and conservation for sustainable development. Industrial Crops Improvement: Biotechnological Approaches for Sustainable Agricultural Development. Springer Nature Switzerland, Cham, pp 183\u0026ndash;204. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/978-3-031-75937-6_11\u003c/span\u003e\u003cspan address=\"10.1007/978-3-031-75937-6_11\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSilalahi M, Walujo EB, Supriatna J, Mangunwardoyo W (2015) The local knowledge of medicinal plants trader and diversity of medicinal plants in the Kabanjahe traditional market, North Sumatra, Indonesia. J Ethnopharmacol 175:432\u0026ndash;443. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jep.2015.09.009\u003c/span\u003e\u003cspan address=\"10.1016/j.jep.2015.09.009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSiswina T, Rustama MM, Sumiarsa D, Apriyanti E, Dohi H, Kurnia D (2023) Antifungal constituents of \u003cem\u003ePiper crocatum\u003c/em\u003e and their activities as ergosterol biosynthesis inhibitors discovered via in silico study using ADMET and drug-likeness analysis. Molecules 28(23):7705. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/molecules28237705\u003c/span\u003e\u003cspan address=\"10.3390/molecules28237705\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSukmarini L, Atikana A, Hertiani T (2024) Antibiofilm activity of marine microbial natural products: potential peptide-and polyketide-derived molecules from marine microbes toward targeting biofilm-forming pathogens. J Nat Med 78(1):1\u0026ndash;20. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11418-023-01754-2\u003c/span\u003e\u003cspan address=\"10.1007/s11418-023-01754-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eViret O (1993) Infection of beech leaves by the endophyte \u003cem\u003eDiscula umbrinella\u003c/em\u003e (Berk. \u0026amp; Br.) Morelet teleomorph: \u003cem\u003eApiognomonia errabunda\u003c/em\u003e (Rob.) H\u0026ouml;hnel: an ultrastructural study [dissertation]. ETH Zurich, Zurich\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYan L, Zhu J, Zhao X, Shi J, Jiang C, Shao D (2019) Beneficial effects of endophytic fungi colonization on plants. Appl Microbiol Biotechnol 103(8):3327\u0026ndash;3340. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00253-019-09713-2\u003c/span\u003e\u003cspan address=\"10.1007/s00253-019-09713-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYu G, Smith DK, Zhu H, Guan Y, Lam TTY (2017) ggtree: an R package for visualization and annotation of phylogenetic trees with their covariates and other associated data. Methods Ecol Evol 8(1):28\u0026ndash;36. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/2041-210X.12628\u003c/span\u003e\u003cspan address=\"10.1111/2041-210X.12628\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYuan ZL, Zhang CL, Lin FC, Kubicek CP (2010) Identity, diversity, and molecular phylogeny of the endophytic mycobiota in the roots of rare wild rice (\u003cem\u003eOryza granulate\u003c/em\u003e) from a nature reserve in Yunnan, China. Appl Environ Microbiol 76(5):1642\u0026ndash;1652. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/AEM.01911-09\u003c/span\u003e\u003cspan address=\"10.1128/AEM.01911-09\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"endophytic fungi, anticariogenic, numerical taxonomy, ITS sequencing","lastPublishedDoi":"10.21203/rs.3.rs-8006293/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8006293/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe 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 \u003cem\u003ePiper crocatum\u003c/em\u003e 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 \u003cem\u003eP. crocatum\u003c/em\u003e 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 (\u003cem\u003eOryza sativa\u003c/em\u003e var. andel abang), followed by methanolic maceration. Antimicrobial activity was tested against \u003cem\u003eStreptococcus mutans\u003c/em\u003e using the disc diffusion (Kirby\u0026ndash;Bauer) method with 2% chlorhexidine as a positive control. Isolates displaying significant activity were further identified via ITS1\u0026ndash;4 rDNA sequencing and phylogenetic analysis. Among the 10 representative isolates, four demonstrated notable inhibitory activity against \u003cem\u003eS. mutans\u003c/em\u003e. Molecular characterization revealed that Isolate 059 clustered with \u003cem\u003eColletotrichum truncatum\u003c/em\u003e, Isolate 062 with \u003cem\u003eColletotrichum cliviae\u003c/em\u003e, Isolate 064 with \u003cem\u003eTorula canangae\u003c/em\u003e, and Isolate 007 with \u003cem\u003eAspergillus rhizopodus\u003c/em\u003e. Phylogenetic analysis confirmed their taxonomic placement with strong bootstrap support. Notably, bioactive derived from Isolate 059 (\u003cem\u003eC. truncatum\u003c/em\u003e) exhibited the highest antimicrobial potential. This study demonstrates that endophytic fungi from \u003cem\u003eP. crocatum\u003c/em\u003e 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, \u003cem\u003eC. truncatum\u003c/em\u003e (Isolate 059) emerges as a compelling candidate for further fractionation, bioactive compound isolation, and development into alternative therapeutics targeting oral infectious diseases.\u003c/p\u003e","manuscriptTitle":"Identification of Endophytic Fungi from Red Betel (Piper crocatum Ruiz \u0026amp; Pav.): Exploration of Anticariogenic Bioactive Against Oral Pathogens","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-05 14:30:41","doi":"10.21203/rs.3.rs-8006293/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fcfc5bdb-dee0-4507-8e7d-d67e1d3e1a65","owner":[],"postedDate":"November 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-05T14:30:43+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-05 14:30:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8006293","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8006293","identity":"rs-8006293","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}