Screening and molecular identification of biosurfactant-producing endophytes from Quillaja lancifolia D. Don

Screening and molecular identification of biosurfactant-producing endophytes from Quillaja lancifolia D. 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Don Fabio Antonio Antonelo, Eliane Zachert, Yve Verônica da Silva Magedans, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8253976/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 Endophytes are widespread microorganisms that colonize plant tissues without causing apparent harm. These organisms possess diverse metabolic repertoires and can be alternatives or complementary sources of high-value bioactive compounds. Saponins stand out due to their surfactant, foaming, and immunoadjuvant properties. Triterpene saponins from Quillaja spp. are used to enhance both humoral and cellular responses in human and veterinary vaccines. Herein, screening and identification of biosurfactant-producing endophytes from Q. lancifolia tissues were carried out. The aim of the study was to contribute to the characterization of endophyte microbial and biochemical diversity in Q. lancifolia . A total of 42 endophytes (22 fungi and 20 bacteria) were isolated from Q. lancifolia plantlets, callus, cell suspensions and seeds, followed by microbial culture, and screening for extracellular foam production. Four fungi isolates were selected as foam-producing strains and were taxonomically identified using ITS-1 DNA sequencing. Foam production tests were performed using extracts from the liquid filtrate of cultures for subsequent triterpene screening by thin layer chromatography. Select extracts were purified and concentrated by solid phase extraction. Antibacterial activity was screened through agar diffusion and microdilution tests. Diplodia sp., Acremonium sp., Fusarium sp. and Aureobasidium sp. were identified as natural foam-producing endophytes. Liquid chromatography-mass spectrometry analyses revealed major differences between plant and fungi purified fractions. This work constitutes the first report of endophytic microorganisms associated with Q. lancifolia . It not only provides insights on the metabolic potential of these beings but also buttresses future investigations on their biotechnological applications. Biotechnology and Bioengineering Mycology Plant Physiology and Morphology bioprospecting specialized metabolites microorganisms foam production plant endophytes Figures Figure 1 Figure 2 Figure 3 Figure 4 Highlights • This is the first report of endophytic microorganisms in • A total of 42 cultured endophytes were screened for extracellular foam production • sp., sp., sp. and sp. yielded foam • Fungal biosurfactants differed markedly from those of the plant host Introduction Quillaja lancifolia D. Don, synonymous with Quillaja brasiliensis (A.St.-Hil. & Tul.) Mart., is a tree species indigenous to Brazil, Uruguay, and Argentina. It is known as a natural source of triterpene saponins that can be used in the production of pharmaceuticals, food, and cosmetics (Luebert, 2014; Fleck et al. 2019 ). Like the stem bark of Q. saponaria Molina, the leaves of Q. lancifolia produce triterpene saponins derived from quillaic acid, known for their notable immunoadjuvant properties (De Costa et al. 2011 ). Mechanistically, Quillaja spp. saponins enhance and prolong cellular and humoral immune responses as adjuvants in various vaccines, including Polio, Zika, SARS-CoV-2, and Influenza viruses (De Costa et al. 2014 ; Cibulski et al. 2021 ; Kumar et al. 2022 ; Silveira et al. 2023 ). Saponins, a class of bioactive compounds, exhibit surfactant properties, generating persistent foam in water, and consist of steroidal (C27) or triterpene (C30) structures (Ferraiuolo et al., 2022 ). Within Quillaja triterpene saponins, quillaic acid (C 30 H 46 O 5 ) serves as the principal aglycone, contributing to the formation of immunoadjuvant saponins like QS-21 (C 92 H 148 O 46 ). This saponin is approved for human vaccine adjuvant use, being primarily sourced from Q. saponaria bark. However, QS-21 is also found in Q. lancifolia leaves, being a component of an immunoadjuvant saponin-enriched fraction named QB-90 (Magedans et al. 2019 ) or its equivalent, fraction B (Wallace et al. 2019 ). The unsustainable nature of commercially extracting these compounds from natural Quillaja populations has spurred interest in bioprospecting alternative sources for triterpene saponins production within the biotechnological sector. Current commercial strategies include cloning of superior tree genotypes in plantations and miniclonal gardens (Magedans et al. 2019 ). Endophytic microorganisms possess the unique capability to inhabit plant tissues without inducing pathogenicity under homeostatic conditions (Petrini, 1991 ; Gupta et al. 2019), even in in vitro culture systems. The specialized metabolism of these endophytes represents a promising source of bioactive compounds (Gakuubi et al. 2021 ; Numan et al. 2022 ), owing to the inherent metabolic diversity of each species and the interaction with specialized metabolic pathways from their host plants. This symbiotic relationship can offer advantages such as differential growth, protection against various stresses and diseases, and chemical elicitation of specialized metabolism (Rabiey et al. 2019 ; Khan et al. 2021 ; Wu et al. 2021 ). Microorganisms exhibit biotechnological potential for sustainable and scalable production of specialized metabolites from plants (Wawrosch and Zotchev, 2021 ). Consequently, exploring endophytic microorganisms associated with Q. lancifolia presents a potential new avenue for discovering strains capable of producing biosurfactants with significant biological activities. The primary aim of this study was to isolate and characterize the main endophytes of Q. lancifolia and examine their biotechnological potential as new sources of bioactive compounds, focusing on biossurfactant-like molecules. Material and methods Plant material and isolation of microorganisms Seeds of Q. lancifolia were obtained from field-grown plants located in the rural area of Canguçu, Rio Grande do Sul, Brazil (31°05'14.6"S, 52°50'02.0"W) in April 2023. Genetic heritage access was registered in the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (SisGen), under registration number A98B7B1. Plant vouchers were deposited in the ICN Herbarium of the Botany Department at the Federal University of Rio Grande do Sul (identification numbers ICN 207362, 207363, and 207367). To ensure asepsis and eliminate epiphytic microorganisms from the seed coat surface, a disinfection procedure was carried out using commercial antifungal Dithane® at 0.5% for 20 minutes, followed by washing with 70% ethanol for 1 minute and a subsequent wash with a 2% active chlorine sodium hypochlorite solution for 10 minutes. Following asepsis, the seeds underwent three rinses with sterile distilled water and were then placed in individualized vials containing 5 mL of sterile culture medium (0.5 × Murashige and Skoog medium, pH 5.8, 0.7% agar). The flasks were maintained in an in vitro cultivation room at 22 ± 2 ºC, photoperiod of 16 hours, and an average irradiance of 30 µmol m⁻² s⁻¹. After 14 days from sowing, fungi and bacteria were isolated from surface-disinfested Q. lancifolia ungerminated seeds. Seedlings from the germinated seeds were submitted to a new surface disinfection with 0.1% HgCl₂ for 10 minutes before endophyte growth induction (Boiu-Sicuia and Cornea, 2019 ). Microorganisms that became visible up until 14 days were isolated. Endophytes were also directly isolated from previously established in vitro cultures of calli and cell suspensions of Q. lancifolia . Cultivation conditions of isolates Bacterial isolates were cultivated in Tryptone Soy Broth (TSB) medium, a nutrient-rich medium that supports bacterial growth and promotes the production of extracellular metabolites (Arivo et al., 2023 ; Linh and Duc, 2025 ), while fungal isolates were cultivated in Czapeck medium (Guimarães et al., 2009 ), both media at pH 7. For liquid culture, sterile flasks (100 mL) were inoculated and incubated at 28 ºC under constant agitation (150 rpm), for both fungi and bacteria. After cultivation for 5 days (bacteria) or 7 days (fungi), the culture broths were collected, centrifuged at 1,720 g for 30 minutes at room temperature, filtered, and freeze-dried (Guimarães et al., 2009 ; Jayasekara et al., 2022 ). The resulting powder was used for biosurfactant-like screening through differential foam formation test and Thin Layer Chromatography (TLC) analyses focusing on the detection of triterpenes. Biosurfactant-like screening For the foam formation test, solutions of lyophilized extracts were prepared at a concentration of 500 mg/mL in ultrapure water. Following vigorous manual shaking, these solutions were visually compared with the corresponding sterile nutrient broth at an identical concentration (based on the guidelines of the Brazilian Sanitary Vigilance Agency - Agência Nacional de Vigilância Sanitária, 2019 ). For Thin Layer Chromatography, C18 silica plates were the stationary phase. BAW (butanol:acetic acid:water, 10:2:8, v/v/v) was the mobile phase. Samples were applied and run in a chromatographic chamber for 40 minutes. To identify the presence of triterpenes, plates were developed with sulfuric anisaldehyde, followed by drying at 105°C. Both Q. lancifolia leaf extract and Q. saponaria Quil-A fraction were used as standards. Bands that stained purple, blue, and green were considered positive for triterpenes; media extracts yielding bands with Rfs comparable to those of plant extracts were also considered of interest for the study (Agatonovic-Kustrin et al. 2019 ; Marques et al. 2023 ). Metabolite purifications For Solid Phase Extraction (SPE), samples were loaded in C18 silica columns (5g), followed by elution with increasing methanol:water gradients (0, 10, 20, 30, 40, 50, 60, 70, and 100%). This procedure allowed the concentration of target compounds and removal of non-target ones. The 70–100% methanol fractions were lyophilized and weighed for quantification. Equation 1 was employed to determine the yield of biossurfactant-like agents. $$\:S\left(\%\right)=\frac{SPEm\:.\:100}{ce}$$ 1 Where \(\:S\left(\%\right)\:\) is the yield of total biossurfactant-like agents, \(\:SPEm\:\) is the mass of purified extract after SPE extraction and \(\:ce\) is the mass of crude extract loaded into the SPE column. Molecular Identification of Select Endophytes DNA Extraction For the extraction of DNA from select filamentous fungi, strains initially underwent cultivation on solid PDA medium in Petri dishes at 28 ºC. Young mycelia were obtained from colonies after 7 days. In 2 mL microtubes, 100 mg of mycelium, and 1 mL of extraction buffer (100 mM Tris-HCl (pH 7.5), 25 mM EDTA, 1.5 M NaCl, 2% (w/v) CTAB - cetyltrimethylammonium bromide) at 65 ºC were added, and the mixture was macerated using a Beader with 0.2 mL of sterile glass pearls for 1.5 minutes at 3,000 rpm. Subsequently, β-mercaptoethanol was added to a final concentration of 3% (v/v), followed by incubation at 65 ºC for 30 minutes in a water bath. Samples were then centrifuged for 5 minutes at 5,000 x g, the supernatant was collected into a new tube and partitioned with chloroform:isoamyl alcohol (24:1, v/v). The DNA, treated with RNAse A for 15 minutes at 37 ºC, underwent another partition with chloroform:isoamyl alcohol (24:1, v/v). The DNA was then precipitated with 95% ethanol for approximately 1h at -20°C and subsequently washed with 70% ethanol. After drying, the pellet was resuspended in TE buffer (100 mM Tris-HCl pH 7.5 and 25 mM EDTA). The final DNA concentration was adjusted to 15 ng/µL. Polymerase Chain Reaction (PCR) For PCR reactions, recombinant Taq DNA polymerase kit (Invitrogen, Waltham, MA, USA) was used. Sterile 0.2 mL microtubes were prepared containing 10 µL of 10X buffer, 3 µL of 50 mM MgCl 2 solution, 2 µL of 10 mM dNTP mixture, 5 µL of 10 µM primer mixture (F and R), 0.2 µL of recombinant Taq DNA Polymerase (Invitrogen), and 5 µL of DNA template at a concentration of 15 ng/µL. The ITS-1 oligonucleotides were employed for the taxonomic identification of the fungi (ITS1-1F-F 3’-CTTGGTCATTTAGAGGAAGTAA and ITS1-1F-R 3’-GCTGCGTTCTTCATCGATGC) (White, 1990 ). The final reaction volume was 25 µL, and PCR was conducted over 35 cycles, involving denaturation at 95 ºC for 30 s, annealing at 55 ºC for 45 s, and extension at 72 ºC for 1 min and 30 s. The amplification process commenced with an initial denaturation step of 3 min at 95 ºC and ended with a final extension step of 5 min at 72 ºC. Subsequently, the amplified products were subjected to electrophoresis in a 0.8% agarose gel, using FluoSafe as a nucleic acid dye. Electrophoresis was conducted at 100 mA for 45 minutes and bands were visualized under UV light. DNA Sequencing and Data Analysis Sequencing was carried out utilizing the Sanger sequencing method. The resulting data were compiled and utilized for species identification through the Basic Local Alignment Search Tool (BLASTn) with a public library available at https://www.ncbi.nlm.nih.gov/ . Sequences for phylogenetic analysis were also obtained from the same database. The obtained DNA sequences were deposited in the NCBI database under accession numbers PX072001-PX072004 (Supplemental data S1). Phylogenetic Analysis To construct the phylogenetic tree, a data matrix was generated based on the lowest E-value results obtained from the NCBI's BLASTn tool ( https://blast.ncbi.nlm.nih.gov/ ) for our sequences. The data matrix in .FASTA format underwent multiple sequence alignment using MAFFT, followed by an Alignment Curation using BMGE, a Tree Inference by PhyML + SMS and Tree Rendering by Newick Display ( https://ngphylogeny.fr/ ) (Lemoine, et al.2019). Tree visualization and editing was performed using iTol ( https://itol.embl.de/ ) (Letunic and Bork, 2021 ). HPLC and LCMS Purified fractions obtained from Diplodia sp. and Aureobasidium sp. were analyzed for presence of QS-21 and related saponins. High-Performance Liquid Chromatography (HPLC) was carried out using an external standard curve prepared with authentic QS-21 (Creative Biolabs, NY, USA) or reference QB-90 purified from Q. lancifolia leaves. Mobile phase consisted of a combination of acetonitrile (phase A) and Milli-Q water (phase B), both containing 0.1% formic acid. Gradient started at 25% of B changing to 50% of B within 20 min. A C18 RP column (250 mm x 4.6 mm (5µm) – Agilent Eclipse - XDB) was the stationary phase. Analyses were carried out at 214 nm in a Shimadzu Prominence chromatograph equipped with UV-Vis DAD detector. Liquid Chromatography-Mass Spectrometry (LCMS) measurements were performed using the LCMS Amazon speed Iontrap (Bruker Daltonics Inc., MA, USA), following a previously established protocol (Wallace et al. 2019 ). Samples were injected at a concentration of 10 mg/mL (20 µL) for preliminary evaluation. Authentic QS-21 (Creative BioLabs, NY, United States) was used as standard. Additionally, Q. lancifolia leaf extract was injected at 10 mg/mL to serve as a reference control for triterpene saponins. The liquid chromatography was conducted using a C18 column (250 mm x 4.6 mm, 5 µm). The mobile phase consisted of water with 0.1% formic acid (v/v) for phase A and acetonitrile with 0.1% formic acid (v/v) for phase B. The gradient began with 15% B, increasing to 65% B over 50 minutes. The flow rate was maintained at 0.6 mL/min. Spectral data were collected in negative mode, with a full scan performed from 800 to 2500 m/z at a resolution speed of 8.1 m/z per second. The most abundant ion was isolated in the Ion Trap for MS2 fragmentation. MS2 data were manually deconvoluted to identify the target compounds. Antimicrobial activity The antimicrobial activity of fungal extracts, as well as crude and purified fractions of Q. lancifolia , was assessed using the agar diffusion method. Briefly, standardized inoculum at 0.5 Mc Farland scale (OD 600 0.08–0.1) of Staphylococcus aureus ATCC 35591, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 23893 were plated on TSA (Tryptone Soy Agar) using a sterile swab. Then, 20 µL of each extract or disk with standardized antimicrobial were directly applied to the surface of agar plates. Petri dishes were kept at 37 ºC for 24 hours, until inhibition zones were measured (Kimura et al. 2006 ; Motta and Brandelli, 2002 ). Based on agar diffusion tests, select bacteria ( P. aeruginosa ) and extracts (SPE Diplodia and QB-90) were submitted to the microdilution test using the 96 well plate in TSB. Concentrations ranged from 2.5–0.001 mg/mL for purified Diplodia extract fraction, and from 25–0.012 mg/mL for QB-90. Chloramphenicol was used as standard drug (320 to 0.15 µg/mL). After incubation, OD 600 was recorded for each well for growth assessment. Percentage inhibition was calculated as response parameter for anti- Pseudomonas activity (adapted from Methodology for Agent Sensitivity Tests Antimicrobials by Dilution for Bacteria Aerobic Growth, M7-A6 standard, by NCCLS - National Committee for Clinical Laboratory Standards). Results and discussion Isolation and selection of microorganisms Twenty-two fungal and twenty bacterial strains were isolated from Q. lancifolia . Among these, four fungal strains were selected based on their capacity to produce biosurfactants, as evidenced by differential foam formation and the presence of bands stained on TLC (strains 12PFR4, 06SFR4, and 12PFR1) using sulfuric anisaldehyde. No bacterial strain showed intense foam formation. Additionally, fungal strain 22PFR4 (2) exhibited bands like some of the those of Q. lancifolia as assessed by TLC (Table 1 , Fig. 1 ), albeit apparently not of triterpene nature. Table 1 General characteristics of isolates positive for biosurfactants in foam and/or TLC tests. Strain Foam TLC Yield (%, w/w) Colony Source F5_12PFR4 (1)* ++ Green/purple 3.0 Grey/black Seedling F9_22PFR4 (2) + Yellow 0.74 Grey/black Seedling F21_06SFR4 (3) + Bluish pink/purple 0.40 White/pink Seed F23_12PFR1 (4) ++ Yellow/pink 6.28 Black/brown Seedling *(1) Diplodia sp.; (2) Acremonium sp.; (3) Fusarium sp.; and (4) Aureobasidium sp. Molecular Identification and Yield Taxonomic identification conducted by sequencing of the ITS-1 region and local alignment analysis revealed the presence of Diplodia sp. (12PFR4), Acremonium sp. (22PFR4), Fusarium sp. (06SFR4), and Aureobasidium sp. (12PFR1). The total exuded fraction for these species was quantified using a triterpene saponin-specific purification method, resulting in yields of 3.0%, 0.74%, 0.40%, and 6.28% (w/w), respectively (Table 1 ). The macro and micromorphology of the colonies of the selected species are shown in Fig. 2 . In related studies focused on the bioprospecting of endophytes with saponin production, species such as Aspergillus terreus , A. flavus , Penicillium sp., and Talaromyces pinophilus have been isolated from the roots of Asparagus racemosus Willd., a saponin-producing plant naturally occurring in Africa, Asia, and Oceania (Rani et al. 2022). Besides, F. oxysporum and A. niger have been prospected from Panax notoginseng (Burk), which is naturally rich in ginsenosides. Saponins similar in character to those of the host plant have been elucidated, and the purified metabolites exhibited antimicrobial activity (Jin et al. 2017 ). Moreover, F. tricinctum , Alternaria sp., Chaetosphaeronema sp., Mucor sp., and Trichoderma sp. were isolated from Lithospermum officinale L., a plant producing shikonin - an anticancer compound (Mollaei et al. 2019 ). The same study also recorded this alkaloid in the endophyte F. tricinctum , suggesting the fungal strain could be able to produce plant-like specialized metabolites. Phylogenetic Analysis The phylogenetic tree illustrates the grouping of the select strains into four distinct clades. These separate groups represent the different genera, Aureobasidium sp., Diplodia sp., Acremonium sp. and Fusarium sp. (Fig. 3 ). Diplodia sp., (Fig. 2 – a, b), appeared to be a predominant endophytic fungus in Q. lancifolia , as evidenced by its consistent re-isolation during the in vitro processing of cells and tissues from the same species. Diplodia belongs to the order Botryosphaeriales and is primarily associated with diseases in woody plant species and major crops, such as maize (Yang et al. 2017 ). This genus is affiliated with Rosaceae, and its species may or may not induce pathological conditions, like stem rot and leaf spots (Philips et al. 2012). Diplodia is considered a facultatively associated taxon with pathosystems but is also conclusively identified as a component of plant endophytic communities (White et al. 2016 ). Plant-associated Diplodia sp. have been shown to produce an array of specialized metabolites, mainly polyketide - and terpenoid - derived compounds. The most notable bioactivities of these fungi are antimicrobial and phytotoxic (Salvatore et al. 2025 ). Despite some species of Fusarium being recognized for their negative effects in crops, species isolated from endophytic communities of medicinal plants show considerable potential for obtaining bioactive products from specialized metabolism, including some related to those of the plant hosts (Mollaei et al. 2019 ). For instance, Fusarium sp. isolated from P. notoginseng yielded several ginsenosides (Wu et al. 2013 ; Jin et al. 2017 ). Moreover, Fusarium isolated from Dioscorea nipponica was identified as a species of significance for the biotransformation and diversification of saponins from Paris polyphylla (Chen et al. 2022 ). Acremonium , known for its phylogenetic complexity, is a genus associated with food contamination and a causal agent of plant diseases (Teixeira and Machado, 2003 ; Summerbell et al. 2011 ). It includes strains with endophytic character, such as Acremonium sp., isolated from Lilium davidii , which exhibited plant growth stimulation and control activity against phytopathogenic species (Khan et al. 2021 ). In another study, Acremonium strictum showed the ability to biotransform ginsenoside Rb1 from P. ginseng (Chen et al. 2008 ). Aureobasidium sp., a dimorphic fungus, is considered of interest in the biotechnology sector due to A. pullulans production of industrial enzymes, antimicrobials, single-cell proteins, pullulans, leamokines with antimicrobial and cytotoxic effects on tumor cell lines, iron-chelating siderophores, and β-glucans (Chi et al. 2009 ; Manitchotpisit et al. 2011; Prasongsuk et al. 2017). A. pullulans A11211-4-57 produced two new biosurfactants, pullusurfactans F and G. These myo-inositol lipids exhibited strong surfactant activity at 1.0 mg/L, making them suitable for cleaning applications (Kim et al. 2022 ). Although the production of complex plant specialized metabolites by fungal endophytes has been often reported, there have been several legitimate challenges to the validity of some long-term results involving the topic, at least in the case of taxol (Gärditz and Czesnick, 2024 ; Stadler and Kolarik, 2024 ). Indeed, in the present work, QS-21 could not be detected in the endophyte cultures. In addition, preliminary attempts to amplify from fungal DNA a key gene of QS-21 biosynthesis encoding beta-amyrin synthase proved unsuccessful (data not shown). HPLC and LCMS results Purified fractions from Diplodia sp. and Aureobasidium sp. aqueous extracts produced foam in water. Therefore, these samples were chemically analyzed for the presence of triterpene saponins. Initial HPLC data suggested the presence of saponin-like molecules in fungal extracts with peak retention times resembling those of QS-21 and related saponins found in plants. However, LCMS 2 did not confirm any triterpenoid saponins in fungi samples (Fig. 4 ). In contrast, the expected presence of QS-21 in Q. lancifolia extract was confirmed, as previously reported (Wallace, 2019). Antimicrobial activity Potential inhibitory activity of plant and fungal extracts was tested against pathogenic Gram-positive and Gram-negative bacteria. Agar diffusion tests showed bioactivity of SPE purified fraction from Diplodia (5 mg/mL) and Q. lancifolia (QB-90, 50 mg/mL) against P. aeruginosa , with inhibition zones of 14.3 and 11.4 mm in diameter, respectively. Norfloxacin (30 µg/mL), a standard drug against Gram-negative bacteria, presented an inhibition diameter of 14.3 mm (Supplemental data S2). Maximum growth inhibition has been obtained from broth microdilution tests, with 51.4% for SPE fraction from Diplodia sp. at 2.5 mg/mL and 42.2% for QB-90 ( Q. lancifolia purified saponin fraction) at 25 mg/mL (Supplemental data S3). Despite the presence of various peaks on Diplopia sp. purified fraction chromatogram, indicating the presence of various compounds, an activity of more than 50% of inhibition was recorded against P. aeruginosa , an important Gram-negative pathogenic bacterium. Hence, a synergistic mechanism of action might be possible. Previous studies from endophytic fungi from Kigelia africana Lam. (sausage tree) showed remarkable anti- Pseudomonas activity of Neofusicoccum luteum fractions with minimal inhibitory concentration of 156.50 µg/mL after seven days of fungal cultivation, and 19.53 µg/mL after 30 days of static fermentation. A purified compound, a C16-terpene dilactone presented MIC value of 0.61 µg/mL, playing a role in the antimicrobial activity (Bodede et al. 2022 ). Despite the inhibitory activity exhibited by the Diplodia purified fraction in the first fermentation batch, a second independent experiment, conducted under identical culture media, temperature, agitation, and incubation time, yielded a different HPLC profile (Supplemental Figure S4). Although maintaining foaming capacity relative to the culture media control, bioactivity assessment by agar diffusion assay revealed no inhibitory activity of the purified fraction of the second batch against P. aeruginosa . This variability of chemical profile and associated bioactivity is likely due to metabolic changes in endophyte cultures over time, particularly following isolation from plant tissues. In fact, this is not surprising given the specific microenvironments, nutrient and energy sources, signal exchange molecules, and impact of seasonal variations of the in planta conditions, all of which differ markedly from in vitro media cultivation. Even within the endophytic environment, the temporal and spatial dynamics of fungal composition, abundance and interaction with host plants is quite significant (Crasta and Raveesha, 2024 ; Ullah et al. 2025 ). Conclusion This is the first report of endophytic microorganisms in Q. lancifolia . Four strains of fungi producing biosurfactants-like foaming agents were prospected from different plant sources and identified by molecular techniques, confirming the presence of Diplodia sp., Fusarium sp., Acremonium sp. and Aureobasidium sp. Despite the absence of key plant compounds in the most promising select endophytes, purified fractions showed overt foaming properties. Endophytic species of Q. lancifolia may constitute a platform to produce compounds of biotechnological interest. Declarations Declaration of generative AI and AI-assisted technologies in the writing process During the drafting stage of the manuscript, the author(s) used OpenAI's ChatGPT in order to assist with language refinement, and organization of the text. After using this tool, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the publication. Funding This research was funded by grants from FAPERGS (Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul 2019/15477-3; 24/2551-0001321-0; 24/2551-0000697-4), CNPq (National Council for Scientific and Technological Development 42430/2-21-5; 310775/2021-3), and CAPES (Coordination for Improvement of Higher Education Personnel- finance code 001). Acknowledgments The authors are grateful to Prof. Gilson R. Pires Moreira (Dept of Zoology, UFRGS) for access to trees growing on his private property. CRediT authorship contribution statement Fábio Antônio Antonelo: Conceptualization, Investigation, Methodology, Writing – original draft. Eliane Zachert: Methodology, Writing – review & editing. Yve Verônica Magedans: Methodology,Data curation, Writing – review & editing. Anna Alves Yendo: Methodology. Cibele Tesser: Methodology. Validation. Alice Elvira Teixeira dos Santos: Methodology. Fernanda Cortez Lopes: Investigation, Methodology, Validation, Writing – review & editing. Marilene Henning Vainstein: Resources. Charley Christian Staats: Writing – review & editing. Juliana Morini Küpper Cardoso: Writing – review & editing. Arthur Germano Fett-Neto: Investigation, Resources, Supervision, Project administration, Validation, Writing – review & editing. References Agatonovic-Kustrin S, Kustrin E, Gegechkori V, Morton DW, 2019. High-performance thin-layer chromatography hyphenated with microchemical and biochemical derivatizations in bioactivity profiling of marine species. 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Frontiers in Plant Science 12: 646146. https://doi.org/10.3389/fpls.2021.646146 Yang T, Groenewald JZ, Cheewangkoon R, Jami F, Abdollahzadeh J, Lombard L, Crous PW, 2017. Families, genera, and species of Botryosphaeriales . Fungal Biology 121(4): 322-346. https://doi.org/10.1016/j.funbio.2016.11.001 Additional Declarations The authors declare no competing interests. Supplementary Files Supplementaldata.docx 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. 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07:03:21","extension":"html","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":151451,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8253976/v1/c40d35715af996b39bf2927f.html"},{"id":97317378,"identity":"f2bf9d93-7cec-412b-a644-d279d7e26140","added_by":"auto","created_at":"2025-12-03 07:03:20","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":59872,"visible":true,"origin":"","legend":"\u003cp\u003eThin Layer Chromatography of fungal and plant extracts. Fungal culture extracts ((1)\u003cem\u003e Diplodia \u003c/em\u003esp., (2)\u003cem\u003e Acremonium \u003c/em\u003esp., (3)\u003cem\u003e Fusarium \u003c/em\u003esp., and (4)\u003cem\u003e Aureobasidium \u003c/em\u003esp.), as well as extracts from \u003cem\u003eQ. saponaria\u003c/em\u003e (5) and \u003cem\u003eQ. lancifolia\u003c/em\u003e (6) were resolved in TLC plates. Triterpenes were visualized using sulfuric anisaldehyde.\u003c/p\u003e","description":"","filename":"Fig1TLC.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8253976/v1/d5f657524d02740125141699.jpg"},{"id":97317384,"identity":"2cbb439a-50a8-4f67-91c5-e97cfc9f434c","added_by":"auto","created_at":"2025-12-03 07:03:20","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":849845,"visible":true,"origin":"","legend":"\u003cp\u003eMacro and micromorphology of \u003cem\u003eDiplodia \u003c/em\u003esp. F5_12PFR4 (A, B), \u003cem\u003eAcremonium \u003c/em\u003esp. F9_22PFR4 (C, D), \u003cem\u003eFusarium \u003c/em\u003esp.\u003cem\u003e \u003c/em\u003eF21_06SFR4 (E, F), and \u003cem\u003eAureobasidium \u003c/em\u003esp. - 12PFR1 (G, H)\u003c/p\u003e","description":"","filename":"Fig2Fungi.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8253976/v1/e8e7c3f4ec5eb09482a9f7e0.jpg"},{"id":97368981,"identity":"31813258-c529-4a86-8ce0-1266a0e4fb57","added_by":"auto","created_at":"2025-12-03 16:23:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":50760,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree of foam-producing endophytic isolates of \u003cem\u003eQ. lancifolia\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Fig3phylogtree.png","url":"https://assets-eu.researchsquare.com/files/rs-8253976/v1/469de2d5a6bd336ff9939cda.png"},{"id":97317376,"identity":"2ea2219f-24b6-40ec-bf7d-26ca12ed5744","added_by":"auto","created_at":"2025-12-03 07:03:20","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":112354,"visible":true,"origin":"","legend":"\u003cp\u003eLCMS\u003csup\u003e2\u003c/sup\u003e and most abundant fragment from QS-21 standard (A), \u003cem\u003eDiplodia\u003c/em\u003e sp. purified fraction (B), \u003cem\u003eAureobasidium \u003c/em\u003esp. purified fraction (C) and \u003cem\u003eQ. lancifolia \u003c/em\u003eseedlings\u003cem\u003e \u003c/em\u003epurified fraction (D).\u003c/p\u003e","description":"","filename":"Fig4.2LCMS.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8253976/v1/3cef890fa6d4df5e56c5841f.jpg"},{"id":97664647,"identity":"15258cdb-ec48-4cab-b5e7-62d1f932e6c5","added_by":"auto","created_at":"2025-12-08 09:12:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2157030,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8253976/v1/83618980-8a8c-44bb-8187-4dd7343a3444.pdf"},{"id":97317380,"identity":"5ba60a9b-9a84-45ba-a304-38085b949a56","added_by":"auto","created_at":"2025-12-03 07:03:20","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":332999,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaldata.docx","url":"https://assets-eu.researchsquare.com/files/rs-8253976/v1/c07ead7cf89efb3d1a21aa9c.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eScreening and molecular identification of biosurfactant-producing endophytes from \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eQuillaja lancifolia\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e D. Don\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Highlights","content":"\u003cp\u003e\u0026bull; This is the first report of endophytic microorganisms in \u003c/p\u003e\u003cp\u003e\u0026bull; A total of 42 cultured endophytes were screened for extracellular foam production\u003c/p\u003e\u003cp\u003e\u0026bull; sp., sp., sp. and sp. yielded foam\u003c/p\u003e\u003cp\u003e\u0026bull; Fungal biosurfactants differed markedly from those of the plant host\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eQuillaja lancifolia\u003c/em\u003e D. Don, synonymous with \u003cem\u003eQuillaja brasiliensis\u003c/em\u003e (A.St.-Hil. \u0026amp; Tul.) Mart., is a tree species indigenous to Brazil, Uruguay, and Argentina. It is known as a natural source of triterpene saponins that can be used in the production of pharmaceuticals, food, and cosmetics (Luebert, 2014; Fleck et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eLike the stem bark of \u003cem\u003eQ. saponaria\u003c/em\u003e Molina, the leaves of \u003cem\u003eQ. lancifolia\u003c/em\u003e produce triterpene saponins derived from quillaic acid, known for their notable immunoadjuvant properties (De Costa et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Mechanistically, \u003cem\u003eQuillaja\u003c/em\u003e spp. saponins enhance and prolong cellular and humoral immune responses as adjuvants in various vaccines, including Polio, Zika, SARS-CoV-2, and Influenza viruses (De Costa et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Cibulski et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kumar et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Silveira et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSaponins, a class of bioactive compounds, exhibit surfactant properties, generating persistent foam in water, and consist of steroidal (C27) or triterpene (C30) structures (Ferraiuolo et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Within \u003cem\u003eQuillaja\u003c/em\u003e triterpene saponins, quillaic acid (C\u003csub\u003e30\u003c/sub\u003eH\u003csub\u003e46\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e) serves as the principal aglycone, contributing to the formation of immunoadjuvant saponins like QS-21 (C\u003csub\u003e92\u003c/sub\u003eH\u003csub\u003e148\u003c/sub\u003eO\u003csub\u003e46\u003c/sub\u003e). This saponin is approved for human vaccine adjuvant use, being primarily sourced from \u003cem\u003eQ. saponaria\u003c/em\u003e bark. However, QS-21 is also found in \u003cem\u003eQ. lancifolia\u003c/em\u003e leaves, being a component of an immunoadjuvant saponin-enriched fraction named QB-90 (Magedans et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) or its equivalent, fraction B (Wallace et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe unsustainable nature of commercially extracting these compounds from natural \u003cem\u003eQuillaja\u003c/em\u003e populations has spurred interest in bioprospecting alternative sources for triterpene saponins production within the biotechnological sector. Current commercial strategies include cloning of superior tree genotypes in plantations and miniclonal gardens (Magedans et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eEndophytic microorganisms possess the unique capability to inhabit plant tissues without inducing pathogenicity under homeostatic conditions (Petrini, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Gupta et al. 2019), even in \u003cem\u003ein vitro\u003c/em\u003e culture systems. The specialized metabolism of these endophytes represents a promising source of bioactive compounds (Gakuubi et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Numan et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), owing to the inherent metabolic diversity of each species and the interaction with specialized metabolic pathways from their host plants. This symbiotic relationship can offer advantages such as differential growth, protection against various stresses and diseases, and chemical elicitation of specialized metabolism (Rabiey et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Khan et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Wu et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMicroorganisms exhibit biotechnological potential for sustainable and scalable production of specialized metabolites from plants (Wawrosch and Zotchev, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Consequently, exploring endophytic microorganisms associated with \u003cem\u003eQ. lancifolia\u003c/em\u003e presents a potential new avenue for discovering strains capable of producing biosurfactants with significant biological activities. The primary aim of this study was to isolate and characterize the main endophytes of \u003cem\u003eQ. lancifolia\u003c/em\u003e and examine their biotechnological potential as new sources of bioactive compounds, focusing on biossurfactant-like molecules.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePlant material and isolation of microorganisms\u003c/h2\u003e\u003cp\u003eSeeds of \u003cem\u003eQ. lancifolia\u003c/em\u003e were obtained from field-grown plants located in the rural area of Cangu\u0026ccedil;u, Rio Grande do Sul, Brazil (31\u0026deg;05'14.6\"S, 52\u0026deg;50'02.0\"W) in April 2023. Genetic heritage access was registered in the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (SisGen), under registration number A98B7B1. Plant vouchers were deposited in the ICN Herbarium of the Botany Department at the Federal University of Rio Grande do Sul (identification numbers ICN 207362, 207363, and 207367).\u003c/p\u003e\u003cp\u003eTo ensure asepsis and eliminate epiphytic microorganisms from the seed coat surface, a disinfection procedure was carried out using commercial antifungal Dithane\u0026reg; at 0.5% for 20 minutes, followed by washing with 70% ethanol for 1 minute and a subsequent wash with a 2% active chlorine sodium hypochlorite solution for 10 minutes. Following asepsis, the seeds underwent three rinses with sterile distilled water and were then placed in individualized vials containing 5 mL of sterile culture medium (0.5 \u0026times; Murashige and Skoog medium, pH 5.8, 0.7% agar). The flasks were maintained in an in vitro cultivation room at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2 \u0026ordm;C, photoperiod of 16 hours, and an average irradiance of 30 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1;.\u003c/p\u003e\u003cp\u003eAfter 14 days from sowing, fungi and bacteria were isolated from surface-disinfested \u003cem\u003eQ. lancifolia\u003c/em\u003e ungerminated seeds. Seedlings from the germinated seeds were submitted to a new surface disinfection with 0.1% HgCl₂ for 10 minutes before endophyte growth induction (Boiu-Sicuia and Cornea, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Microorganisms that became visible up until 14 days were isolated. Endophytes were also directly isolated from previously established \u003cem\u003ein vitro\u003c/em\u003e cultures of calli and cell suspensions of \u003cem\u003eQ. lancifolia\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCultivation conditions of isolates\u003c/h3\u003e\n\u003cp\u003eBacterial isolates were cultivated in Tryptone Soy Broth (TSB) medium, a nutrient-rich medium that supports bacterial growth and promotes the production of extracellular metabolites (Arivo et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Linh and Duc, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), while fungal isolates were cultivated in Czapeck medium (Guimar\u0026atilde;es et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), both media at pH 7. For liquid culture, sterile flasks (100 mL) were inoculated and incubated at 28 \u0026ordm;C under constant agitation (150 rpm), for both fungi and bacteria. After cultivation for 5 days (bacteria) or 7 days (fungi), the culture broths were collected, centrifuged at 1,720 g for 30 minutes at room temperature, filtered, and freeze-dried (Guimar\u0026atilde;es et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Jayasekara et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The resulting powder was used for biosurfactant-like screening through differential foam formation test and Thin Layer Chromatography (TLC) analyses focusing on the detection of triterpenes.\u003c/p\u003e\n\u003ch3\u003eBiosurfactant-like screening\u003c/h3\u003e\n\u003cp\u003eFor the foam formation test, solutions of lyophilized extracts were prepared at a concentration of 500 mg/mL in ultrapure water. Following vigorous manual shaking, these solutions were visually compared with the corresponding sterile nutrient broth at an identical concentration (based on the guidelines of the Brazilian Sanitary Vigilance Agency - Ag\u0026ecirc;ncia Nacional de Vigil\u0026acirc;ncia Sanit\u0026aacute;ria, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFor Thin Layer Chromatography, C18 silica plates were the stationary phase. BAW (butanol:acetic acid:water, 10:2:8, v/v/v) was the mobile phase. Samples were applied and run in a chromatographic chamber for 40 minutes. To identify the presence of triterpenes, plates were developed with sulfuric anisaldehyde, followed by drying at 105\u0026deg;C. Both \u003cem\u003eQ. lancifolia\u003c/em\u003e leaf extract and \u003cem\u003eQ. saponaria\u003c/em\u003e Quil-A fraction were used as standards. Bands that stained purple, blue, and green were considered positive for triterpenes; media extracts yielding bands with Rfs comparable to those of plant extracts were also considered of interest for the study (Agatonovic-Kustrin et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Marques et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eMetabolite purifications\u003c/h3\u003e\n\u003cp\u003eFor Solid Phase Extraction (SPE), samples were loaded in C18 silica columns (5g), followed by elution with increasing methanol:water gradients (0, 10, 20, 30, 40, 50, 60, 70, and 100%). This procedure allowed the concentration of target compounds and removal of non-target ones. The 70\u0026ndash;100% methanol fractions were lyophilized and weighed for quantification.\u003c/p\u003e\u003cp\u003eEquation \u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e was employed to determine the yield of biossurfactant-like agents.\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:S\\left(\\%\\right)=\\frac{SPEm\\:.\\:100}{ce}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eWhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:S\\left(\\%\\right)\\:\\)\u003c/span\u003e\u003c/span\u003eis the yield of total biossurfactant-like agents, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:SPEm\\:\\)\u003c/span\u003e\u003c/span\u003eis the mass of purified extract after SPE extraction and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:ce\\)\u003c/span\u003e\u003c/span\u003e is the mass of crude extract loaded into the SPE column.\u003c/p\u003e\n\u003ch3\u003eMolecular Identification of Select Endophytes\u003c/h3\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eDNA Extraction\u003c/h2\u003e\u003cp\u003eFor the extraction of DNA from select filamentous fungi, strains initially underwent cultivation on solid PDA medium in Petri dishes at 28 \u0026ordm;C. Young mycelia were obtained from colonies after 7 days. In 2 mL microtubes, 100 mg of mycelium, and 1 mL of extraction buffer (100 mM Tris-HCl (pH 7.5), 25 mM EDTA, 1.5 M NaCl, 2% (w/v) CTAB - cetyltrimethylammonium bromide) at 65 \u0026ordm;C were added, and the mixture was macerated using a Beader with 0.2 mL of sterile glass pearls for 1.5 minutes at 3,000 rpm.\u003c/p\u003e\u003cp\u003eSubsequently, β-mercaptoethanol was added to a final concentration of 3% (v/v), followed by incubation at 65 \u0026ordm;C for 30 minutes in a water bath. Samples were then centrifuged for 5 minutes at 5,000 x g, the supernatant was collected into a new tube and partitioned with chloroform:isoamyl alcohol (24:1, v/v). The DNA, treated with RNAse A for 15 minutes at 37 \u0026ordm;C, underwent another partition with chloroform:isoamyl alcohol (24:1, v/v). The DNA was then precipitated with 95% ethanol for approximately 1h at -20\u0026deg;C and subsequently washed with 70% ethanol. After drying, the pellet was resuspended in TE buffer (100 mM Tris-HCl pH 7.5 and 25 mM EDTA). The final DNA concentration was adjusted to 15 ng/\u0026micro;L.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePolymerase Chain Reaction (PCR)\u003c/h3\u003e\n\u003cp\u003eFor PCR reactions, recombinant Taq DNA polymerase kit (Invitrogen, Waltham, MA, USA) was used. Sterile 0.2 mL microtubes were prepared containing 10 \u0026micro;L of 10X buffer, 3 \u0026micro;L of 50 mM MgCl\u003csub\u003e2\u003c/sub\u003e solution, 2 \u0026micro;L of 10 mM dNTP mixture, 5 \u0026micro;L of 10 \u0026micro;M primer mixture (F and R), 0.2 \u0026micro;L of recombinant \u003cem\u003eTaq\u003c/em\u003e DNA Polymerase (Invitrogen), and 5 \u0026micro;L of DNA template at a concentration of 15 ng/\u0026micro;L.\u003c/p\u003e\u003cp\u003eThe ITS-1 oligonucleotides were employed for the taxonomic identification of the fungi (ITS1-1F-F 3\u0026rsquo;-CTTGGTCATTTAGAGGAAGTAA and ITS1-1F-R 3\u0026rsquo;-GCTGCGTTCTTCATCGATGC) (White, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). The final reaction volume was 25 \u0026micro;L, and PCR was conducted over 35 cycles, involving denaturation at 95 \u0026ordm;C for 30 s, annealing at 55 \u0026ordm;C for 45 s, and extension at 72 \u0026ordm;C for 1 min and 30 s. The amplification process commenced with an initial denaturation step of 3 min at 95 \u0026ordm;C and ended with a final extension step of 5 min at 72 \u0026ordm;C. Subsequently, the amplified products were subjected to electrophoresis in a 0.8% agarose gel, using FluoSafe as a nucleic acid dye. Electrophoresis was conducted at 100 mA for 45 minutes and bands were visualized under UV light.\u003c/p\u003e\n\u003ch3\u003eDNA Sequencing and Data Analysis\u003c/h3\u003e\n\u003cp\u003eSequencing was carried out utilizing the Sanger sequencing method. The resulting data were compiled and utilized for species identification through the Basic Local Alignment Search Tool (BLASTn) with a public library available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Sequences for phylogenetic analysis were also obtained from the same database. The obtained DNA sequences were deposited in the NCBI database under accession numbers PX072001-PX072004 (Supplemental data S1).\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003ePhylogenetic Analysis\u003c/h2\u003e\u003cp\u003eTo construct the phylogenetic tree, a data matrix was generated based on the lowest E-value results obtained from the NCBI's BLASTn tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://blast.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://blast.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for our sequences. The data matrix in .FASTA format underwent multiple sequence alignment using MAFFT, followed by an Alignment Curation using BMGE, a Tree Inference by PhyML\u0026thinsp;+\u0026thinsp;SMS and Tree Rendering by Newick Display (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ngphylogeny.fr/\u003c/span\u003e\u003cspan address=\"https://ngphylogeny.fr/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Lemoine, et al.2019). Tree visualization and editing was performed using iTol (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://itol.embl.de/\u003c/span\u003e\u003cspan address=\"https://itol.embl.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Letunic and Bork, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eHPLC and LCMS\u003c/h2\u003e\u003cp\u003ePurified fractions obtained from \u003cem\u003eDiplodia\u003c/em\u003e sp. and \u003cem\u003eAureobasidium\u003c/em\u003e sp. were analyzed for presence of QS-21 and related saponins. High-Performance Liquid Chromatography (HPLC) was carried out using an external standard curve prepared with authentic QS-21 (Creative Biolabs, NY, USA) or reference QB-90 purified from \u003cem\u003eQ. lancifolia\u003c/em\u003e leaves. Mobile phase consisted of a combination of acetonitrile (phase A) and Milli-Q water (phase B), both containing 0.1% formic acid. Gradient started at 25% of B changing to 50% of B within 20 min. A C18 RP column (250 mm x 4.6 mm (5\u0026micro;m) \u0026ndash; Agilent Eclipse - XDB) was the stationary phase. Analyses were carried out at 214 nm in a Shimadzu Prominence chromatograph equipped with UV-Vis DAD detector.\u003c/p\u003e\u003cp\u003eLiquid Chromatography-Mass Spectrometry (LCMS) measurements were performed using the LCMS Amazon speed Iontrap (Bruker Daltonics Inc., MA, USA), following a previously established protocol (Wallace et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Samples were injected at a concentration of 10 mg/mL (20 \u0026micro;L) for preliminary evaluation. Authentic QS-21 (Creative BioLabs, NY, United States) was used as standard. Additionally, \u003cem\u003eQ. lancifolia\u003c/em\u003e leaf extract was injected at 10 mg/mL to serve as a reference control for triterpene saponins. The liquid chromatography was conducted using a C18 column (250 mm x 4.6 mm, 5 \u0026micro;m). The mobile phase consisted of water with 0.1% formic acid (v/v) for phase A and acetonitrile with 0.1% formic acid (v/v) for phase B. The gradient began with 15% B, increasing to 65% B over 50 minutes. The flow rate was maintained at 0.6 mL/min. Spectral data were collected in negative mode, with a full scan performed from 800 to 2500 m/z at a resolution speed of 8.1 m/z per second. The most abundant ion was isolated in the Ion Trap for MS2 fragmentation. MS2 data were manually deconvoluted to identify the target compounds.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eAntimicrobial activity\u003c/h2\u003e\u003cp\u003eThe antimicrobial activity of fungal extracts, as well as crude and purified fractions of \u003cem\u003eQ. lancifolia\u003c/em\u003e, was assessed using the agar diffusion method. Briefly, standardized inoculum at 0.5 Mc Farland scale (OD\u003csub\u003e600\u003c/sub\u003e 0.08\u0026ndash;0.1) of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 35591, \u003cem\u003eEnterococcus faecalis\u003c/em\u003e ATCC 29212, \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC 25922 and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e ATCC 23893 were plated on TSA (Tryptone Soy Agar) using a sterile swab. Then, 20 \u0026micro;L of each extract or disk with standardized antimicrobial were directly applied to the surface of agar plates. Petri dishes were kept at 37 \u0026ordm;C for 24 hours, until inhibition zones were measured (Kimura et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Motta and Brandelli, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2002\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eBased on agar diffusion tests, select bacteria (\u003cem\u003eP. aeruginosa\u003c/em\u003e) and extracts (SPE \u003cem\u003eDiplodia\u003c/em\u003e and QB-90) were submitted to the microdilution test using the 96 well plate in TSB. Concentrations ranged from 2.5\u0026ndash;0.001 mg/mL for purified \u003cem\u003eDiplodia\u003c/em\u003e extract fraction, and from 25\u0026ndash;0.012 mg/mL for QB-90. Chloramphenicol was used as standard drug (320 to 0.15 \u0026micro;g/mL). After incubation, OD\u003csub\u003e600\u003c/sub\u003e was recorded for each well for growth assessment. Percentage inhibition was calculated as response parameter for anti-\u003cem\u003ePseudomonas\u003c/em\u003e activity (adapted from Methodology for Agent Sensitivity Tests Antimicrobials by Dilution for Bacteria Aerobic Growth, M7-A6 standard, by NCCLS - National Committee for Clinical Laboratory Standards).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eIsolation and selection of microorganisms\u003c/h2\u003e\u003cp\u003eTwenty-two fungal and twenty bacterial strains were isolated from \u003cem\u003eQ. lancifolia\u003c/em\u003e. Among these, four fungal strains were selected based on their capacity to produce biosurfactants, as evidenced by differential foam formation and the presence of bands stained on TLC (strains 12PFR4, 06SFR4, and 12PFR1) using sulfuric anisaldehyde. No bacterial strain showed intense foam formation. Additionally, fungal strain 22PFR4 (2) exhibited bands like some of the those of \u003cem\u003eQ. lancifolia\u003c/em\u003e as assessed by TLC (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), albeit apparently not of triterpene nature.\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\u003eGeneral characteristics of isolates positive for biosurfactants in foam and/or TLC tests.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\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=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStrain\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFoam\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTLC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eYield (%, w/w)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eColony\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eSource\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eF5_12PFR4 (1)*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e++\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGreen/purple\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eGrey/black\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eSeedling\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eF9_22PFR4 (2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eYellow\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eGrey/black\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eSeedling\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eF21_06SFR4 (3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBluish pink/purple\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eWhite/pink\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eSeed\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eF23_12PFR1 (4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e++\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eYellow/pink\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e6.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eBlack/brown\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eSeedling\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003e*(1) \u003cem\u003eDiplodia\u003c/em\u003e sp.; (2) \u003cem\u003eAcremonium\u003c/em\u003e sp.; (3) \u003cem\u003eFusarium\u003c/em\u003e sp.; and (4) \u003cem\u003eAureobasidium\u003c/em\u003e sp.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eMolecular Identification and Yield\u003c/h2\u003e\u003cp\u003eTaxonomic identification conducted by sequencing of the ITS-1 region and local alignment analysis revealed the presence of \u003cem\u003eDiplodia\u003c/em\u003e sp. (12PFR4), \u003cem\u003eAcremonium\u003c/em\u003e sp. (22PFR4), \u003cem\u003eFusarium\u003c/em\u003e sp. (06SFR4), and \u003cem\u003eAureobasidium\u003c/em\u003e sp. (12PFR1). The total exuded fraction for these species was quantified using a triterpene saponin-specific purification method, resulting in yields of 3.0%, 0.74%, 0.40%, and 6.28% (w/w), respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The macro and micromorphology of the colonies of the selected species are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn related studies focused on the bioprospecting of endophytes with saponin production, species such as \u003cem\u003eAspergillus terreus\u003c/em\u003e, \u003cem\u003eA. flavus\u003c/em\u003e, \u003cem\u003ePenicillium\u003c/em\u003e sp., and \u003cem\u003eTalaromyces pinophilus\u003c/em\u003e have been isolated from the roots of \u003cem\u003eAsparagus racemosus\u003c/em\u003e Willd., a saponin-producing plant naturally occurring in Africa, Asia, and Oceania (Rani \u003cem\u003eet al.\u003c/em\u003e 2022). Besides, \u003cem\u003eF. oxysporum\u003c/em\u003e and \u003cem\u003eA. niger\u003c/em\u003e have been prospected from \u003cem\u003ePanax notoginseng\u003c/em\u003e (Burk), which is naturally rich in ginsenosides. Saponins similar in character to those of the host plant have been elucidated, and the purified metabolites exhibited antimicrobial activity (Jin et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Moreover, \u003cem\u003eF. tricinctum\u003c/em\u003e, \u003cem\u003eAlternaria\u003c/em\u003e sp., \u003cem\u003eChaetosphaeronema\u003c/em\u003e sp., \u003cem\u003eMucor\u003c/em\u003e sp., and \u003cem\u003eTrichoderma\u003c/em\u003e sp. were isolated from \u003cem\u003eLithospermum officinale\u003c/em\u003e L., a plant producing shikonin - an anticancer compound (Mollaei et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The same study also recorded this alkaloid in the endophyte \u003cem\u003eF. tricinctum\u003c/em\u003e, suggesting the fungal strain could be able to produce plant-like specialized metabolites.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003ePhylogenetic Analysis\u003c/h2\u003e\u003cp\u003eThe phylogenetic tree illustrates the grouping of the select strains into four distinct clades. These separate groups represent the different genera, \u003cem\u003eAureobasidium\u003c/em\u003e sp., \u003cem\u003eDiplodia\u003c/em\u003e sp., \u003cem\u003eAcremonium\u003c/em\u003e sp. and \u003cem\u003eFusarium\u003c/em\u003e sp. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eDiplodia\u003c/em\u003e sp., (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e \u0026ndash; a, b), appeared to be a predominant endophytic fungus in \u003cem\u003eQ. lancifolia\u003c/em\u003e, as evidenced by its consistent re-isolation during the \u003cem\u003ein vitro\u003c/em\u003e processing of cells and tissues from the same species. \u003cem\u003eDiplodia\u003c/em\u003e belongs to the order Botryosphaeriales and is primarily associated with diseases in woody plant species and major crops, such as maize (Yang et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This genus is affiliated with Rosaceae, and its species may or may not induce pathological conditions, like stem rot and leaf spots (Philips et al. 2012). \u003cem\u003eDiplodia\u003c/em\u003e is considered a facultatively associated taxon with pathosystems but is also conclusively identified as a component of plant endophytic communities (White et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Plant-associated \u003cem\u003eDiplodia\u003c/em\u003e sp. have been shown to produce an array of specialized metabolites, mainly polyketide - and terpenoid - derived compounds. The most notable bioactivities of these fungi are antimicrobial and phytotoxic (Salvatore et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDespite some species of \u003cem\u003eFusarium\u003c/em\u003e being recognized for their negative effects in crops, species isolated from endophytic communities of medicinal plants show considerable potential for obtaining bioactive products from specialized metabolism, including some related to those of the plant hosts (Mollaei et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). For instance, \u003cem\u003eFusarium\u003c/em\u003e sp. isolated from \u003cem\u003eP. notoginseng\u003c/em\u003e yielded several ginsenosides (Wu et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Jin et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Moreover, \u003cem\u003eFusarium\u003c/em\u003e isolated from \u003cem\u003eDioscorea nipponica\u003c/em\u003e was identified as a species of significance for the biotransformation and diversification of saponins from \u003cem\u003eParis polyphylla\u003c/em\u003e (Chen et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eAcremonium\u003c/em\u003e, known for its phylogenetic complexity, is a genus associated with food contamination and a causal agent of plant diseases (Teixeira and Machado, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Summerbell et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). It includes strains with endophytic character, such as \u003cem\u003eAcremonium\u003c/em\u003e sp., isolated from \u003cem\u003eLilium davidii\u003c/em\u003e, which exhibited plant growth stimulation and control activity against phytopathogenic species (Khan et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In another study, \u003cem\u003eAcremonium strictum\u003c/em\u003e showed the ability to biotransform ginsenoside Rb1 from \u003cem\u003eP. ginseng\u003c/em\u003e (Chen et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eAureobasidium\u003c/em\u003e sp., a dimorphic fungus, is considered of interest in the biotechnology sector due to \u003cem\u003eA. pullulans\u003c/em\u003e production of industrial enzymes, antimicrobials, single-cell proteins, pullulans, leamokines with antimicrobial and cytotoxic effects on tumor cell lines, iron-chelating siderophores, and β-glucans (Chi et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Manitchotpisit et al. 2011; Prasongsuk et al. 2017). \u003cem\u003eA. pullulans\u003c/em\u003e A11211-4-57 produced two new biosurfactants, pullusurfactans F and G. These myo-inositol lipids exhibited strong surfactant activity at 1.0 mg/L, making them suitable for cleaning applications (Kim et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAlthough the production of complex plant specialized metabolites by fungal endophytes has been often reported, there have been several legitimate challenges to the validity of some long-term results involving the topic, at least in the case of taxol (G\u0026auml;rditz and Czesnick, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Stadler and Kolarik, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Indeed, in the present work, QS-21 could not be detected in the endophyte cultures. In addition, preliminary attempts to amplify from fungal DNA a key gene of QS-21 biosynthesis encoding beta-amyrin synthase proved unsuccessful (data not shown).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eHPLC and LCMS results\u003c/h2\u003e\u003cp\u003ePurified fractions from \u003cem\u003eDiplodia\u003c/em\u003e sp. and \u003cem\u003eAureobasidium\u003c/em\u003e sp. aqueous extracts produced foam in water. Therefore, these samples were chemically analyzed for the presence of triterpene saponins. Initial HPLC data suggested the presence of saponin-like molecules in fungal extracts with peak retention times resembling those of QS-21 and related saponins found in plants. However, LCMS\u003csup\u003e2\u003c/sup\u003e did not confirm any triterpenoid saponins in fungi samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In contrast, the expected presence of QS-21 in \u003cem\u003eQ. lancifolia\u003c/em\u003e extract was confirmed, as previously reported (Wallace, 2019).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eAntimicrobial activity\u003c/h2\u003e\u003cp\u003ePotential inhibitory activity of plant and fungal extracts was tested against pathogenic Gram-positive and Gram-negative bacteria. Agar diffusion tests showed bioactivity of SPE purified fraction from \u003cem\u003eDiplodia\u003c/em\u003e (5 mg/mL) and \u003cem\u003eQ. lancifolia\u003c/em\u003e (QB-90, 50 mg/mL) against \u003cem\u003eP. aeruginosa\u003c/em\u003e, with inhibition zones of 14.3 and 11.4 mm in diameter, respectively. Norfloxacin (30 \u0026micro;g/mL), a standard drug against Gram-negative bacteria, presented an inhibition diameter of 14.3 mm (Supplemental data S2).\u003c/p\u003e\u003cp\u003eMaximum growth inhibition has been obtained from broth microdilution tests, with 51.4% for SPE fraction from \u003cem\u003eDiplodia\u003c/em\u003e sp. at 2.5 mg/mL and 42.2% for QB-90 (\u003cem\u003eQ. lancifolia\u003c/em\u003e purified saponin fraction) at 25 mg/mL (Supplemental data S3). Despite the presence of various peaks on \u003cem\u003eDiplopia\u003c/em\u003e sp. purified fraction chromatogram, indicating the presence of various compounds, an activity of more than 50% of inhibition was recorded against \u003cem\u003eP. aeruginosa\u003c/em\u003e, an important Gram-negative pathogenic bacterium. Hence, a synergistic mechanism of action might be possible.\u003c/p\u003e\u003cp\u003ePrevious studies from endophytic fungi from \u003cem\u003eKigelia africana\u003c/em\u003e Lam. (sausage tree) showed remarkable anti-\u003cem\u003ePseudomonas\u003c/em\u003e activity of \u003cem\u003eNeofusicoccum luteum\u003c/em\u003e fractions with minimal inhibitory concentration of 156.50 \u0026micro;g/mL after seven days of fungal cultivation, and 19.53 \u0026micro;g/mL after 30 days of static fermentation. A purified compound, a C16-terpene dilactone presented MIC value of 0.61 \u0026micro;g/mL, playing a role in the antimicrobial activity (Bodede et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDespite the inhibitory activity exhibited by the \u003cem\u003eDiplodia\u003c/em\u003e purified fraction in the first fermentation batch, a second independent experiment, conducted under identical culture media, temperature, agitation, and incubation time, yielded a different HPLC profile (Supplemental Figure S4). Although maintaining foaming capacity relative to the culture media control, bioactivity assessment by agar diffusion assay revealed no inhibitory activity of the purified fraction of the second batch against \u003cem\u003eP. aeruginosa\u003c/em\u003e. This variability of chemical profile and associated bioactivity is likely due to metabolic changes in endophyte cultures over time, particularly following isolation from plant tissues. In fact, this is not surprising given the specific microenvironments, nutrient and energy sources, signal exchange molecules, and impact of seasonal variations of the \u003cem\u003ein planta\u003c/em\u003e conditions, all of which differ markedly from \u003cem\u003ein vitro\u003c/em\u003e media cultivation. Even within the endophytic environment, the temporal and spatial dynamics of fungal composition, abundance and interaction with host plants is quite significant (Crasta and Raveesha, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Ullah et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis is the first report of endophytic microorganisms in \u003cem\u003eQ. lancifolia\u003c/em\u003e. Four strains of fungi producing biosurfactants-like foaming agents were prospected from different plant sources and identified by molecular techniques, confirming the presence of \u003cem\u003eDiplodia\u003c/em\u003e sp., \u003cem\u003eFusarium\u003c/em\u003e sp., \u003cem\u003eAcremonium\u003c/em\u003e sp. and \u003cem\u003eAureobasidium\u003c/em\u003e sp. Despite the absence of key plant compounds in the most promising select endophytes, purified fractions showed overt foaming properties. Endophytic species of \u003cem\u003eQ. lancifolia\u003c/em\u003e may constitute a platform to produce compounds of biotechnological interest.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDeclaration of generative AI and AI-assisted technologies in the writing process\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring the drafting stage of the manuscript, the author(s) used OpenAI's ChatGPT in order to assist with language refinement, and organization of the text. After using this tool, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by grants from FAPERGS (Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul 2019/15477-3; 24/2551-0001321-0; 24/2551-0000697-4), CNPq (National Council for Scientific and Technological Development 42430/2-21-5; 310775/2021-3), and CAPES (Coordination for Improvement of Higher Education Personnel- finance code 001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are grateful to Prof. Gilson R. Pires Moreira (Dept of Zoology, UFRGS) for access to trees growing on his private property.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFábio Antônio Antonelo:\u0026nbsp;\u003c/strong\u003eConceptualization, Investigation, Methodology, Writing – original draft. \u003cstrong\u003eEliane Zachert:\u0026nbsp;\u003c/strong\u003eMethodology, Writing – review \u0026amp; editing. \u003cstrong\u003eYve Verônica Magedans:\u003c/strong\u003e Methodology,Data curation, Writing – review \u0026amp; editing.\u003cstrong\u003eAnna Alves Yendo:\u0026nbsp;\u003c/strong\u003eMethodology. \u003cstrong\u003eCibele Tesser:\u0026nbsp;\u003c/strong\u003eMethodology. Validation. \u003cstrong\u003eAlice Elvira Teixeira dos Santos:\u003c/strong\u003e Methodology.\u003cstrong\u003eFernanda Cortez Lopes:\u0026nbsp;\u003c/strong\u003eInvestigation, Methodology, Validation, Writing – review \u0026amp; editing. \u003cstrong\u003eMarilene Henning Vainstein:\u003c/strong\u003e Resources.\u003cstrong\u003e\u0026nbsp;Charley Christian Staats:\u0026nbsp;\u003c/strong\u003eWriting – review \u0026amp; editing. \u003cstrong\u003eJuliana Morini Küpper Cardoso:\u0026nbsp;\u003c/strong\u003eWriting – review \u0026amp; editing.\u003cstrong\u003e\u0026nbsp;Arthur Germano Fett-Neto:\u0026nbsp;\u003c/strong\u003eInvestigation, Resources, Supervision, Project administration, Validation, Writing – review \u0026amp; editing.\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003eAgatonovic-Kustrin S, Kustrin E, Gegechkori V, Morton DW, 2019. 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Families, genera, and species of \u003cem\u003eBotryosphaeriales\u003c/em\u003e. \u003cem\u003eFungal Biology\u003c/em\u003e \u003cstrong\u003e121(4):\u003c/strong\u003e 322-346. https://doi.org/10.1016/j.funbio.2016.11.001\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Universidade Federal do Rio Grande do Sul (UFRGS)","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":"bioprospecting, specialized metabolites, microorganisms, foam production, plant endophytes","lastPublishedDoi":"10.21203/rs.3.rs-8253976/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8253976/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEndophytes are widespread microorganisms that colonize plant tissues without causing apparent harm. These organisms possess diverse metabolic repertoires and can be alternatives or complementary sources of high-value bioactive compounds. Saponins stand out due to their surfactant, foaming, and immunoadjuvant properties. Triterpene saponins from \u003cem\u003eQuillaja\u003c/em\u003e spp. are used to enhance both humoral and cellular responses in human and veterinary vaccines. Herein, screening and identification of biosurfactant-producing endophytes from \u003cem\u003eQ. lancifolia\u003c/em\u003e tissues were carried out. The aim of the study was to contribute to the characterization of endophyte microbial and biochemical diversity in \u003cem\u003eQ. lancifolia\u003c/em\u003e. A total of 42 endophytes (22 fungi and 20 bacteria) were isolated from \u003cem\u003eQ. lancifolia\u003c/em\u003e plantlets, callus, cell suspensions and seeds, followed by microbial culture, and screening for extracellular foam production. Four fungi isolates were selected as foam-producing strains and were taxonomically identified using ITS-1 DNA sequencing. Foam production tests were performed using extracts from the liquid filtrate of cultures for subsequent triterpene screening by thin layer chromatography. Select extracts were purified and concentrated by solid phase extraction. Antibacterial activity was screened through agar diffusion and microdilution tests. \u003cem\u003eDiplodia\u003c/em\u003e sp., \u003cem\u003eAcremonium\u003c/em\u003e sp., \u003cem\u003eFusarium\u003c/em\u003e sp. and \u003cem\u003eAureobasidium\u003c/em\u003e sp. were identified as natural foam-producing endophytes. Liquid chromatography-mass spectrometry analyses revealed major differences between plant and fungi purified fractions. This work constitutes the first report of endophytic microorganisms associated with \u003cem\u003eQ. lancifolia\u003c/em\u003e. It not only provides insights on the metabolic potential of these beings but also buttresses future investigations on their biotechnological applications.\u003c/p\u003e","manuscriptTitle":"Screening and molecular identification of biosurfactant-producing endophytes from Quillaja lancifolia D. 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