Introduction

Endophytic fungi reside within healthy plant tissues and play a crucial role in the plant’s microecosystem. Their population dynamics are influenced by factors such as the genetic makeup, age, and environmental conditions of the host plant1,2. These fungi establish symbiotic relationships with their hosts, producing important bioactive compounds such as alkaloids, diterpenes, flavonoids, and isoflavonoids. These compounds contribute to plant growth, enhance resilience, and strengthen defense mechanisms against environmental stressors, pests, and diseases while also promoting the accumulation of secondary metabolites3,4.

Many bioactive compounds produced by endophytic fungi are chemically identical or similar to those synthesized by their host plants5. Examples include Taxol6,7, capsaicin8, and piperine9. The in vitro cultivation of endophytes capable of producing these metabolites presents a sustainable alternative to harvesting plants from natural populations, reducing environmental impact, and ensuring consistent production irrespective of climatic conditions10,11. Therefore, endophytic fungi have gained significant attention in biotechnology and industry for their applications as biocontrol agents, antimicrobials, antitumor agents, antioxidants, antidiabetics, antibiotics, and insecticides12,13.

Regarding antibacterial activity, alkaloids, terpenoids, polyketides, lactones, and phenolic substances produced by endophytic fungi target the bacterial cell walls, alter membrane permeability, or inhibit essential enzymes. For example, an endophyte isolated from Azadirachta indica produces alkaloids and lactones that inhibit S. aureus and B. subtilis14. While Gram-negative bacteria are generally more resistant due to their outer membrane, fungi like Colletotrichum produce compounds that penetrate this barrier, effectively inhibiting E. coli and P. aeruginosa15. Some fungi, such as Beauveria bassiana, exhibit broad-spectrum activity, producing compounds like beauvericin that inhibit both Gram-positive and Gram-negative bacteria5.

Endophytic fungi are also a rich source of antifungal agents. Their metabolites demonstrate potent activity against human and plant fungal pathogens16. These mechanisms include the production of primary metabolites, secondary metabolites, and volatile organic compounds (VOCs) that limit pathogen growth. For instance, the fungus Trichoderma spp. produces peptaibols and trichodermolides, which effectively inhibit phytopathogenic fungi of the genera Fusarium and Botrytis5. Similarly, the fungus Colletotrichum exhibits antifungal activity against Phytophthora infestans, the causal agent of late blight. Other genera, such as Aspergillus and Penicillium, are well-known for their production of secondary metabolites, including alkaloids and volatile compounds, which possess strong antifungal properties. Additionally, Phomopsis spp., an endophytic fungus isolated from medicinal plants, demonstrates antifungal activity against human pathogens Candida albicans and Aspergillus fumigatus17,18.

Criteria include plants with ethnobotanical significance, which are known to have traditional medicinal uses, and those growing in biodiversity hotspots. Tropical rainforests, with their intense competition and evolutionary pressures, offer a high likelihood of yielding novel molecular structures and bioactive compounds14,19.

The use of biological control for managing plant pests has recently gained significant attention. Many biological control agents, particularly endophytic fungi, are naturally associated with crops and play a crucial role in suppressing harmful pathogens20. Fungal pathogens can severely impact plant physiology, triggering host defense mechanisms in response to infection. In Bolivia, potato cultivation is essential for food security but faces significant threats from fungal diseases such as Phytophthora infestans (late blight), Fusarium solani, and Alternaria solani. These pathogens can cause yield losses of 30–50%, depending on management practices. National efforts, led by organizations like the PROINPA Foundation, focus on identifying these diseases and developing integrated control strategies, including resistant crop varieties and improved cultural practices21.

The plant species used in this study were selected and collected for their traditional medicinal uses (Table 1). In particular, species from the Piper genus have long been used to treat a range of conditions, including urological problems, skin, liver, and stomach disorders, and to support wound healing. They are also known for their antipyretic (fever-reducing) and anti-inflammatory effects. Additionally, Piper species show promise as natural antioxidants and antimicrobial agents for food preservation. Research indicates that their phytochemicals and essential oils, especially phenolic compounds, exhibit strong antioxidant activity, sometimes even outperforming synthetic alternatives. These compounds, along with monoterpenes and sesquiterpenes, also contribute to their antibacterial and antifungal properties against human pathogens22.

Table 1 Medicinal plants of the Valle Del sacta in Cochabamba, Bolivia.
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Plants from the Peperomia genus (within the Piperaceae family) are also recognized for their medicinal potential, with effects such as antihypertensive, anti-inflammatory, pain-relieving (antinociceptive), antiplasmodial, and osteogenic activity. Among them, Peperomia pellucida stands out for its strong antioxidant activity, largely due to its high levels of terpenoids, phenolic compounds, and flavonoids23.

Similarly, the species of the Faramea genus have been traditionally used to treat wounds, fevers, and digestive issues, thanks to their anti-inflammatory, analgesic, and antimicrobial properties. These effects are mainly attributed to the presence of bioactive compounds like alkaloids, flavonoids, and terpenoids, common in members of the Rubiaceae family. Some research suggests that related species possess antimicrobial activity, which may also be true for Faramea multiflora24.

Finally, Dictyoloma vandellianum A. Juss., from the Rutaceae family, has been traditionally used to relieve inflammation and pain. Its extracts contain bioactive compounds such as alkaloids, coumarins, and terpenoids that have demonstrated antimicrobial effects against various bacteria and fungi. In addition, its flavonoids and phenolic compounds contribute significantly to its antioxidant capacity25. In traditional Bolivian medicine, the leaves are widely used, especially in infusions, with the understanding that these are synthesis sites for a variety of active compounds26,27.

In this study, four medicinal plants from the Amazon region of Cochabamba, Bolivia, were collected. Four endophytic fungal species were isolated from their leaves and identified through 18S ribosomal DNA sequencing, phylogenetic analysis using NCBI-BLAST, and morphological characterization. The crude extracts from these fungi were tested for antibacterial activity using a minimum inhibitory concentration (MIC) assay, demonstrating strong efficacy against Gram-positive bacteria. Additionally, their antagonistic potential was assessed through confrontation with major phytopathogenic fungi affecting potato crops using the dual culture plate assay, revealing high antifungal activity. These findings highlight endophytic fungi as promising sources of biologically active secondary metabolites with potential applications in medicine, pharmaceuticals, agriculture, and environmental biotechnology.

Methods

Materials

Media used for the cultivation of Endophytic fungi and phytopathogenic fungi were Potato Dextrose Agar (PDA) and Potato Dextrose Broth (PDB), prepared following standard protocols28. For bacterial cultures, Mueller-Hinton Agar (MH), Trypticasein Soy Broth (TSB), and Trypticasein Soy Agar (TSA) (Oxoid, UK) were used. DNA extraction was performed using the Quick-DNA Fungal/Bacterial Miniprep Kit (Zymo Research, Irvine, CA, USA). PCR products were purified with the GeneJET PCR Purification Kit (Thermo Scientific, Waltham, MA, USA). For polymerase chain reaction (PCR) amplification, 18S rDNA-specific primers (EF4f and EF3r) from Integrated DNA Technologies (IDT, Coralville, IA, USA) were utilized. Chemical extraction and separation processes involved ethyl acetate, ethanol, sodium hypochlorite, p-anisaldehyde, ferric chloride, and aluminum chloride, which were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Isolation of endophytic fungi from Amazonian plants

Four Amazonian plant species were collected in Valle del Sacta, located within Carrasco Province, Cochabamba, Bolivia. The collection took place on the Universidad Mayor de San Simón property with the necessary permits granted by the authorities and in compliance with legal regulations (Table 1).

Plant identification was conducted by a professional expert using taxonomic keys for Amazonian and regional flora. Verification was carried out at the “Martín Cárdenas” National Forest Herbarium (BOLV), where a voucher specimen of each species was deposited. The collected plant material, consisting of small branches with leaves, was stored in refrigerated containers at 4 °C for transport to the laboratory19.

Endophytic fungi were isolated from healthy leaves. To do this, the leaf surface was disinfected by sequential washing with running water, 70% ethanol (2 min), 1% sodium hypochlorite (1 min), and sterilized water (2 min)14,19,29,30,31,32. The plant material was subsequently cut into fragments of approximately 5 mm² and plated on Petri dishes with PDA medium supplemented with chloramphenicol (100 µg/mL), using 8–10 fragments per plate. The plates were incubated at 22 °C for 15 days. The emerging fungal mycelium was transferred to plates with fresh medium for purification, obtaining axenic strains that were stored for subsequent characterization. For long-term conservation, the purified strains were suspended in PDB with 10% glycerol as a cryoprotectant, placed in sterile cryovials, and stored at −20 °C, to preserve their viability and ensure their stability for future studies33,34. All isolated fungal strains were deposited in the Microbiology Laboratory of the Centro de Tecnología Agroindustrial (CTA) at Universidad Mayor de San Simón, Cochabamba, Bolivia.

Identification of endophytic fungi

Four endophytic fungal strains were isolated from the leaves of four medicinal plant species, including Piper heterophyllum Ruiz & Pav. (Paichané negro), Peperomia sp., Faramea multiflora A. Rich. ex DC.(Yuracaré), and Dictyoloma vandellianum A. Juss. (Sombrerillo) (Table 1). Identification of endophytic isolates was carried out using both classical taxonomic methods based on morphological characteristics and molecular techniques.

For the macromorphological characteristics, after seven days of culturing at 30 °C on PDA in 10 × 100 mm Petri dishes, macroscopic vegetative traits were examined. These included colony characteristics such as color, texture, topography, diffuse pigmentation, and colony borders. Microscopic features, including hyphal structures and reproductive elements, were assessed using the microculture technique on PDA and yeast extract sucrose (YES) media for 7–10 days. Fungal samples were stained with Lactophenol blue and observed under an optical microscope (LRI - Olympus-100×/0.65, Tokyo, Japan). Observations were then compared with established taxonomic keys35,36,37.

The molecular identification was performed by amplifying the 18S rDNA gene using specific PCR primers listed in Table 2. DNA template was obtained through the collection of 50–100 mg wet weight of fungal samples from fresh cultures grown on PDA plates at 28–30 °C for 7–15 days. Thereafter, genomic DNA extraction was performed using the Quick-DNA Fungal/Bacterial Miniprep Kit (Zymo Research, Irvine, CA, USA), following the manufacturer’s protocol38and stored at −20 °C in 100 µL of DNA elution buffer.

For PCR amplification, EF4f and EF3r primers (Table 2) were used to target the 18S rDNA gene from fungal DNA (SMB-18, SMB-20, SMB-22, and SMB-28). PCR reactions were performed in a T100 Thermal Cycler (Bio-Rad Laboratories Inc., Hercules, CA, USA) under the conditions outlined in Table 2. Following PCR, the products were purified and sequenced, and phylogenetic trees were constructed using the methodology described by Mendieta-Brito et al.32 for the identification of the fungal strains. In all obtained phylogenetic trees for the four fungal species, the scale bar indicates nucleotide substitutions per site, using the neighbor-joining method. The number of nodes indicates the bootstrap values of 1000 replicates. The model used was Jukes-Cantor (JC).

Table 2 Molecular markers and their PCR primers and programs used.
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Chemical profile of crude extract from endophytic fungi

For the preparation of crude extracts, five 5 × 5 mm mycelium fragments from each endophytic fungal strain (SMB-18, SMB-20, SMB-22, and SMB-28) were excised from PDA plates and transferred to 500 mL Erlenmeyer flasks containing 200 mL of PDB. The cultures were incubated in a shaker incubator for 15 days at 30 °C and 200 rpm.

After incubation, the fungal biomass was harvested using vacuum filtration through a Büchner funnel fitted with standard filter paper. For the liquid-medium fraction, a liquid–liquid extraction technique was employed using ethyl acetate with a volume ratio of 1:1, and three extractions were performed. The remaining mycelium portion underwent maceration with twice its volume of ethyl acetate for 24 h. Subsequently, the extracted fractions from both mycelium and broth were combined, and the solvent was evaporated using a rotary evaporator under vacuum, with the water bath set at 30℃. Thin-layer chromatography (TLC) was employed to analyze the chemical profile of these crude extracts. TLC plates were developed using suitable eluents hexane: ethyl acetate (1:1); and developer solutions to identify and compare the compounds present in the extracts. The Retention factor (Rf) of each compound was calculated as the ratio of the distance migrated by the compound to the distance migrated by the eluent front, according to the methodology described by Mendieta-Brito et al.32.

Antibacterial test: determination of minimum inhibitory concentration (MIC)

The antimicrobial activity of the crude fungal extracts was assessed by determining the minimum inhibitory concentration (MIC) using a microdilution method, as described by the Clinical and Laboratory Standards Institute40,41, with slight modifications. Since in pre-test on disc diffusion assay, the sample dried from aquouse phase wasn’t shown the activity against to gram negative and positive mircroorganisms, ethylacetate extracts were evaluated on MIC. The MIC was evaluated against the following five bacterial strains that were obtained from the Korean culture collections (KCTC): Staphylococcus aureus (KCTC 3881, Gram-positive), Escherichia coli (KCTC 1039, Gram-negative), Pseudomonas aeruginosa (KCTC 1637, Gram-negative), Enterococcus faecalis (KCTC 2011, Gram-positive), and Propionibacterium acnes (KCTC 6919, Gram-positive).

Bacterial cultures were grown under specific conditions: E. coli, S. aureus, and P. aeruginosa were incubated aerobically at 37 °C in Nutrient Broth (MBcell, South Korea), E. faecalis and P. acnes were cultured at 37 °C under facultative anaerobic conditions in Brain Heart Infusion (BHI) Broth (MBcell, South Korea). The bacterial suspensions were diluted until the absorbance at 660 nm reached 0.03, corresponding to a concentration of approximately 1–2 × 107 CFU/mL.

The test was performed in a sterilized 96-well microplate (Falcon, Dublin, OH, USA), with 1 mL of fungal extract serially diluted in each well. The initial concentration of the extracts was adjusted to 1000 µg/mL, followed by serial twofold dilutions to 500, 250, 125, 62.5, 31.3, and 15.6 µg/mL. To each well, 10 µL of bacterial culture medium was added. The microplates were incubated for 12 h at 37 °C with shaking (150 rpm). The bacterial growth inhibition was quantified by measuring the optical density at 620 nm (OD620) using a spectrophotometer (BIO-RAD Laboratories Inc., USA).

Ampicillin was used as a positive control, with known MIC values: 1.25 µg/mL for E. coli and E. faecalis, and a range of 20–0.625 µg/mL for S. aureus (MIC90 not determined). The MIC ranges of 1.0–0.05 µg/mL for P. aeruginosa and P. acnes were also determined based on ampicillin’s known activity.

Antifungal test: direct confrontation—dual culture plate assay

Phytopathogenic fungi, including Helminthosporium sp., Fusarium oxysporum, and Fusarium solani, which are of agronomic importance to potato crops, were obtained from the microbiology and phytopathology laboratories of the PROINPA Foundation.

The antagonistic activity of the endophytic fungi, Aspergillus sp. SMB-18, Fusarium sp. SMB-20, Aspergillus sp. SMB-22, and Alternaria sp. SMB-28 was evaluated against the phytopathogenic fungi, using a dual culture assay. In the assay, the inoculation of the endophytic fungi and the pathogen in a paired fashion on the surface of PDA or YES plates was done. Where each fungus (endophyte and pathogen) was inoculated 2.1 cm apart from the perimeter of the Petri dish42.

The inoculation was performed in triplicate. Also, a control plate containing only the pathogen was included to monitor its uninhibited growth. All plates were incubated in a New Brunswick Innova 4200 Incubator (Edison, NJ, USA), under standardized conditions (natural light/dark cycles, humidity 30–40%, temperature 23–25 °C) to support optimal microorganism growth.

Pathogen growth was measured at regular intervals (7, 10, 14, and 18 days), depending on the maximum growth observed in the control plates. The pathogen inhibition was calculated by comparing the radial growth of the pathogen on treated plates (facing the endophyte) with its growth on control plates43. The percentage of mycelial growth inhibition was determined using the following formula:

$$\:Inhibition\:Percent\:\left(I\right)\:=\:\left[\right(R1\:-\:R2)\:\div\:R1]\:\times\:\:100,$$

where R1 = radial growth of the pathogen in control and R2 = radial growth of the pathogen in treatment44.

Additionally, the interaction between the endophytic fungi and phytopathogens was categorized according to established classification systems (Table 3) based on the observed inhibitory effects against the growth of the pathogens.

Table 3 Types of the interaction categories between endophytic and pathogenic fungi.
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Results

Isolation and molecular identification of endophytic fungi

Figure 1 presents an image of the collected host plants from the Amazon region of Bolivia. These plants were identified by Mgr. Modesto Zarate, an associate researcher at the Herbarium, and their identification was corroborated by reference to the “Martín Cárdenas” National Forest Herbarium (BOLV).

Fig. 1
Fig. 1
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Collection of plant species in the Amazon region called Valle del Sacta at an altitude of 240 m above sea level, Cochabamba-Bolivia (A) Piper heterophyllum Ruiz & Pav. (17°06’06"S and 64°46’54"W) (B) Peperomia sp. (17°06’06"S and 64°46’54"W) (C) Faramea multiflora A. Rich. Ex DC. (17°06’00"S and 64°46’54"W) and (D) Dictyoloma vandellianum A. Juss. (17°06’31"S and 64°46’40"W), to the right of the photo of each plant is the sample assembled in a Micro-herbarium.

The species, known in their regions of origin for their medicinal uses (Table 1), were identified as Piper heterophyllum Ruiz & Pav., Peperomia sp., Faramea multiflora A. Rich. ex DC., and Dictyoloma vandellianum A. Juss. Voucher specimens are available under the following accession numbers: MZ6727, MZ6732, MZ6725, and MZ7738, respectively.

Four endophytic fungi were successfully isolated from the leaves of the four medicinal plants (Figs. 2, 3, 4, 5 and 6). The fungal isolates were subjected to both morphological and molecular identification processes to determine their taxonomic classification (Figs. 2, 3, 4,5 and 6, and Tables 4 and 5).

Fig. 2
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Phylogenetic tree based on the partial sequence of a small-subunit ribosomal RNA gene of endophytic fungus Aspergillus sp. SMB-18 (accession no. PQ490368) obtained with EF4f/Fung5r, showing its relationship via neighbor-joining with other closely related taxa from NCBI GenBank.

Fig. 3
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Phylogenetic tree based on the partial sequence of a small-subunit ribosomal RNA gene of endophytic fungus Fusarium sp. SMB-20 (accession no. PQ483103) obtained with EF4f/EF3r, showing its relationship via neighbor-joining with other closely related taxa from NCBI GenBank.

Fig. 4
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Phylogenetic tree based on the partial sequence of a small-subunit ribosomal RNA gene of endophytic fungus Aspergillus sp. SMB-22 (accession no. PQ483115) obtained with EF4f/EF3r, showing its relationship via neighbor-joining with other closely related taxa from NCBI GenBank.

Fig. 5
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Phylogenetic tree based on the partial sequence of a small-subunit ribosomal RNA gene of endophytic fungus Alternaria sp. SMB-28 (accession no. PQ483124) was obtained with EF4f/EF3r, showing its relationship via neighbor-joining with other closely related taxa from NCBI GenBank.

Fig. 6
Fig. 6
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Macro- and micromorphological characteristics of endophytic fungi: Colonies on PDA upper (A) and reverse side (B) after 7 days at 30 °C. Colonies on YES upper (C) and reverse side (D) after 7 days at 30 °C. Generative hyphae and mycelium (E) and conidiophores and conidia (F). Samples stained with Lactophenol blue, observed under an LRI-Olympus microscope at 100×/0.65 magnification, and a scale of 1:200.

Table 4 GenBank accession numbers for the isolated endophytic fungi.
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Table 5 Cultural and morphological characteristics of 4 endophytic fungal isolates from Amazonian Bolivian plants.
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Molecular identification was conducted by amplifying the fungal 18S rDNA with the EF4f/EF3r primers, which successfully amplified the target sequences for all four fungal strains (SMB-18, SMB-20, SMB-22, and SMB-28). BLAST analysis of the resulting sequences identified these fungi as belonging to three distinct genera: Fusarium (SMB-20), Aspergillus (SMB-18, SMB-22), and Alternaria (SMB-28), confirming their classification as endophytic fungi (Figs. 2, 3, 4 and 5). Based on partial sequences of the 18S rDNA gene, phylogenetic analysis revealed that the isolated strains are closely related to other fungal species. However, species-level identification remained challenging due to the limited availability of comparable molecular data. The phylogenetic trees, shown in Figs. 2, 3, 4 and 5, were constructed using the Neighbor-Joining method, selecting the most suitable model based on sequence similarity. The fungal sequences obtained in this study were deposited in GenBank (Table 4). Given the low probability and the limited approach to the genus level, it is evident that an alternative locus is required for proper molecular identification and to expand the sequence data (Figs. 2, 3, 4 and 5).

Morphological characteristics of endophytic fungi

The description of cultural and morphological characteristics of fungal endophytes, along with microphotographs of their morphological structures, is presented in Table 5; Fig. 6.

The morphological identification of the different strains of endophytic fungi in this study is primarily based on distinct characteristics, including the shape and size of macro- and microconidia, the presence or absence of chlamydospores, as well as colony appearance, pigmentation, and growth rate on agar media45,46,47.

Figure 6 presents the cultural and morphological characteristics of the studied endophytic fungi, including mycelial growth on PDA and YES, along with microphotographs of their hyphae, conidiophores, and conidia. Colonies of endophytic fungi typically exhibit rapid growth on PDA media at 30 °C, ranging between 3 and 7 days. However, growth is comparatively slower in YES culture media, taking between 7 and 10 days. Table 5 presents the macro- and microscopic characteristics of the species with strong similarities in comparison to the studied strains. Specifically, Aspergillus niger was used for comparison with Aspergillus sp. SMB-18 and SMB-22, Fusarium oxysporum, were compared with Fusarium sp. SMB-20 and Alternaria alternata were compared with Alternaria sp. SMB-28.

In Aspergillus species, the size and morphology of ascospores, particularly diagnostic features such as rugosity, margins, wings, and grooves, are essential for species identification45,46. Aspergillus strains SMB-18 and SMB-22 produce abundant black conidia on the surface and lack a cottony texture when grown on PDB (Fig. 6A, B). In contrast, sporulation was reduced on YES medium (Fig. 6C, D), where the strains exhibited improved growth, making them suitable for microscopic analysis. These strains form filamentous hyphae resembling miniature plants (Fig. 6E) (Table 5). Initially, the mycelium appeared white but turned black within two days on PDA, coinciding with conidial spore production. The reverse side of the colony displayed a light-yellow coloration. Notably, the conidiophores and conidia observed in both strains (Fig. 6E, F) are characteristic of the genus Aspergillus and closely resemble those of Aspergillus niger. Fusarium species were primarily identified based on distinctive characteristics, including the shape and size of macro- and microconidia, colony morphology, pigmentation, and growth rate on agar media47. The Fusarium sp. SMB-20 exhibited greater growth in PDA than in YES, showing different mycelial characteristics. On PDA, color development was more intense, pink-purple, and the texture appeared more cottony (Fig. 6A–D). Microscopic analysis revealed abundant filamentous hyphae on both PDA and YES, elongated conidiophores with rounded ends, and oval conidia, typical of the Fusarium genus (Fig. 6E, F, and Table 5). The genus Alternaria comprises primary conidiophores that can be straight or curved, varying in length from short to long, and may be either simple or branched, with one or multiple apical conidiophores37. In Alternaria alternata, the conidia are oval, elongated ellipsoid, small, and septate, resembling those observed in our study strain, Alternaria sp. SMB-28. This strain forms slow-growing colonies that produce few spores, yet these spores share key characteristics with A. alternata. Additionally, the similarity extends to other features, such as colony color on PDA (Fig. 6A-F; Table 5).

Chemical profile of endophytic fungi extracts

The fungi Fusarium sp. SMB-20, Aspergillus sp. SMB-18, Aspergillus sp. SMB-22, and Alternaria sp. SMB-28 were cultivated in 200 mL of potato dextrose broth (PDB) for 15 days. Following cultivation, secondary metabolites were extracted from both the liquid media and fungal biomass using ethyl acetate. The yields of the extracts were 24.4 mg, 94.9 mg, 159.3 mg, and 36.1 mg for Fusarium sp. SMB-20, Aspergillus sp. SMB-18, Aspergillus sp. SMB-22, and Alternaria sp. SMB-28, respectively.

To identify the main groups of chemical compounds of the extracts, thin-layer chromatography (TLC) was employed, along with various staining techniques (Fig. 7A-E). The results from TLC analysis showed the presence of flavonoids, terpenoids, and phenolic compounds with visible chromophores under normal light for Aspergillus sp. SMB-18, SMB-22, and Alternaria sp. SMB-28 (Fig. 7A). However, no visible chromophores or highly unsaturated compounds were detected for Fusarium sp. SMB-20 under the same conditions.

Fig. 7
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Thin-layer chromatography (TLC) of extracts from endophytic fungi strains, SMB-18, SMB-20, SMB-22, and SMB-28 that were shown in the TLC plate as 18, 20, 22, and 28, respectively. Compounds with chromophores or high unsaturation observed under visible light (A). The presence of conjugated double bonds observed under UV light at 254 nm (B). The presence of flavonoids stained with aluminum chloride and illuminated under UV light at 365 nm (C). Compounds stained with p-anisaldehyde, indicating the presence of terpenes (purple spots) and flavonoids (red spots) (D). Phenolic compounds stained with ferric chloride (E).

Upon examination of the samples under UV light at 254 nm, conjugated double bonds were observed in all fungal extracts (Fig. 7B). Flavonoid compounds, which were stained with aluminum chloride and confirmed in all the extracts when exposed to UV light at 365 nm (Fig. 7C). Additionally, after developing the TLC plates with p-anisaldehyde, all four extracts exhibited purple spots with Rf values of 0.96 and 0.88, indicating the presence of terpenoid compounds. Red spots located at Rf values of 0.53 and 0.35 were identified in extracts from SMB-18 and SMB-22, suggesting the presence of flavonoids (Fig. 7D). These flavonoids were further corroborated by their appearance under UV light at 254 nm (Fig. 7B), confirming the presence of conjugated double bonds. Furthermore, the development of brown spots with ferric chloride suggested the presence of phenolic compounds, particularly in SMB-18, SMB-22, and SMB-28, with the latter showing a variety of phenolic compounds (Fig. 7E).

Antimicrobial activity of crude Ethyl acetate extracts from endophytic fungi

The antimicrobial properties of crude extracts from endophytic fungi were evaluated against five bacterial strains: Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis, and Propionibacterium acnes. The minimum inhibitory concentrations (MICs) were determined for each ethyl acetate extract, providing insights into their efficacy against these pathogens.The findings showed that the extracts exhibited strong antibacterial activity against Gram-positive bacteria, with MICs ranging from 15.6 to 100 µg/mL, as shown in Tables 5 and 6. In contrast, no antimicrobial effect was observed against the Gram-negative strains, suggesting a selective antibacterial profile targeting Gram-positive bacteria. This differential activity suggests that the extracts could be specifically effective for applications targeting Gram-positive bacterial infections.

Table 6 Minimum inhibitory concentration (MIC) and Inhibition activity at various concentrations of crude extract prepared from endophytic fungi against 5 bacteria.
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A closer look at the individual extracts revealed distinct patterns of inhibition. For instance, Aspergillus sp. SMB-18 exhibited strong antibacterial effects, where complete inhibition of E. faecalis was observed at a concentration of 1,000 µg/mL. It also significantly inhibited S. aureus, achieving substantial inhibition (++) down to 125 µg/mL. Notably, the extract demonstrated complete inhibition of P. acnes at 125 µg/mL and maintained strong inhibition down to 15.6 µg/mL, suggesting potential for use against P. acnes-related bacteria.

Fusarium sp. SMB-20 also demonstrated promising antibacterial properties. It completely inhibited E. faecalis and P. acnes at concentrations as low as 62.5 µg/mL, with substantial inhibition of S. aureus observed at 125 µg/mL. This indicates that Fusarium sp. SMB-20 may hold high promise in combating a range of Gram-positive bacterial pathogens.

The Aspergillus sp. SMB-22 extract was highly effective, particularly against S. aureus and P. acnes. A complete inhibition (+++) of E. faecalis and P. acnes was observed at 500 µg/mL. Moreover, substantial inhibition (++) of S. aureus was achieved at concentrations as low as 62.5 µg/mL. This suggests that Aspergillus sp. SMB-22 may be an excellent candidate for further exploration in the development of antibacterial agents. These results underscored the potential of Aspergillus sp. SMB-22 as a source of potent antimicrobial agents and highlighted the need for further investigation into the specific compounds responsible for this pronounced antibacterial activity.

Alternaria sp. SMB-28 also demonstrated antibacterial activity. The crude extract demonstrated inhibition of S. aureus across a range of concentrations, achieving complete inhibition by more than 90% (++) at 1,000 µg/mL, 500 µg/mL, and 250 µg/mL. The Alternaria sp. SMB-28 extract also showed antibacterial activity against P. acnes at 250 µg/mL, which further highlights its potential as a source for antibacterial agents.

Table 7 Summary of minimum inhibitory concentration (MIC) and Inhibition activity at 125 µg/mL of crude extract prepared from endophytic fungi.
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Antifungal test: direct confrontation—dual culture plate assay

The antagonistic potential of four endophytic fungi was evaluated through direct confrontation with phytopathogenic fungi using the dual culture plate assay. The inhibition of pathogen growth was quantified by measuring the radial mycelial extension in the confrontation assays (Fig. 8).

Fig. 8
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Direct confrontation of the endophytes Aspergillus sp. SMB-18, Fusarium sp. SMB-20, Aspergillus sp. SMB-22 and Alternaria sp. SMB-28 on the phytopathogens Helminthosporium, sp. (H), Fusarium oxysporum (Fo) and Fusarium solani (Fs). Confrontation of Fusarium sp. SMB-20 on PDA, on H (I), on Fo (V) and on Fs (IX). Confrontation of Alternaria sp. SMB-28 on PDA, on H (II), on Fo (VI) and on Fs (X). Confrontation of Aspergillus sp. SMB-18 on YES, on H (III), on Fo (VII) and on Fs (XI). Confrontation of Aspergillus sp. SMB-22 on YES, on H (IV), on Fo (VIII) and on Fs (XII).

Aspergillus sp. SMB-18 exhibited substantial inhibition of Helminthosporium sp., Fusarium oxysporum, and Fusarium solani by 62%, 42%, and 51%, respectively. Similarly, Fusarium sp. SMB-20 also showed notable antagonistic effects, with inhibition rates of 58% against Helminthosporium sp., 50% against F. oxysporum, and 57% against F. solani (Fig. 9).

Fig. 9
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Percentage of inhibition of phytopathogen growth by endophytic fungi.

Among the tested isolates, Aspergillus sp. SMB-22 exhibited the highest inhibition against Helminthosporium sp., achieving an impressive 80% inhibition. It also inhibited F. oxysporum and F. solani by 37% and 56%, respectively. In contrast, Alternaria sp. SMB-28 demonstrated relatively lower antagonistic activity, with inhibition percentages of 38% for Helminthosporium sp., 27% for F. oxysporum, and 20% for F. solani (Fig. 9).

These results indicate that Aspergillus sp. SMB-18 and SMB-22 exhibit strong potential as biocontrol agents against phytopathogenic fungi, particularly Helminthosporium sp., while Alternaria sp. SMB-28 showed weaker inhibitory effects.

Based on the dual culture plate assay, six distinct types of interactions (A-F) (Table 3) were identified between the four endophytic fungi and three potato crop pathogens (Table 8): Helminthosporium sp., Fusarium oxysporum, and Fusarium solani (Fig. 8). All endophytes exhibited some level of inhibition against the pathogens, with no confrontations categorized as Type A (mutual growth without inhibition). Type B interactions were observed in the confrontations of Fusarium sp. SMB-20 with F. oxysporum (Fig. 8-Fo (V)), Aspergillus sp. SMB-22 with F. oxysporum (Fig. 8-Fo (VIII)), and Alternaria sp. SMB-28 with F. solani (Fig. 8-Fs (X)), where a minor inhibition zone was observed between the interacting fungi.

Table 8 Types of confrontation as a result of dual tests between endophytic and phytopathogenic fungi.
Full size table

Type C interactions, characterized by moderate inhibition, were noted between Fusarium sp. SMB-20 and Helminthosporium sp. (Fig. 8-H (I)), where the pathogen’s growth was slightly hindered in the presence of the endophyte. Type D interactions were recorded for Alternaria sp. SMB-28 against both Helminthosporium sp. (Fig. 8-H (II)) and F. oxysporum (Fig. 8-Fo (VI)), where inhibition zones of varying sizes were observed but not as prominent as in Type E or F interactions.

Types E and F represented the interactions with the highest inhibition levels of the pathogen. These interactions were predominantly observed in the Aspergillus endophytes, SMB-18 and SMB-22. Type E interactions, which displayed more substantial inhibition, were seen in the confrontations of Aspergillus sp. SMB-18 with F. oxysporum (Fig. 8-Fo (VII)), Aspergillus sp. SMB-18 with F. solani (Fig. 8-Fs (XI)), and Aspergillus sp. SMB-22 with F. solani (Fig. 8-Fs (XII)). Type F, which indicated the strongest inhibition, was observed in the interactions of Aspergillus sp. SMB-18 with Helminthosporium sp. (Fig. 8-H (III)) and Aspergillus sp. SMB-22 with Helminthosporium sp. (Fig. 8-H (IV)), where the pathogens showed considerable growth reduction.

The results from the interaction classifications highlight the varying degrees of antagonistic activity exhibited by the endophytes, with Aspergillus sp. strains SMB-18 and SMB-22 showing the most potent antagonistic effects against the potato crop pathogens.

Discussion

The Amazon region of Bolivia is recognized as one of the most biodiverse areas in the world, as highlighted by Ibisch and Mérida48. Despite Bolivia’s biodiversity being underexplored, its immense natural wealth presents untapped potential for medicinal research. Nevertheless, the existing baseline data and their interpretation highlight the significance of Bolivia’s biodiversity at both national and international levels. This remarkable biodiversity underscores the region’s immense potential as a source of bioactive compounds for medicinal research. The deep connection between biodiversity and indigenous plant use creates a unique opportunity for discovering novel bioactive compounds from native plants, as well as their associated endophytic fungi. These microorganisms are known to produce a wide variety of bioactive metabolites that can be explored for promising applications in agriculture, medicine, and biotechnology. In this context, the traditional use of the plants and their ecological habitats are crucial factors to consider when isolating endophytes.

Our study, which focused on the isolation of endophytic fungi from plants in the Bolivian Amazon, aligns with the idea that regions with high biodiversity as likely to harbor unique microbial communities. Four fungal isolates, Aspergillus sp. SMB-18, Aspergillus sp. SMB-22, Fusarium sp. SMB-20, and Alternaria sp. SMB-28, were successfully isolated from the leaves of medicinal plants (Piper heterophyllum Ruiz & Pav., Peperomia sp., Faramea multiflora A. Rich. Ex DC., and Dictyoloma vandellianum A. Juss.). These results are consistent with previous studies emphasizing the rich phylogenetic diversity of endophytic fungi in tropical ecosystems, driven by the high diversity of host plants and environmental factors14,49.

Notably, while Aspergillus sp. SMB-18 and SMB-22 belong to the same genus; they exhibited distinct antagonistic profiles, supporting the hypothesis that even closely related species may evolve different adaptations based on their host environments. The isolation of Fusarium and Alternaria strains further supports the growing evidence of these genera being prevalent in tropical endophytic fungal communities50,51.

The molecular identification and phylogenetic analysis of these fungi demonstrate the need for improved species-level resolution. As we observed, utilizing a single genetic marker for identification may not be sufficient, particularly when distinguishing between closely related fungal species35,52. Chemical profiling of the fungal extracts revealed a wide array of secondary metabolites, such as flavonoids, terpenes, and phenolic compounds. The identification of these metabolites through TLC provides strong evidence for the biochemical potential of these endophytic fungi as sources of bioactive compounds5,15. Flavonoids and terpenes are well-known for their antimicrobial, antioxidant, and anti-inflammatory properties, which may explain the observed antimicrobial activities of the fungal extracts5. The presence of these compounds, particularly flavonoids with conjugated double bonds and terpenes observed under UV light, aligns with the antimicrobial activities demonstrated by these extracts. Moreover, the phenolic compounds detected, which are known for their ability to disrupt bacterial cell membranes, further support the idea that these extracts could have therapeutic value, particularly in antibacterial applications53.

The antimicrobial activity of the crude fungal extracts demonstrated selective efficacy against Gram-positive bacteria, including S. aureus, E. faecalis, and P. acnes (Table 6). This selective activity is likely due to the simpler cell wall structure of Gram-positive bacteria compared to Gram-negative bacteria, which may allow for better penetration and inhibition by the bioactive compounds54,55,56. The observed MICs ranged from 15.6 to 500 µg/mL. Aspergillus sp. SMB-18 and SMB-22 exhibited particularly strong activity against S. aureus and E. faecalis, with inhibition percentages reaching as high as 97.7% at 125 µg/mL. Although the MICs of the fungal extracts were less potent than the positive control, ampicillin, these results suggest that the fungi have considerable antibacterial properties, especially at higher concentrations. Fusarium sp. SMB-20 exhibited noteworthy activity with a MIC of 31.3 µg/mL to E. faecalis, making it a particularly promising candidate for future antibacterial research. Against P. acnes, a Gram-positive bacterium of dermatological relevance, Aspergillus sp. SMB-18, Fusarium sp. SMB-20, and Aspergillus sp. SMB-22 achieved 100% inhibition at 125 µg/mL. This highlights the potential of these fungi in addressing skin-related bacterial infections57. These findings demonstrate that certain endophytic fungi, particularly Aspergillus and Fusarium species, possess strong antibacterial properties. Future studies should focus on isolating and characterizing the specific bioactive metabolites responsible for these activities, which could lead to the development of novel antibiotics for clinical use.

The dual culture plate assay, used to assess the antifungal properties of the endophytes, revealed diverse antagonistic interactions between the fungi and three potato crop pathogens (Helminthosporium sp., Fusarium oxysporum, and Fusarium solani). Five distinct interaction types (B-F) were identified, reflecting the range of inhibitory responses exhibited by the fungi. Type E interactions, which showed the most strong inhibition, were observed in confrontations between Aspergillus sp. SMB-18 and both F. oxysporum and F. solani, as well as between Aspergillus sp. SMB-22 and F. solani. These results highlight the potential of these fungi, particularly Aspergillus species, as biocontrol agents for managing phytopathogenic fungi. Additionally, Fusarium sp. SMB-20 demonstrated Type C and B interactions, indicating its versatility in targeting both Helminthosporium sp. and F. oxysporum. The lower inhibition observed with Alternaria sp. SMB-28 suggests that its antifungal potential may be less pronounced, though it still exhibited some activity against F. solani.

Overall, the diverse interaction types and the promising antimicrobial activities demonstrated by these endophytic fungi suggest that they could serve as valuable sources of secondary metabolites with potential applications in medicine, agriculture, and biocontrol.

Conclusions

In this study, the isolation and identification of four endophytic fungi, Fusarium sp. SMB-20, Aspergillus sp. SMB-18 and SMB-22, and Alternaria sp. SMB-28, were successfully achieved from the leaves of different Amazonian medicinal plants in Bolivia: Piper heterophyllum Ruiz & Pav., Peperomia sp., Faramea multiflora A. Rich. ex DC., and Dictyoloma vandellianum A. Juss. The crude extracts from these fungi, cultured in PDB, demonstrated strong antibacterial activity against S. aureus, E. faecalis, and P. acnes, with MIC values ranging from 15.6 to 500 µg/mL. This selective efficacy against Gram-positive bacteria highlights the potential of these fungi as sources of bioactive compounds with antimicrobial properties. In antifungal assays, the dual culture plate method revealed varying degrees of antagonistic interactions between the isolated endophytes and the phytopathogens Helminthosporium sp., Fusarium oxysporum, and Fusarium solani. Notably, Aspergillus sp. SMB-18 and SMB-22 exhibited the highest inhibitory effects, classified under interaction types E and F, demonstrating strong potential as biocontrol agents against plant pathogens.

These findings underscore the significance of endophytic fungi as promising reservoirs of biologically active secondary metabolites with potential applications in medicine, pharmaceuticals, agriculture, and environmental biotechnology. Further studies should focus on the isolation, structural characterization, and mode of action of these bioactive compounds to harness their full potential for therapeutic and agricultural innovations.