The emergence of new diseases and the development of drug-resistant pathogenic microorganisms pose a serious problem to humankind. To resolve this issue, search for efficient antimicrobial agents is required. For thousands of years, natural products of plant origin have been used in traditional medicine to cure diseases in humans (Mahajan et al., 2013). Endophytes are considered potential sources of bioactive natural products (Hagag et al., 2022). Fungal endophytes, the microorganisms that reside in the internal tissues of plants without causing any apparent disease, are known to produce useful bioactive compounds, including antibacterial, antiviral, antioxidant, and anticancer ones, as well as industrially important hydrolytic enzymes (Wilson, 1995; Verma et al., 2014; Kharwar et al., 2014; Gupta and Chaturvedi, 2019). Endophytic fungi that living inside healthy plant tissues effectively protect their hosts from biotic and abiotic stresses (Omacini et al., 2001; Redman et al., 2002). Since endophytes occupy unique biological niches in plants growing in diverse environments, they may be considered a source of novel bioactive natural compounds (Strobel et al., 2003; Verma et al., 2009). Recent findings also suggest that endophytic fungi may mimic the synthesis of host-origin metabolites (Singh et al., 2021). Since the discovery of penicillin from Penicillium notatum, fungi have been a great source of bioactive metabolites (Berdy, 2005), with even higher chances of finding novel compounds in endophytic fungi, since they colonize a special niche (Gupta and Chaturvedi, 2019). This prediction led to the discovery of Paclitaxel (Taxol) from Taxus brevifolia (Stierle et al., 1993). Taxol is the world’s first billion-dollar anticancer drug, the main source of which is Taxus spp. Successful identification of the fungal taxol has led to discovery of other bioactive compounds, such as vincristine, vinblastine, piperine, and azadirachtin from the endophytic fungi of their original hosts (Kusari et al., 2013; Singh et al., 2021). Bioactive secondary metabolites of endophytic origin are a good alternative to existing synthetic drugs, which provides more resistance to pathogens. The plant targeted for the endophyte isolation in this study was Ocimum sanctum L. (holy basil, member of the family Lamiaceae), a well-known medicinal plant. It is native to the Indian subcontinent, is widely cultivated throughout the Southeast Asian tropics, and is known for its religious and versatile medicinal properties (Mahajan et al., 2013). The leaves of O. sanctum are well-known for their antibacterial, antifungal, antiulcerogenic, antistress, anticancer, analgesic, antipyretic, anti-inflammatory, antihypertensive, radioprotective, and antitumor activities (Mahajan et al., 2013; Cohen, 2014). In India, the leaves of this plant are generally used for the treatment of fever and allergies. Keeping in mind the medicinal importance of this plant, the goal of the present work was to isolate the endophytic fungi from O. sanctum leaf and stem tissues and to characterize their biological activities, including antibacterial, antioxidant, and anticancer ones, as well as extracellular enzyme production and to further characterize their bioactive molecules.

MATERIALS AND METHODS

Plant selection site. Young and healthy leaves and stems were collected from disease-free plants in the botanical garden of the Banaras Hindu University (BHU), Varanasi, U.P., India, in a sterile polyethylene bag and immediately transported to the laboratory in an icebox. The samples were stored at 4°C and processed for isolation within 24 h. A total of 100 explants (50 from the leaves and 50 from the stem segments) were randomly cut and surface-sterilized for the isolation of endophytic fungi.

Surface treatment. The collected plant parts were rinsed in running tap water for 15‒20 min an then in double distilled water to remove soil particles from the surface of the plant segments. Surface sterilization was performed as described previously (Petrini et al., 1993; Sahu et al., 2022), with minor modifications. For that purpose, a sterile pinch cutter was used to cut the stems into 0.25-cm thick sections and the leaves into the segments of 0.5 × 0.5 cm2 using. The segments were immersed in 70% ethanol for 2–3 min, followed by sterilization with 4% aqueous sodium hypochlorite (NaOCl) for 2 min; the segments were then rinsed several times with sterile distilled water, in 70% ethanol for 1‒2 min and finally the tissues were rinsed 5 times in sterile double distilled water and allowed to surface dry under aseptic conditions.

Isolation and morphological identification of the endophytic fungal isolates. The segments of sterilized leaf and stem tissues were placed on potato dextrose agar (PDA) amended with streptomycin sulfate (100 μg mL–1). A total of 100 segments (50 leaves and 50 stems) were used to observe the emergence of endophytic fungi. The plates were sealed with parafilm to avoid contamination and incubated at 27 ± 2°C for 15 days. The plates were observed regularly at alternate days for the emergence of endophytic fungi. The emerging fungal hyphae around the tissues were transferred to fresh PDA plates and purified. The endophytic fungi were grouped and identified based on morphological characteristics, including colony color, morphology, and microscopic observations of fruiting bodies and spore/conidia structures. The standard taxonomic manuals were used to identify the fungal genera and species (Ellis, 1971; Barnett and Hunter, 1972; Ainsworth et al., 1973; Von and Schipper, 1978). All isolated endophytic fungi were maintained in cryovials layered with 20% glycerol at –20°C in a deep freezer (Blue Star) in our laboratory in the Department of Botany, Banaras Hindu University, India.

Molecular identification and phylogeny of the endophytic fungal isolates. For molecular identification, genomic DNA was extracted from pure cultures of endophytic fungi following the protocol described by Sim et al. (2010). The universal primers ITS1: 5′TCCGTAGGTGAACCTGCGG3′ and ITS4: 5′TCCTCCGCTTATTGATAGC3′ (GeNei) (White et al., 1990) were used to amplify the 5.8S rDNA and two ITS regions between the 18S and 28S rDNA. The PCR mixture (25 µL) contained 1 µL DNA template, 1 µL each primer, 0.33 µL (3-unit µL–1) Taq polymerase, 1.5 µL of 25 mM MgCl2, 0.25 µL dNTPs, buffer (10×) 2.5 µL and 17.42 µL MQ water. PCR reactions were performed in a My-cycler (Bio-Rad, Hercules, CA, United States) under the following conditions: pre-denaturation at 94°C for 4 min, 35 cycles at 94°C (denaturation) for 1 min, 55°C (annealing) for 1 min, 72°C (extension) for 1 min, and final extension for 5 min at 72°C. Amplified PCR products were resolved by electrophoresis in a 1.5% (wt/vol) agarose gel followed by staining with ethidium bromide (0.5 µg mL–1) for visual examination. PCR products were sequenced by AgriGenome Labs Pvt. Ltd. (Cochin, Kerala, India). The amplified rDNA sequences obtained were used to retrieve similar sequences from the NCBI GenBank sequence database using the NCBI BLAST program. The sequences were submitted to the NCBI GenBank database and accession numbers were obtained for each isolate. MEGA version 7 software was used to align the sequence with the ClustalW function, and the phylogenetic tree was constructed using the maximum-likelihood method with closely related sequences of all nine isolates, retrieved from the NCBI database (two or three sequences for each i.e., a total of 32 and 1 outgroup).

Colonization frequency (%CF) of endophytic fungi. The collective colonization frequency (%CF) of the endophytic fungi was calculated using the formula given by Hata and Futai (1995): %CF = (Ncol/Nt) × 100, where Ncol is the number of segments of plant tissue colonized by each fungus and Nt is the total number of segments of plant tissue studied.

Extracellular enzyme production activity. A qualitative assay for the extracellular production of enzymes by the fungal endophytes was performed by digestion of suspended substrate in basal agar medium on petri plates. For that, mycelial plugs of each isolate (5 mm in diameter) were inoculated in the center of already prepared plates and incubated for 7 days at 27 ± 2°C in a BOD incubator. The clear zone around the fungal colony was measured as positive a test for the enzyme activity. In brief, an amylase assay was performed on glucose yeast extract peptone (GYP) agar medium (Hankin and Anagnostakis, 1975) composed of 1 g glucose, 0.1 g yeast extract, 0.5 g peptone, 15 g agar, and 1 L distilled water, pH 6 and supplemented with 2% soluble starch. After seven days of incubation, the plates were flooded with 1% iodine in 2% potassium iodide. Clear zones surrounding the colonies were measured. Cellulase activity was observed on yeast extract peptone agar medium (Lingappa, 1962) composed of 0.1 g yeast extract, 0.5 g peptone, 15 g agar, 1 L distilled water, pH 6 and supplemented with 0.5% Na-carboxymethyl cellulose (CMC). After seven days of incubation, the plates were flooded with 0.2 aqueous solutions of Congo red for 10 min and destained with 1 M NaCl for 15 min. The clear zones surrounding the fungal colonies were measured. Pectinase activity was determined using a slightly modified protocol described by Aguilar and Huitron (1990). For that, pectin agar was prepared containing 5.0 g yeast extract, 5.0 g pectin, and 15 g agar in 1 L distilled water with pH 6. The endophytic fungi were inoculated onto pectin agar plates and incubated for 7 days. After incubation, the plates were flooded with a freshly prepared 1% aqueous cetyltrimethyl ammonium bromide (CTAB) solution. The clear zone around the fungal colony was measured.

Production and extraction of secondary metabolites from the fungal endophytes. For the production of secondary metabolites, the actively growing mycelial discs of each pure fungal endophyte were cut and inoculated in 500 mL of potato dextrose broth (PDB). After 21 days of incubation at 27 ± 2°C, the mycelium was removed by filtration through four layers of muslin cloth, and the filtrate was poured into a separating funnel. For the extraction of secondary metabolites, the filtrate was mixed well with an equal volume of ethyl acetate. The solvent layer was collected separately, and the filtrate was subjected to extraction twice with an equal volume of ethyl acetate. The extracted secondary metabolites were concentrated and evaporated in a rotary vacuum evaporator (IKA, Germany). The concentrated secondary metabolites obtained from each isolate were transferred in glass vials separately for further evaporation at room temperature. After complete dryness, metabolites were stored in a refrigerator at 4°C for further study.

Antibacterial activity of secondary metabolites of the fungal endophytes. The fungal secondary metabolites were evaluated for their antibacterial activity against three strains of human pathogenic bacteria, Enterococcus faecalis (EF), Klebsiella pneumoniae (KP), and methicillin-resistant Staphylococcus aureus (MRSA) procured from the Institute of Medical Sciences (IMS) BHU, Varanasi, India using the disc diffusion assay (Hudzicki and Bauer, 2009). Pre-weighed crude metabolite extracts were dissolved in methanol and diluted to 10 mg mL–1 for the assay. A sterile paper disc (5 mm diameter, Whatman no. 1) was impregnated with 20 µL of the methanol extract using a micropipette and dried under a laminar hood for 20 min. Air-dried paper discs containing 0.2 mg crude metabolite extract from each isolate were placed separately on nutrient agar plates lawn-inoculated with pathogenic bacterial cultures. The disc containing only 20 µL of methanol was used as the control. The plates were incubated at 30 ± 2°C for 24 h. The zones of inhibition around the discs were measured for each extract. All experiments were performed in triplicates.

Antioxidant activity of secondary metabolites of the fungal endophytes. Crude metabolites of endophytic fungi were screened for free radical scavenging activity by the DPPH (2,2-diphenyl 2-picryl hydrazyl) method. DPPH, a violet-colored compound, is a stable free radical that accepts an electron or a hydrogen atom to form a stable diamagnetic molecule, which is yellow in color. This de-colorization is showing a positive test for anti-oxidant activity. Crude extract of endophytic fungi (100 µL) dissolved in methanol (1 mg mL-1) were mixed with 2900 µL DPPH solution (100 µM) in methanol and incubated in the dark for 30 min. A change in the color of DPPH was observed as a positive test.

X-ray crystallography. The crude metabolite extract of the isolate SL2 was fractionated with hexane, and the remaining extract was dissolved in ethyl acetate and slowly evaporated at 20°C in glass vials. After 2‒3 days of evaporation, fine crystals were formed. The crystals were cleaned with ethyl acetate and analyzed by X-ray crystallography. Data collection was performed using an Xcalibur Eos CCD (OXFORD) single-crystal diffractometer at room temperature (20°C). Graphite-monochromated MoKα radiation (γ = 0.71073 Å) was used for diffraction. The structures of the successfully diffracted crystals were analyzed.

NMR spectroscopy. The 1H and 13C NMR spectra were recorded on a JNM-ECZ500R/S1 Spectrometer with an operating field strength of 500 MHz. The 1H NMR spectra of the crystals were analyzed in DMSO (d6). The NMR software was imported into Mest ReNova (version 12.0), and manual baseline correction and automatic phase correction were performed. The tetramethylsilane (TMS) proton signal was calibrated to 0.00 ppm. The peaks of –CH3 (a, b, c) were recorded in the range of 1–3 ppm. The peak appeared at 3.1 ppm may be the (d) proton. Due to deshielding of oxygen atom proton of (e and f) appeared at 4.9 and 8.5 ppm. Phenolic group (–OH) proton appeared at 15.4 ppm and carboxylic acid proton (–COOH) appeared at 16.3 ppm (Fig. 5).

On the other hand, 13C-NMR spectroscopy provides information about the carbon environment in a molecule. This is particularly useful for determining the connectivity and carbon framework of organic compounds. The carbon skeleton and arrangement of functional groups can be identified by analyzing the chemical shifts of the carbon atoms in the crystal. In 13C-NMR of the crystal, the –CH3 carbon peak appeared in the range of 10‒20 ppm. The ketonic (‒C=O) carbon appeared at 187 ppm and carboxylic carbon (–COOH) appeared at 170 ppm. Further, carbon peaks appeared at 76, 131, 137, 110, and 162 ppm respectively (Fig. S1).

Anticancer activity of the pure compound. The anticancer activity of this pure compound was assessed by the MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl tetrazolium bromide) assay, as described previously by Jaiswara et al. (2021) on the Dalton’s Lymphoma (DL) cell line. DL is a murine T-cell lymphoma used to evaluate the antitumor potential of various synthetic and natural compounds both in vitro and in vivo. DL cells (1 × 106 cells mL–1) were inoculated into 96-well culture plates and treated with the indicated concentrations of the pure compound for 24 and 48 h. After the indicated time periods, 25 µL of the MTT solution (5 mg MTT in 1 mL PBS) was added to each well of the 96 well culture plates containing DL cells and incubated at 37°C for 4 h in a humidified CO2 incubator. Thereafter, the pellet of formazan crystals was collected by centrifugation (3000 rpm for 15 min) and dissolved in 50 µL of acidified isopropanol followed by measurement of absorbance at 540 nm by using an ELISA plate reader (ERBA LISA SCAN, Germany).

RESULTS

Isolation, identification, and phylogenetic analysis of endophytic fungi. Nine morphologically distinct endophytic fungal isolates were recovered from 100 segments of the leaf and stem tissues of Ocimum sanctum (Table 1). Based on microscopic studies and the ITS rDNA sequencing, the nine isolates were identified as Corynespora cassiicola (SS1), Alternaria alternata (SS2), Pleosporales sp. (SS4), Alternaria longipes (SS5), Alternaria tenuissima (SS6), and Aspergillus clavatonanicus (SS7) from the stem, and Penicillium citrinum (SL1), Aspergillus allahabadii (SL2), and Cochliobolus hawaiiensis (SL3) from the leaf samples (Table 1). Sequences of all the isolates were submitted to the NCBI database, and the accession numbers obtained along with the closest match from the NCBI database with their similarity query coverage (%QC) are listed in Table 1. A phylogenetic tree was constructed using the maximum-likelihood method implemented in the Mega 7 software, which provided more accurate descriptions of the patterns of relatedness, and all nine strains clustered at strongly supported and consistent nodes inside their respective clusters. Bootstrap values for all pertinent nodes were consistent and robust. The rest of their related taxa were clustered at individual well-supported nodes as shown in Fig. 1. Out of nine isolates, Aspergillus clavatonanicus (SS7) showed the highest CF% (16%) among the stem isolates, followed by Corynespora cassiicola (SS1) (10%) and Alternaria tenuissima (SS6) (10%). However, the highest CF% in leaf samples were found for Cochliobolus hawaiiensis (SL3) (14%), followed by Penicillium citrinum (SL1) (8%) (Table 1).

Table 1. Morphological and molecular (based on 18S-rDNA ITS1 sequencing) identification, closest match from the NCBI database with their accession number and similarity/query coverage (%QC), and total colonization frequency of endophytic fungi isolated from stems and leaves of Ocimum sanctum
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Fig. 1.
figure 1

Phylogenetic tree constructed by maximum-likelihood method using 18S rDNA ITS sequences generated during this study (as TGE) and sequences retrieved from GenBank using p-distance for nucleotides and the pair-wise gap deletion choice.

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Extracellular enzyme production. Of the nine isolates tested, seven of showed positive results in one or more enzyme production tests. Aspergillus allahabadii (SL2) showed positive results in all three enzymatic assays. Cochliobolus hawaiiensis (SL3) showed the highest amylase activity, with a clear zone of >10 mm, whereas SS2 and SS4 did not show any enzymatic activity (Table 2). Cellulase activity was observed in the isolates of Aspergillus allahabadii (SL2), Alternaria tenuissima (SS6), and Aspergillus clavatonanicus (SS7). Three isolates, Penicillium citrinum (SL1), Aspergillus allahabadii (SL2), and Aspergillus clavatonanicus (SS7), also showed pectinase activity (Table 2).

Table 2. Enzymatic activity of endophytic fungi isolated from stem and leaves of Ocimum sanctum
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Extraction of crude metabolites and antibacterial activity. Crude metabolites were successfully extracted from 21 day-old broth cultures of all fungal isolates using ethyl acetate. The antibacterial activity of crude metabolite extracts was tested against the three standard bacterial pathogens, Klebsiella pneumoniae (KP), Enterococcus faecalis (EF), and MRSA using the disc diffusion assay. Of the nine isolates, the crude extracts of five isolates showed strong antibacterial activity against one or more of the tested pathogens (Fig. 2 and Table 3). The inhibition zone diameters varied from 6 to >10 mm. The extracts from Aspergillus allahabadii (SL2) and Aspergillus clavatonanicus (SS7) were effective against all tested bacterial pathogens, with an inhibition zone of 10 mm or more (except SL2 against MRSA). Penicillium citrinum (SL1) was effective against two bacterial pathogens. Alternaria longipes (SS5) and Alternaria tenuissima (SS6) were only effective against one bacterial pathogen each. Crude extracts of SL3, SS1, SS2, and SS4 did not show any antibacterial activity against the tested pathogens.

Fig. 2.
figure 2

Antibacterial activity of secondary metabolites of endophytic fungi against pathogenic bacteria using disc diffusion assay. (a) Endophytic fungi isolated from leaves where 1—Penicillium citrinum, 2—Aspergillus allahabadii, 3—Cochliobolus hawaiiensis, (b) Endophytic fungi isolated from stem where 1—Corynespora cassiicola, 2—Alternaria alternata, 4—Pleosporales sp., 5—Alternaria longipes, 6—Alternaria tenuissima; 7—Aspergillus clavatonanicus and c—control.

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Table 3. Antibacterial activity of crude extracts of endophytic fungi of Ocimum sanctum using disc diffusion assay. Each disc was loaded with 200 µg of extract
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Antioxidant activity. DPPH, a stable free radical, was used to study the radical-scavenging effects of the extracts as antioxidant activity. The scavenging effects of the samples were evaluated based on the extent of decolorization. The crude fungal metabolite extracts of six out of nine isolates showed positive results. The fungal extract of Cochliobolus hawaiiensis (SL3) showed maximum decolorization of the DPPH radical. The extracts from Penicillium citrinum (SL1), Aspergillus allahabadii (SL2), Corynespora cassiicola (SS1), Alternaria alternata (SS2), and Aspergillus clavatonanicus (SS7) also showed significant antioxidant activities. However, SS4, SS5, and SS6 did not exhibit any antioxidant activity (Fig. 3).

Fig. 3.
figure 3

Antioxidant activity of crude metabolites of isolated endophytic fungi using DPPH free radical scavenging method. A change in the color of DPPH from violet to colourless to yellow was observed as a positive test. Where, C is control and ‘–’ means negative test.

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Characterization of the pure compound from the endophytic fungus Aspergillus allahabadii (SL2). Fine crystals were formed from partially purified metabolites of A. allahabadii under slow evaporation in ethyl acetate. Microscopic examination confirmed the purity of the crystals. X-ray crystallography confirmed the structure of the compound as citrinin (Fig. 4). Moreover, the 1H-NMR (Fig. 5) and 13C-NMR (Fig. S1) spectra also confirmed that the purified compound was citrinin.

Fig. 4.
figure 4

XRD-Crystallographic and chemical structure of compound isolated from Aspergillus allahabadii and identified as Ci-trinin.

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Anticancer activity of the pure compound citrinin. The anticancer activity of citrinin was assessed using the MTT assay, as discussed in materials and methods. The results are shown in Fig. 6. Our results showed that citrinin exhibited a significant antitumor potential in a dose-dependent manner after 48 h with the IC50 value ~150 µM, although low cytotoxic activity was noted after 24 h.

Fig. 5.
figure 5

1H-NMR spectroscopic image of the isolated pure compound Citrinin isolated from Aspergillus allahabadii.

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Fig. 6.
figure 6

Assessment of antitumor activity of citrinin extracted from Aspergillus allahabadii. Dalton’s lymphoma cells-DL (1 × 106 cells mL–1) were treated with mentioned concentrations of citrinin (0, 50, 100, 150, 200 and 400 μM) for 24 and 48 h followed by estimation of percent cytotoxicity by MTT assay as described in materials and methods. The values shown are the mean ± SD of three independent experiments done in triplicate. * p < 0.05 vs. cells treated with citrinin for 24 h.

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DISCUSSION

Since the discovery of penicillin from Penicillium notatum, fungi have been recognized as a great source of a plethora of bioactive compounds. Because of their specialized niche, endophytic fungi of medicinal plants have shown the potential to produce novel bioactive compounds that have greatly attracted the attention of researchers (Gupta and Chaturvedi, 2019; Singh, 2019; Jiang et al., 2022). The selection of medicinal plants for the isolation of endophytes is influenced by such factors as chemo-diversity, the traditional use of the plants, and the region from which the plant is obtained (Verpoorte, 1998). Given the growing need for novel compounds with antimicrobial, antioxidant, and anticancer potential, this study focuses on exploring the diversity of endophytic fungi derived from the O. sanctum plant and investigating their potential as a source of compounds with antibacterial, antioxidant, and anticancer properties.

In this study, a total of nine endophytic fungi were isolated from leaf and stem tissues of the plant. The colonization frequency data suggest that different endophytic fungal strains colonize different plant tissues (such as stem and leaves) to a different extent. This suggests the varying degree of affinity of the isolated strains towards different tissues of the host plant. Aspergillus clavatonanicus was found to be the most prevalent species in stem tissue, whereas Cochliobolus hawaiiensis predominated in the leaf tissue. These isolates were identified using a combination of morphological and molecular methods (Table 1). A combined approach using microscopic spore structures and molecular ITS rDNA sequencing methods is considered a reliable method for the identification of culturable fungi at the species level (Verma, 2014; Singh et al., 2018). Advancements in sequencing technologies, such as next-generation sequencing, have enabled the exploration of the functional roles of non-culturable microorganisms (Tao et al., 2008; Maghembe et al., 2020). Species of Aspergillus, Cochliobolus, Alternaria, and Corynespora were the most common isolates in our study. These species have also been isolated as endophytes from other medicinal plants, including Aegle marmelos, Azadirachta indica, Adenocalymma alliaceum, and Tectona grandis (Verma et al., 2007; Kharwar et al., 2010, 2011; Singh et al., 2018).

In the antibacterial assay against three human pathogenic bacteria, Enterococcus faecalis, Klebsiella pneumoniae, and MRSA, more than 55% of the isolates significantly inhibited one, two, or all three pathogens tested in the disc diffusion assay (Table 3, Fig. 2). Gong and Guo (2009) reported that only 8.3% of the fungal isolates of Dracaena cambodiana and Aquilaria sinensis showed antibacterial activity. However, in our previous study, 58.33% of Madhuca indica fungal isolates screened positive for antibacterial activity against the tested pathogens (Verma et al., 2014). This suggests that these endophytic fungi have the potential to combat such drug-resistant bacteria as MRSA. Interestingly, two endophytic fungi, Aspergillus allahabadii and Aspergillus clavatonanicus, inhibited the growth of all three human pathogens tested, showing broad-spectrum antibacterial activity (Table 3, Fig. 2). The crude extracts of these two endophytic fungi also showed strong antioxidant activity (Fig. 3). Many studies have reported significant antioxidant activity of many endophytic fungi (Huang et al., 2007; Sadananda et al., 2011; Mishra et al., 2018). Hence, these can be used as a source of natural antioxidants and further efforts in identification and isolation of the bioactive constituents can lead to the development of antioxidant-based drugs or dietary supplements.

Fungi have been a great source of industrially important enzymes, and many endophytic fungi have also been reported to successfully produce hydrolytic enzymes, including amylase, cellulase, protease, and pectinase, which probably help the endophytes to successfully colonize their host plants (Karnchanatat et al., 2007; Caroll and Pertini, 2014; Verma and White, 2018). In this study, out of nine isolates, seven showed positive test results for one or more enzymes, in which A. allahabadii tested positive for all enzymes (amylase, cellulase, and pectinase) (Table 2). The amylase activity can help the fungus to break down the starch into simpler forms of sugar, which it may utilize for growth and development during unfavourable conditions (Sunitha et al., 2012). Furthermore, the high amylase activity in endophytic fungi may also have industrial applications, such as in food and pharmaceutical industries. Similarly, cellulases can play important roles in lignocellulosic biomass degradation, bioremediation, and industrial applications (Robl et al., 2013). Finally, pectinases play a crucial role in the degradation of pectin, the polysaccharide present in plant cell walls, and hence can act as an important tool in the decomposition of host tissue and the establishment of symbiosis between endophytic fungi and their plant host (Rahul et al., 2015; Hawar, 2022).

The crude metabolite extract of one isolate, A. allahabadii, showed strong antioxidant and antibacterial activities. During the purification process, fine crystals were formed and X-ray crystallography confirmed the identity of the pure crystal as citrinin (Fig. 4). Citrinin is a low molecular weight (250.25 g mol−1) polyketide compound produced by many fungi, including the Penicillium and Aspergillus species, a known mycotoxin contaminant of several food grains (Dunn, 1994). Despite its toxicity, citrinin exhibits a range of biological activities, including antibacterial, antifungal, and cytotoxic ones (Mazumder et al., 2002; Park et al., 2008, Filho et al., 2017; Wang et al., 2019). Previous studies reported that citrinin extracted from Penicillium citrinum inhibited the growth of many bacterial pathogens including gram-positive (Staphylococcus aureus, Bacillus pumilus, Klebsiella pneumoniae, and Streptococcus pneumoniae) and gram-negative ones (Escherichia coli, Shigella boydii, and Salmonella typhimurium) (Mazumder et al., 2002; Wang et al., 2019). Recently, a few studies have shown the cytotoxic action of citrinin against different cancers, such as breast and colon cancer, and reported the antitumor activity of citrinin possibly due to induction of apoptosis (Filho et al., 2021). A study also reported that citrinin produced by the fungus P. citrinum increased the motility of the rhizospheric bacteria Paenibacillus polymyxa (Park et al., 2008).

In the present study, we found that the crude extract of A. allahabadii containing citrinin showed strong antibacterial activity against all tested pathogens (Fig. 2 and Table 3). Interestingly, recent studies have demonstrated the anticancer potential of citrinin against colon and breast cancers (Salah et al., 2017; Filho et al., 2021; Menezes et al., 2023). However, to date, the antitumor potential of citrinin has not yet been examined in hematological malignancies such as T-cell lymphoma, which is considered a highly complex neoplastic disorder. Moreover, to establish citrinin as a promising anticancer drug, it is essential to test its tumoricidal activity against a wide range of cancers. In our study, citrinin exerted a significant cytotoxic effect in a dose-dependent manner after 48 h, although a low cytotoxic activity was noted after 24 h. The results of the present study suggest that fungal endophytes are a great source of bioactive molecules having great therapeutic potential (Kharwar et al., 2011; Singh et al., 2018). Further studies are required to characterize the pure compounds isolated from other active fungal isolates. Moreover, investigations are also warranted to explore the mechanisms underlying these bioactivities and their potential applications in drug development and plant-protection strategies.

ABBREVIATIONS AND NOTATION

ITS

internal transcribed spacer

PDA

potato dextrose agar

CF

colonization frequency

DMSO

dimethyl sulfoxide

MRSA

methicillin-resistant Staphylococcus aureus

CTAB

cetyltrimethyl ammonium bromide

TMS

tetramethyl silane

DPPH

(2,2-diphenyl 2-picryl hydrazyl)

MTT

(3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl

tetrazolium bromide)

DL

Dalton’s lymphoma

PBS

phosphate buffer saline

IC50

half maximum inhibitory concentration