Plant materials collection and endophytes isolation
The undamaged and disease-free plant specimens were gathered and carefully sealed within zip-lock polythene bags in Tapobon, Paschim Medinipur, West Bengal, India. The region is characterized by a tropical, warm, and humid climate, featuring an average temperature of 34°C. Tapobon is situated between latitudes 22°25′ to 22°57′ North and longitudes 87°11′ East, with an altitude of 23 meters. The primary plant parts utilized for extracting endophytic fungi were the leaves and stems. To eradicate the epiphytic microbes surface sterilization was done using sodium hypochlorite and 70% alcohol. In water agar plates the thin (1.0 cm x 1.0 cm) segments of the sterile samples were placed and incubated at 25°C in a BOD incubator. Within 72-96 hours of incubation, fungal hyphae from most of the samples emerged which were then transferred to PDA (potato dextrose agar) plates.

Identification of isolated endophytes
After fifteen days of incubation, endophytic fungi were characterized through an assessment of their visible and internal features and examined under a light compound microscope (Leica DM 3000). Fungal genera were identified by macroscopic and microscopic morphology. Each of the isolated and recognized endophytic fungi was designated with unique codes and used for further studies. Isolate APL11 was subjected to molecular identification following the standard protocols and the obtained nucleotide sequence was submitted to GenBank. A phylogenetic tree compared with other isolates was prepared for the identification of APL11.⁶ Calculation of the colonization frequency of each isolate, in addition to assessing diversity using indices such as Shannon–Wiener, Simpson’s dominance, Simpson’s diversity, and species richness. These calculations were performed in accordance with established methodologies as outlined in Santra and Banerjee.⁷

Assay for the production of extracellular enzymes
The screening process involved two methods: a qualitative approach, known as the agar plate method, and a quantitative method, referred to as the liquid culture method. To assess the functional role of extracellular enzymes produced by fungal endophytes, the fungi were cultivated on potato dextrose agar (PDA) for a period of 6-7 days, with incubation at 25°C. Subsequently, 5 mm mycelial plugs were placed on solid media. Following this incubation, at room temperature, the extent of enzyme activity surrounding the fungal colony was measured, as described by Krishnan et al. in 2016.⁸ The procedure used for both the qualitative and quantitative estimation of amylolytic, proteolytic, lipase, laccase, and tannase activity is detailed below.

Screening of fungal endophytes for amylase production and estimation of amylase activity
For the qualitative analysis of amylase production, fungal isolates were cultivated on GYP (Glucose Yeast Extract Peptone Agar) medium containing soluble starch at a concentration of 0.2% and adjusted to pH 6.0. Following 72 hours of incubation, the plates were flooded with a solution composed of 1% iodine and 2% potassium iodide. The presence of a clear zone surrounding the fungal colonies served as an indicator of amylolytic activity. For quantitative analysis, the isolates that exhibited positive outcomes in the iodine test on solid media were chosen. These selected isolates were cultured in 80 mL of basal media within Erlenmeyer flasks, which were supplemented with 1.5% starch. Flasks were sterilized and cooled, and 5 mm² PDA plugs containing fungal mycelia were inoculated into each flask. An uninoculated flask with the same media composition served as the control. Liquid samples were collected from the flasks every 24 hours for a period of 5 days in 2 mL Eppendorf tubes, starting from 48 hours of incubation

Completing the incubation phase, the broth was first filtered using Whatman No. 1 filter paper and subsequently subjected to centrifugation at 5000 revolutions per minute (rpm) for 15 minutes at a temperature of 40°C. The supernatant, which contained liberated reducing sugars, was used for further analysis. The quantification of reducing sugars was performed utilizing the DNS (dinitrosalicylic acid) method.⁹

To determine the amylase activity, a reaction mixture was prepared using 1 mL of 0.2 molar phosphate buffer (K₂HPO₄ + KH₂PO₄) with a pH of 6.0, 1% starch solution 0.5 mL (w/v) and the crude enzyme extract 0.5 mL. The reaction mixture was then incubated at 37°C for approximately 45 minutes. To stop the reaction, 2 mL of DNS solution was added to each sample. A separate estimation at 0-time was also performed to determine the initial glucose concentration in the solution before the reaction started. The solutions were boiled in a water bath for 15 minutes, resulting in a reddish-brown color due to the release of reducing sugars. The optical density of solutions was measured at a wavelength of 550 nm, employing a UV-spectrophotometer (Shimadzu Corp., Serial AI-4547-0302).

To quantify the amount of sugars produced in the samples, a standard curve was established using a stock solution of glucose with a concentration of 1 mg/mL at various concentrations (20, 40, 60, 80, 100 µg/mL). The absorbance of the samples was then compared to this standard curve to determine the sugar content. In this context, one unit of amylase activity was defined as the quantity of enzyme that releases 1 mole of reducing sugar, such as maltose or glucose, per minute under the specified assay conditions.⁹

Screening of fungal endophytes for protease action and estimation of protease production
Protein breaking ability of endophytic fungus was assayed on a modified Czapek Dox medium with a composition of KH₂PO₄ 1.5 g, (NH₄)₂SO₄ 0.5 g, MgSO₄, 7H₂0 0.06 g and CaCl₂, 2H₂O 0.06 g along with separately sterilized 1 g milk powder in a 100 ml of media composition of pH 7.0.¹⁰ Mycelia plugs from pure fungal cultures were placed on the plates and incubated for 5 days at 25°C. Positive protease producers were identified according to the halo clear zone surrounding the fungal colony. For quantitative analysis, protease-producing fungal isolates were cultured in 250 mL Erlenmeyer flasks, each filled with 80 mL of basal salt media. The composition of the basal salt media per liter included: NaNO₃-2.5 g, KH₂PO₄ – 1 g, MgSO₄·7H₂O-0.5 g, and KCl-0.5 g. and obviously supplemented with 1%(w/v) powder milk. The medium was sterilized, cooled, and aseptically inoculated with the selected fungi and incubated for 7 days at 150 rpm. Starting from 48 hours of incubation, the liquid was collected every 24 hours for the remaining 5 days.

The liquid samples were centrifuged at 8000 rpm for 15 minutes at 4°C in a cooling centrifuge (REMI-AACI-9575; C-2432). The resulting supernatant was utilized for assessing proteolytic activity using casein as the substrate. Specifically, 0.5 mL of the supernatant was mixed with 0.5 mL of a 1% (w/v) casein solution in a 100 mM sodium acetate buffer with a pH of 5.5. This mixture was then incubated for 30 minutes at 45°C, and the reaction was stopped by adding 0.5 mL of trichloroacetic acid (TCA). After a 15-minute incubation at room temperature, the mixture was centrifuged at 6000 rpm for 15 minutes to remove debris. The absorbance of the soluble fraction was measured at 650 nm using a UV-spectrophotometer after the addition of Folin-Ciocalteu’s reagent, and the protease activity was quantified based on a standard curve created using tyrosine solutions with concentrations ranging from 20 to 100 mg/L.¹ The protease activity was expressed as the amount of enzyme releasing 1 µmol of tyrosine per minute according to the standard curve.

Screening of fungal endophytes for lipase Activity and estimation of lipase production
To assess the production of lipolytic enzymes, fungi from pure culture plates were cultivated on Peptone Agar medium with peptone, NaCl, CaCl₂·2H₂O, and agar at a pH of 6.0. This medium was enriched with separately sterilized Tween 20 at a concentration of 1%. A 5 mm² fungal plug was inoculated onto the Peptone Agar plates and incubated for 5 days. At the end of this incubation period, the presence of lipase activity was confirmed by the formation of a visible precipitate around the fungal colony. This precipitate results from the conversion of lauric acid into its calcium salt, which is released by the lipase enzyme. For the quantitative determination of lipase enzyme activity, fungal samples were cultivated in 250 mL flasks, each containing 100 mL of autoclaved medium. This medium included (NH₄)₂SO₄, MgSO₄, NaCl, Ca(NO₃)₂, KH₂PO₄, glucose, and yeast extract, with the pH adjusted to 5.5. Sterilized olive oil was separately added at a rate of 1 mL per 100 mL of sterile medium. The samples were incubated at 30°C and agitated at 110 rpm on a BOD shaker for 4 days. Every 24 hours, 2 mL of samples were collected and employed for the enzyme assay, as described by Geoffry and Schur.¹¹

The collected samples underwent centrifugation at 5000 rpm for 20 minutes, and the resulting supernatant was employed to assess lipase activity. For the lipase assay, an emulsifying agent was created by combining 0.5 mL of olive oil, 0.5 mL of phosphate buffer (0.066M at pH 7), and 0.5 mL of tween 20 in equal proportions. In the lipase assay, 1 mL of the emulsifying agent was mixed with 1 mL of the supernatant and shaken for 3 minutes. This mixture was then placed in an incubator shaker at 37°C and 150 rpm for 5 hours. Following incubation, the optical density (OD) was measured spectrophotometrically at a wavelength of 655 nm. Lipase activity was defined as the amount of enzyme capable of releasing 1µmol of fatty acid at 37°C and pH 7 under the specified assay conditions.¹¹

Screening of fungal endophytes for tannase action and estimation of tannase production
To detect tannase-producing endophytic fungal strains, a plate assay was conducted using the approach outlined in the methodology by Roy et al.¹² Modified Czapek Dox medium was prepared with specific compositions of various ingredients. Separate solutions of 1% tannic acid and the prepared medium were sterilized and mixed under laminar airflow conditions. Plugs of mycelia measuring 5mm² from each fungus were introduced onto Petri dishes filled with the appropriate medium, followed by an incubation period of 6 days at 27°C. The observation of a distinct clear halo surrounding the fungal colony served as confirmation of tannic acid breakdown. For tannase production, 100mL of modified liquid Czapek Dox medium was used, containing tannic acid (1g), NH₄Cl (0.3g), CaCl₂ (0.05g), NaH₂PO₄ (0.05g), Na₂HPO₄ (0.05g), MgSO₄ (0.1g), and yeast extract (0.05g) The media were sterilized in an autoclave and then cooled to room temperature. Mycelia plugs from PDA (Potato Dextrose Agar) were transferred to the liquid media, and the inoculated media were placed in a shaker BOD incubator at 150 rpm for 3 days.

To estimate the tannase activity, the collected liquid containing the crude enzyme was used. The remaining tannic acid was quantified by performing the Bovine Serum Albumin (BSA) precipitation method described by Mahapatra et al.¹³ The following solutions were prepared: a 0.2 M phosphate buffer with a pH of 5.5, a Bovine Serum Albumin (BSA) solution at a concentration of 1 mg/mL, an SDS-triethanolamine solution (containing 1% w/v SDS in 5% v/v triethanolamine), and a 0.13 M FeCl₃ solution. The tannase reaction was initiated by combining 0.1 mL of the enzyme solution with 0.3 mL of a 1% (w/v) tannic acid solution in the 0.2 M phosphate buffer (pH 5.5). The reaction mixture was subsequently incubated at 40°C for duration of 30 minutes. Control estimation without an enzyme was also prepared. To detect the initial tannic acid concentration before the start of the reaction, a 0-time estimation was performed.

Following the incubation, the reaction was halted by introducing 2 mL of Bovine Serum Albumin (BSA) at 0°C. The mixture was subsequently centrifuged at 5000 rpm for 10 minutes, and the supernatant was removed. The precipitate was re-suspended in 2 mL of the SDS-triethanolamine solution. To this resuspended solution, 1 mL of FeCl₃ (0.13 M) was added, and the absorbance of the resulting solution was gauged at 550 nm using a spectrophotometer.

Screening of fungal endophytes for laccase production and estimation of laccase activity
To determine laccase activity in endophytic fungi, they were cultivated on GYP agar medium supplemented with 0.05% α-Napthol. The medium composition consisted of peptone (10 g/L), yeast extract (5 g/L), glucose (20 g/L), and agar (15 g/L) at pH 7.0. The presence of laccase was confirmed by a noticeable color change in the media, shifting from colorless to brownish-violet, signifying the oxidation of colourless α-napthol by the endophytic fungi, following the method described by Umar and Ahmed, 2022. For the quantitative assessment of enzyme activities, Czapex Dox medium was employed as the liquid fermentation medium. A 5 mm² disc of actively growing fungal mycelia from the periphery was aseptically inoculated into the broth. The culture was then incubated at 25°C for 7 days. The liquid was collected and stored at 4°C for subsequent enzymatic assays.

Following the completion of the incubation period, the collected liquids were subjected to centrifugation at 5000 rpm for 15 minutes at 4°C. The resulting supernatant was utilized to assess laccase activity, which was quantified in U/mL. To eliminate the potential influence of hydrogen peroxide (H₂O₂) produced by the fungus, catalase (1,000 U/mL) was introduced to the assay solution and allowed to incubate for 1 hour at 37°C.¹⁴

Laccase activity was performed spectrophotometrically at 420 nm, using 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS) as a substrate. The assay mixture consisted of 150 µL of 0.5 mM ABTS substrate, 2.7 mL of 0.1 M sodium acetate buffer at pH 4.5, and 150 µL of the culture supernatant. The mixture was incubated for 5 minutes, after which the absorbance was measured at 420 nm using a spectrophotometer, referencing an appropriate enzyme and substrate blank. Laccase activity was defined as the quantity of enzyme capable of oxidizing 1 µmol of ABTS per minute, and these activities were expressed in U/mL. All assays were conducted in triplicate.