Isolation and Identification of Endophytic Fungi from Syzygium cumini Linn and Investigation of Their Pharmacological Activities

This study was conducted to isolate and identify the endophytic fungi from the bark and leaves of the Syzygum cumini plant and investigate the pharmacological activities of endophytic fungi along with plant parts. After isolation, endophytic fungi were identified based on morphological characteristics and molecular identification. Antimicrobial, antioxidant, and cytotoxic activities were studied by a disc diffusion method, free radical scavenging DPPH assay, and brine shrimp lethality bioassay, respectively. A total of eight endophytic fungi were isolated and identified up to the genus level based on morphological characteristics and confirmed by molecular identification techniques. Among the eight isolates, three isolates were identified as Colletotrichum sp. (SCBE-2, SCBE-7, and SCLE-9), while the rest of the isolates belonged to Diaporthe sp. (SCBE-1), Pestalotiopsis sp. (SCBE-3), Penicillium sp. (SCBE-4), Phyllosistica sp. (SCLE-7), and Fusarium sp. (SCLE-8). The presence of flavonoids, anthraquinones, coumarins, and isocoumarins was assumed by the preliminary screening of the fungal and plant extracts by a thin-layer chromatographic technique under UV light. Fungal extracts of Pestalotiopsis sp. Penicillium sp. were found sensitive to all test bacteria, but only extracts from the leaf and bark showed significant antifungal activity along with their antimicrobial activity. Penicillium sp. The fungal extract showed the highest free radical scavenging activity (2.43 μg/mL) near that of ascorbic acid (2.42 μg/mL). Some fungal extracts showed cytotoxic activity that, in general, suggests their probable abundance of biological metabolites. This is the first approach to investigate the endophytic fungi of Syzygium cumini Linn. in Bangladesh, to find the pharmacological potential of endophytes, and to explore novel compounds from those endophytes.


Introduction
Mankind always rely on nature, especially when it comes to fnd new drugs to cure diseases [1]. Te need for safe, chemical free, and less toxic treatment options has prompted the search for innovative and enhanced ways to deal with these health-related problems. To serve this purpose, several medicines including antioxidants, antidiabetic drugs, antiinfammatory agents, antiulcer agents, antibiotics, and antifungal agents have been discovered from diferent sources like medicinal plants, edible and medicinal higher fungi (mushrooms), microbial sources like fungus, bacteria, and endophytes [2][3][4][5][6]. Recently, many known as well as new endophytic bioactive metabolites have been identifed as possessing a wide variety of pharmacological activities such as antibiotic, antiviral, anticancer, anti-infammatory, and antioxidant [7]. So, endophytes associated with plants may be considered a source of novel compounds [8]. Endophytes are microbes that are present in internal plant tissues without inducing symptomatic infection in their host plant. Endophytic fungi have contributed to their host plants by developing bioactive compounds that provide protection to the plant [9,10]. In each plant, one or more endophytes can reside, so plants provide a rich microorganism repository [11,12]. In this study, Syzygium cumini has been used for investigation. Syzygium cumini (Syn. Eugenia jambolana), also known as jamun or black plum, belongs to the family Myrtaceae [13]. Te entire plant parts such as seed, fruit, leaves, fower, and bark are widely used in folk medicine like Ayurveda, Unani [14,15]. Studies have shown that various extracts of jamun exhibits pharmacological efects such as anti-infammatory, antifungal, antiviral, anti-infammatory, antiulcerogenic, cardioprotective, antiallergic, anticancer, radio protective, antioxidant, and hepatoprotective properties [8]. So, endophytes residing in Syzygium cumini may contain a lot of biologically active metabolites. Terefore, the present study was designed to investigate the pharmacological potential of endophytic fungi isolated from the Syzygium cumini plant to explore some novel drug candidates as well as to compare them with the plant extract.

Collection of Plant Material and Isolation of Endophytes.
Te plant sample Syzygium cumini Linn was collected from the Jahangirnagar University botanical garden in November, 2018. It was identifed by the taxonomist of the Bangladesh National Herbarium, Dhaka, Bangladesh. A voucher specimen (DACB-47653) has been deposited in the Herbarium for further reference. Leaves and barks from healthy and mature plants were randomly collected for the study. Te plant material was carried to the laboratory in sterile bags and processed within a few hours after sampling. Tey were then cut with an anti-cutter, over a sterile glass plate. Endophytic fungi were isolated from the fresh plant parts following the procedure and suitably modifed [16][17][18].

Morphological Identifcation of Endophytic Fungus.
To identify the isolates of endophytic fungus, slides prepared from cultures were stained with a lactophenol cotton blue reagent and then examined with a bright-feld and phase contrast microscope [19]. Teir morphological identifcation was completed based on the growth pattern, hyphae, the color of the colony and medium, surface texture, margin character, aerial mycelium, sporulation and production of acervuli, the coloration of the medium, and the size and coloration of the conidia using standard identifcation manuals [20].

Molecular Identifcation.
Te molecular identifcation of fungal strains was completed by DNA amplifcation and sequencing of the internal transcribed spacer (ITS) region using the molecular biological protocol [21,22]. A segment of fungal hyphae (0.5-1.0 cm 2 ) was collected from the Petri dish and lyophilized in an Eppendorf tube (2 mL) (Eppendorf, Germany). Te lyophilized fungal mycelia were pulverized and the infection was disrupted. Fungal DNA isolation was achieved according to the manufacturer's protocol by using the DNeasy Plant Mini Package (QIAgen, USA). Te procedures include cell lysis, RNA digestion by RNase A, removal of precipitates and cell waste, DNA shearing, precipitation, and purifcation. Ten, the isolated DNA was amplifed by polymerase chain reaction (PCR). Te PCR was performed using the Master Mix Kit of HotStarTaq (QIAgen, USA). As primers, ITS 1 (with TCCGTAGGTGAACCTGCGG base sequences) and ITS4 (with TCCTCCGCTTATTGATGATGC base sequences) (Invitrogen, USA) were mixed with the HotStarTaq Master Mix Kit and DNA template with a total volume of 50 μL. Te mixture was then added to the thermal cycler using the programmed PCR (BioRad, USA). Te amplifed fungal DNA (PCR product) was then submitted by a commercial service for sequencing and the base sequence was compared using BLAST Algorithm with publicly accessible databases, including GenBank.

Endophytic Fungal Cultivation and Extraction.
Small scale cultivation was performed on Petri dishes for the isolated fungal strains with approximately 1 L of PDA media for each strain. Endophytic isolates were incubated for 21 days at 28°C and the culture media were extracted two times with ethyl acetate to obtain the crude extracts [17]. Te extracts of the fungi were concentrated into a solid residue by evaporation with a rotary vacuum evaporator under reduced pressure to obtain an ethyl acetate crude extract [23].

Preliminary Chemical Analysis.
All the extracts, including fungal and plant, were qualitatively tested for the presence of chemical constituents. Te screening of extracts was performed by visual detection, UV light (both in short at 254 nm and in long at 365 nm), and vanillin-H 2 SO 4 spray reagent [17].
2.6. Antioxidant Activity. Te antioxidant activity of extracts was estimated using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging method. 2.0 mL of methanol solution of the extract at diferent concentrations was mixed with 2.0 mL of a DPPH methanol solution (20 μg/mL). Samples were placed in a dark place at room temperature for 30 min to complete the reaction. Ten, the absorbance was measured at 517 nm against methanol as blank by a UV spectrophotometer using the method described by Brand Williams [24]. For each sample, the result was presented as an IC 50 . Ascorbic acid (AA) and 2-tert-butyl-4-hydroxyanisole (BHA) were used as positive controls.
Inhibition of free radical DPPH in percent (I%) was calculated as follows:  [26]. Vincristine sulfate was used as a positive control and DMSO was used both as a solvent and as a negative control. All the samples were tested in triplicate.

Morphological Identifcation of Fungal Isolates.
A total of eight endophytic fungi were isolated from the bark and leaf parts of the plant Syzygium cumini. Tey have been coded as internal strain numbers, namely, SCBE-1, SCBE-2, SCBE-3, SCBE-4, SCBE-7, SCLE-7, SCLE-8, and SCLE-9. Te microscopic characters of strain SCBE-1 produced woolly to cottony, whitish, pale to light brown mycelium, conidiophores that were simple. Two types of conidia were observed upon microscopic examination. Te alpha (α) conidia were hyaline, fusiform to ovate, straight, aseptate, and frequently biguttulate. Te beta (β) conidia were fliform, sigmoidal, and hyalinase arising from the mycelium. Tese characteristics indicated that the fungus SCBE-1 belongs to Diaporthe sp. (Figure 1). Strains SCBE-2, SCBE-7, and SCLE-9 showed profusely branched, septate, and hyaline mycelium. Simple, elongated, disc-shaped, or cushion-shaped conidiophores were also found. Conidia were hyaline, singlecelled, and ovoid to oblong in shape. Tis fungus developed quickly, usually covering the entire Petri dish in 10 days and sporulation was abundant on PDA media. Tese characteristics indicated that the fungi belong to Colletotrichum sp. (Figure 1). Teir morphology was diferent and supposed to belong to diferent species. Strain SCBE-3 was characterized as Pestalotia sp. for the presence of white colour mycelium and dark, discoid, or cushion-shaped acervuli. Conidiophores (annellides) were present within tight fruiting structures (aecervuli or pycnidia). Spores (conidia) were 4to 5-celled, with two or three dark brown center cells ( Figure 1). Strain SCBE-4 showed hyaline, fufy, colored, branched, and septate mycelium. Conidia was arising singly or less often in synnemata from the mycelium, branched near the apex to form a brash like, ending in phialides, eventually turned into green and granular spores, one celled, ovoid. Tis characteristic indicated that the fungus SCBE-4 belongs to Penicillium sp. (Figure 1). In the case of SCLE-7, pycnidia were dark, ostilate, lenticular to globose, which were immersed in the host tissue. Short or obsolete conidiophores were observed. Conidia were small, 1-celled, hyaline, ovoid to elongated. So, SCLE-7 was characterized as Phyllosistica sp. (Figure 1). SCLE-8 produced sparse to abundant cottony mycelium with white violet mycelium and pigmentation from colorless to pale violet. Conidia or phialo spore's hyaline, two kinds of spores were observed. Macroconidia were multicellular organisms with a pointed end that was slightly curled or bent. Microconidia were onecelled and oblong in shape, with a fufy colony and spores that were cylindrical, septate, and aseptate. So, strain SCLE-8 was identifed as Fusarium sp. (Figure 1). Te high-quality DNA extracted from endophytic fungi used ITS1 and ITS4 as primers for PCR. Te genomic DNA of the fungi that successfully amplifed SCBE-1 and SCBE-2 isolates had a length of 625 bp; the SCBE-3 and SCBE-4 isolates had a length of 600 bp, while the SCBE-7, SCLE-7, SCLE-8, and SCLE-9 isolate had a length of 550 bp (Figure 2). Teir detailed description of GenBank accession number of closely related fungal strains, along with their similarity and family is summarized in Table 1.

Preliminary Screening.
A thin-layer chromatographic technique was conducted to determine the presence of various secondary metabolites. Te screening of extracts was performed by visual detection, UV light (both in short at 254 nm and in long at 365 nm), and vanillin-H 2 SO 4 spray reagent and presented in a tabular form ( Table 2). Tis result indicates the presence of a good number of compounds like favonoids, terpenoids, steroids, coumarins, isocoumarins, anthraquinone, etc., which are depicted in a tabular form.  Table 3.
Extracts from the plant (bark and leaf extract) and fungal strains of SCBE-3, SCBE-4, and SCLE-9 showed antimicrobial activity which indicates that they may produce secondary metabolites with antimicrobial potential.

Antioxidant Activity of Plant and Fungal Extracts.
In the free radical scavenging assay, the extract of the leaf and bark part of the plant showed signifcant antioxidant activity with an IC 50 value of 2.93 and 4.42 μg/mL, respectively. Comparatively, the leaf extract of the plant exhibited better antioxidant activity than the bark extract. Among the endophytic fungi, Penicillium sp. (SCBE-4) showed the highest free radical scavenging activity which the IC 50 value (2.43 μg/mL) was near to that of ascorbic acid (2.42 μg/mL) and butylated hydroxy anisole (BHA) (2.14 μg/mL). It was also impressive that SCBE-4 showed more antioxidant activity than the leaf and bark parts of the plant. Another fungus Fusarium sp. (SCLE-8), Colletotrichum sp. (SCBE-7), and Phyllosistica sp. (SCLE-7) exhibited antioxidant activity with IC 50 values of 42.99, 47.77, and 51.23 μg/mL respectively. However, the IC 50 value was not possible to determine in case of the rest of the fungi because they have not exhibited 50% free radical scavenging activity (Figure 3).

Discussion
Te need for discovering new chemical enteritis to treat diferent ailments is ever increasing as the world is continually facing new threats like cancer, AIDS, COVID-19 etc. Furthermore, the development of resistance of antibiotics and anticancer drugs is a global concern. From the ancient time, plants have served as a source of potential bioactive compounds against diferent ailments. Recently, endophytes associated with plants earned much attention from the researchers as they are ofering metabolites with higher therapeutic potential than plants themselves [27]. Endophytic fungi are amazing organisms colonizing inside the plant tissue and are established as reservoirs of excellent  bioactive metabolites [28]. Tus, the screening of bioactivities of natural products, either medicinal plants or fungi, with the aim to discover potential lead molecules can be underestimated.  -8). Diaporthe species is known as a major plant pathogen causing infections [29]. However, it has been reported to exert potential bioactivity [30]. Colletotrichum sp. is a widely distributed fungal genus reported as plant pathogens, but these fungi have been reported to produce secondary metabolites with diverse bioactivities [31]. Besides these, Colletotrichum sp. has been reported as a novel Te Scientifc World Journal endophytic fungus which is able to produce taxol with excellent cytotoxic activity [32,33]. Penicillium herquei, an endophytic fungus isolated from Cordyceps sinensis, produced three new α-pyrone derivatives [34]. Pestalotiopsis microspora isolated from Terminalia morobensis was enriched to the antioxidant compounds pestacin and isopestacin [11,35]. Phyllosticta capitalensis is an endophyte and weak plant pathogen with a worldwide distribution presently known from 70 plant families [36]. Fusarium wilt is one of the major diseases caused by Fusarium oxysporum pathogenic strains. Some F. oxysporum strains are actually benefcial to the host and can provide protection against root pathogens [37]. Terefore, the goal of this study was to look into the endophytic fungi of Syzygium cumini as a source of bioactive compounds with cytotoxic, antioxidant, and antimicrobial properties. Preliminary screening of fungal extracts of isolated fungi reveals the presence of favonoids, coumarin, isocoumarin, steroids, and many other bioactive phytochemicals.
Extracts of the endophytic fungal isolates and bark and leave extracts were subjected to screen for preliminary antimicrobial study by the disc difusion method, cytotoxicity using the brine shrimp lethality bioassay, and antioxidant activity by DPPH free radical scavenging activity.
Overall extracts of leaf and Pestalotiopsis sp. and Penicillium sp. strain showed signifcant antimicrobial activity against tested human pathogenic bacteria S. typhi, B. megaterium, E. coli, and S. aureus. Only extracts of bark and leaves showed antifungal activity. Te bioactivity screening of fungal crude extracts reveals the presence of strong and specifc antimicrobial activity against diferent bacteria and fungi. Specifc bioactivity may be defned as high inhibition of the growth of one type of target organism with little or no activity against others. Tis phenomenon suggests the presence of compounds that have specifc modes of action as opposed to highly toxic compounds that are often of little use as therapeutics, which is interesting in drug discovery [38].
In the free radical scavenging assay, comparatively, the leaf extract of the plant exhibited better antioxidant activity than the bark extract. Among the isolated endophytic fungi, Penicillium sp. showed the highest free radical scavenging activity (IC 50 of 2.43 μg/mL, near to the IC 50 value of ascorbic acid 2.42 μg/mL) than any other fungal and plant extracts. So, it may be stated that Penicllium sp. may be a prominent source of the novel antimicrobial and antioxidant compound. In the brine shrimp lethality assay, the most potent activity was found from Fusarium sp. (LC 50 of 0.4467 μg/mL) and the least activity from Phyllosistica sp. (LC 50 of 4.3481 μg/mL). Tese results are consistent with other reports [35,[39][40][41][42].
Furthermore, phytochemical screening of the leaf, bark, and fungal extracts reveals the presence of several bioactive metabolites like favonoids, terpenoids, steroids, coumrins, isocoumarins, anthraquinone, etc. Tose classes of phytochemicals are found to exert several pharmacological activities like antibacterial, cytotoxic, antioxidant activity, etc. Tis fnding is in line with the fndings of previous researchers who found a variety of bioactive compounds in endophytic fungal extracts and medicinal plants [43][44][45].

Conclusions
Tis study is an attempt to isolate the endophytic fungus from the Syzygium cumini plant and explore the pharmacological activities of those fungi along with the plant part. Tis study showed that this plant is a plethora of endophytes that are capable of exerting many antioxidants and antimicrobial as well as cytotoxic activities. Plant parts are also bioactive. Tus, this approach may be a way of acquiring novel metabolites having a diverse range of biological activities. However, these endophytic fungi may contain biologically active compounds. Further research can be conducted to isolate the compound and investigate its pharmacological activities.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.

Conflicts of Interest
Te authors declare that they have no conficts of interest regarding the publication.