Antibacterial activity of bioactive compound produced by endophytic fungi isolated from Mangifera casturi Kosterm endemic plant from South Kalimantan, Indonesia

The endophytic fungi that live in endemic plants are a promising bio‐prospect as the producers of antibacterial compounds. This research is aimed to evaluate the endophytic fungi antibacterial compound from Mangifera casturi . The bioactive compounds of 13 endophytic fungi were extracted using ethyl acetate and evaluated for antibacterial activity using disk diffusion assay. The minimum inhibitory concentration (MIC) was measured using the serial broth dilution method. Scanning Electron Microscopy (SEM) was used to examine cell damage because of the extract. The antibacterial compounds were then detected using GC‐MS analysis. The endophytic fungi were identified morphologically and molecularly based on ITS rDNA sequencing Among the 13 isolates, the endophytic fungi identified as Botryosphaeria rhodina AK32 produced the antibacterial compounds that exhibited the highest activity and a broad spectrum. Moreover, they were capable against resistant bacteria, Methicillin Resistant Staphylococcus aureus (MRSA) with an MIC value of 1.56% for all the test bacteria. The mechanism of action of AK32 ethyl acetate extract seemed to affect the condition of bacterial cell walls, causing morphological alteration such as shrinkage of the cell, warted cells, and hollow cells. Based on GC‐MS, the antibacterial compounds of AK32 ethyl acetate extract were di‐n‐octyl


Introduction
Bacteria are one of the causes responsible for many dis eases in the world for a long time ago, and some of the dis eases are lethal to a human being. The scientist has found a solution to this problem with the first discovery of an tibacterial, sulphonamides in 1935 and penicillin in 1940 (Li and Webster 2018). However, bacterial mutations oc cur continuously resulting in resistance to the existing an tibacterial compounds. This situation forced us to looking for the antibacterial compound that is able to overcome the resistant of bacteria (Silver 2011).
One of the sources of organisms that could produce an tibacterial compounds is endophyte, especially endophytic fungi (Deshmukh et al. 2014; Nisa et al. 2015. Fungi were the most diverse and abundant group of endophytic organisms known as the most diverse producer of natural product (Tiwari 2015). From total bioactive compounds produced by microorganisms, as much as 47% were pro duced by fungi (Bills and Gloer 2016). As a microorgan ism that lives within plant tissue, endophytic fungi have known could produce secondary metabolites similar to the host plant (Stierle and Stierle 2015). Endophytic fungi also known work together with the host plant to produce impor tant compound such as antimicrobial compound that help the host plant to improve their diseaseresistance ability (LudwigMüller 2015). Strobel and Daisy (2003) said that there was specific rationale for the collection host plant for endophyte isolation and secondary metabolite discovery. One of the criteria was host plant should be endemic plant that has been known to have a special feature. Endemic plant that occupied an unique habitat seem to be more po tential for endophytic fungal isolate source that produce novel bioactive compound with unique structure (ElDeeb et al. 2013).
antibacterial compound. Many studies have reported that many part of M. casturi contained antibacterial compound. A study conducted by Rosyidah et al. (2010) revealed that M. casturi's bark possesses antibacterial activity against E. coli and S. aureus. Extract and fraction of M. casturi fruit also reported possess antibacterial compound against Pseudomonas aeruginosa, Bacillus cereus (Meliana et al. 2021) and Streptococcus mutant (Suhendar et al. 2019) as well. Moreover, another part of M. casturi such as leaf extract either known to have antibacterial activity against S. mutant (Khairiyah et al. 2019) and Streptococcus san guinis (Sari et al. 2020). For those reasons, M. casturi is a suitable candidate to be explored for its endophytic fungi producing antibacterial compounds. In addition, this ex ploration will also contribute to conservation efforts be cause it can reduce the exploitation of the M. casturi plant, considering that this plant is listed as extinct in the wild by IUCN. To date, there is still no research done for antibac terial compounds produced by endophytic fungi isolated from M. casturi.

Endophytic fungi isolates
The endophytic fungi isolates used in this study were an unidentified endophytic isolates collection of FMIPA UNS Biology Laboratory isolated in April 2019 from M. cas turi's root, stem, and leaves. The plant was obtained from several places at Padang Batung Subdistrict, Kandangan, Hulu Sungai Selatan District, South Kalimantan. A total of 31 isolates of endophytic fungi were sorted and selected based on isolate's growth rate. Thirteen isolates of endo phytic fungi that had quick growth rate were selected and cultured on PDA media and incubated at 28±2°C for fur ther analysis.

Bioactive compounds extraction
Bioactive compounds extraction from endophytic fungi was carried out based on Sharma et al. (2016) with slight modification. The endophytic fungi were cultivated in potato dextrose broth (PDB) and incubated for two weeks at room temperature in a shaker incubator at 90 rpm. Af ter incubation, the culture broths were separated from the mycelium by filtration using Whatman filter paper. Fun gal mycelia were homogenized by breaking the cell using mortar and pestle prior to extraction using ethyl acetate as the organic solvent. The extraction then conducted by adding 100 mL of ethyl acetate to mycelium and 100 mL of ethyl acetate to culture broths, and macerated for 24 h. Fil ter paper then used for filtering and separating Mycelium from ethyl acetate. Ethyl acetate was added into culture broth 1:1 volume ratio in separating funnel, shaken for 10 min and left for 5 min until two separated layers formed. The upper layers that contain bioactive compounds were collected and lower layer were discarded. For maximum extract yield, the extract obtained from mycelia and fer mentation broth then mixed and concentrated by evaporat ing the solvent under a rotary evaporator at 40°C to yield ethyl acetate extract for further assay.

Antibacterial screening
Screening of the antibacterial activity from ethyl acetate extract of endophytic fungi was carried out using the disc diffusion assay method. Crude extracts were evaluated for their antibacterial activity against Gramnegative bacteria (Escherichia coli), Grampositive bacteria (Staphylococ cus aureus), and resistant bacteria (methicillinresistant Staphylococcus aureus). Those were obtained from the microbiology laboratory, Faculty of Medicine, Universi tas Sebelas Maret. The bacteria were grown in Muller Hinton agar (MHA) media. As much as 20 µL of crude extract was dripped to the paper disc using ethyl acetate. Antibacterial activity detected after incubation at 37°C for 24 h. The activity of extract to inhibit bacterial growth was shown with the appearance of the inhibition zone. Chlo ramphenicol (20 mg/mL) and vancomycin (0.03 mg/mL) were used as the positive control, whereas ethyl acetate was used as the negative control.

Determination of minimum inhibitory concentration (MIC)
Ethyl acetate extract possessed the highest diameter of in hibition zone obtained from the preliminary assay was se lected to determine the MIC by the serial broth dilution method. Selected ethyl acetate extract of endophytic fungi was diluted twofold for ten times in sterile tubes asepti cally. Final concentrations of ethyl acetate extract at each tube after dilution were 25% to 0.098% (v/v). Each tube then inoculated with an equal volume of overnight grown bacterial culture and incubated at 37°C for 24 h. The MIC was defined as the lowest concentration at which no visible growth observed after incubation.

Scanning electron microscope (SEM) analysis
Ethyl acetate extract with the highest antibacterial activ ity was investigated its effect on bacteria using SEM anal ysis based on Supaphon et al. (2013). The bacteria were treated with ethyl acetate extract at four times of MIC con centration and incubated for 18 h. After incubation, the pellet was collected and fixed with 2.5% glutaraldehyde (C 3 H 8 O 2 ) in phosphate buffer (PBS) for 2 h, then washed using PBS twice. Pellet then was postfixed in 1% of os mium tetraoxide for 1 h and washed using PBS. After ward, the pellet was dehydrated using alcohol series (50%, 70%, 80%, 90%, and 100%). The pellet then was freeze dried and analyzed using SEM at Research Unit for Nat ural Product Technology, Indonesian Institute of Sciences (LIPI), Yogyakarta.

GC-MS Analysis
The most active extract was further subjected to GCMS analysis to be identified as the chemical composition in the extract. The GCMS analysis of the extract was performed using Shimadzu GCMSQP 2010S Plus fitted with an RTX5 MS (30 m x 0.25 mm x 0.25 mm) capillary col umn in Organic Chemistry Laboratory of Mathematics and Science Faculty, Gadjah Mada University. The instrument temperature program was initially set at 70°C and main tained for 5 min and gradually risen up to 300°C at the end of the period at a rate of an increment by 5°C/min, and maintained for 19 min. Pure helium gas was used as the carrier gas with a flow rate at 0.5 mL/min. Splitless injection port temperature was set and ensured at 300°C. The mass spectrometer was performed in an electron ion ization of 70 eV, while the mass spectral scan range was set at 28600 (m/z). The identification of chemical com pounds present in the extracts was based on their mass spectra with data comparison to NIST12 (National Insti tute of Standards and Technology, US) and WILEY229 libraries.

Morphologically and molecularly identification of endophytic fungi
The endophytic fungal isolate from M. casturi with the strongest antibacterial activity was identified morpholog ically and molecularly. Morphological identification was carried out by observing the macroscopic and microscopic characteristics of the endophytic fungi. Macroscopic char acteristics observation included surface and reverse the colour of the colony. Whereas the microscopic identifica tion was conducted using slide culture method to observe the characteristics for the hypha, conidiophore, conidia, and other certain characteristics. Morphological identifi cation in this study referred to Watanabe (2002). Molecular identification was carried out based on ITS rDNA sequence. Fungal genomic DNA was iso lated using QuickDNATM Fungal / Bacterial Miniprep Kit with cellbreaking treatment using sonication priory and continued by following the manufacturer's procedure. The ITS region was subjected and amplified using the polymerase chain reaction (PCR) with primer pairs ITS1 (5'TCCGTAGGTGAACCTGCGG'3) and ITS4 (5'TC CTCCGCTTGATATGC'3) (White et al. 1990). Targeted Pcr product was 550 bp, validated using electrophore sis with gel agarose and visualized by using GelDoc (GonzálezTeuber et al. 2017). Amplified DNA then was subjected to DNA sequencing fand this DNA sequence was compared with already existing DNA sequences in NCBI GenBank using BLASTN program (http://blast.nc bi.nlm.gov/).

Endophyte from Mangifera casturi
As much as 13 isolates with rapid growth rate were se lected from 31 endophytic fungi of M. casturi isolates col lection. The selected isolates were isolated from different part of M. casturi plant, three from the root, five from the stem, and five were isolated from the leave of M. casturi. Those isolates have a growth range from 39 cm of diam eter when grown in PDA media for 7 d. Among thirteen isolates, each isolate have a different characteristics of the

Antibacterial activity of endophytic fungi
The result exhibited that from 13 selected isolate, four ethyl acetate extract of endophytic fungi were having a broad spectrum antibacterial activity due to their ability to inhibit Grampositive (S. aureus) and Gramnegative (E. coli) test bacteria. Five extracts have narrow spec trum (only against S. aureus) and another four did not showed any antibacterial activity at all. In the ability to inhibit MRSA, among nine extracts which had activity against Grampositive test bacteria, there were eight ex tracts which had the activity to inhibit MRSA, and the highest antibacterial activity of ethyl acetate extract was shown by AK32 ethyl acetate extract which could inhibit all of the test bacteria (Table 1). Ethyl acetate extract from several endophytic fungi showed antibacterial activity by forming an inhibition zone. The inhibition zone formed due to the sensitivity of bacteria to the antibacterial agent within the ethyl acetate extract. The wider zone formed the higher antibacterial activity of the ethyl acetate extract. As shown in Figure 1, the inhibition zone formed by AK32 against MRSA was the widest zone, indicating that AK32 has the highest ac tivity; whereas DK5 has a smaller zone indicated smaller activity and BK31 with no inhibition zone formed indi cated no antibacterial activity.

Minimum inhibitory concentration of endophytic fungal ethyl acetate extract
Ethyl acetate extract of AK32 isolate that has the highest antibacterial activity was assayed for its MIC to test bacte ria. The result exhibited that ethyl acetate extract of AK32 isolate has a MIC value 1.56% (v/v) to all of the test bac teria (Table 2). This information indicates that as much as 1.56% of the ethyl acetate extract of AK32 isolate is more

Antibacterial mechanism of action of the ethyl acetate extract
SEM analysis showed the appearance of test bacterial morphology after treated with ethyl acetate extract of AK32 isolate with four times MIC concentration. The microscopic display indicated morphological differences in treated and untreated of all three test bacteria. In un treated bacteria, all of the test bacteria showed an intact and smooth structure (Fig.2ac). Whereas for bacteria treated with ethyl acetate extract of AK32 isolate, they showed an observable morphological change (Fig.2df). said that the main component of cell wall was peptidogly can which is found in almost of all bacteria and respon sible to maintain the integrity of the cell itself. Damage occurred in that might cause cytoplasmic leakage and lead to cell death (Vollmer et al. 2008).

GC-MS analysis
GCMS chromatogram (Fig. 3) shows the presence of 89 compounds identified on different retention times and % area from AK32 ethyl acetate extract. The results of GC MS analysis show that in AK32 ethyl acetate extract there are various classes of compounds such as fatty acids, phe nolic, lipids, alkaloids, terpenoid, and volatile compounds.

Morphology and Molecular Identity of Potential Ethyl Acetate Endophytic Fungi Isolate
AK32 isolate has a greyishblack surface colour with cottonylike texture, whereas the reverse colour was black  ( Fig. 4ab). Microscopically, AK32 isolate showed ster ile mycelia, so that AK32 isolate was classified as non sporulating isolate (Fig. 4c). Strobel (2018) said that en dophytic fungi frequently showed nonsporulating hyphae on PDA media, this occurred due to the inability of the en dophytic to developed spore in the media. This indicates that AK32 isolate could not be identified based on its mor phological characteristics because sterile mycelia do not show speciesspecific characteristics (Sun and Guo 2012). The molecular identification based on the ITS rDNA region was used to validate the result from morphological identification. The amplicon of the ITS rDNA region was ± 550bp (Fig. 5).
Based on BLAST analysis, the potential antibacterial producing isolates have a similarity percentage of 99.60% with the Gene Bank database ( Table 3). The ≥99% simi larity to the database from Gene Bank indicates similarity at a species level (Liu 2011).

Discussion
Intensive search for new effective antimicrobial agents is required, which is facilitated by exploring new resources, especially endophytic fungi which have not been widely explored (Taufiq and Darah 2019). The antibacterial com pound finding from endophytic fungi associated with en demic plants is a promising endeavor to answer the chal lenge in increasing the need for antibacterial drugs. In this study, nine isolates show antibacterial activity, and four of them (AK32, BK32, AKF1, and DK7) exhibited an an tibacterial activity not only against Grampositive bacte ria, but also against Gramnegative bacteria with strong activity. These broad spectra of activity were affected by the existence and synergistic activity of various chemical compounds within the extract which have various mecha nisms of action against Grampositive and Gramnegative bacteria.
Another five ethyl acetate extracts of endophytic fungi displayed a narrow spectrum which effective only for Grampositive bacteria. This indicated that the compound contained in this extract did not have mechanisms of ac tion against Gramnegative bacteria. The more effective ness of the extract against Grampositive bacteria was also due to its morphological constituent of Grampositive bacteria that have a simpler cell wall structure contained only the peptidoglycan layer as the outer barrier (Breijyeh et al. 2020). On the other hand, Gramnegative bacteria have a more complex barrier with additional protection af forded by the outer membrane contained lipopolysaccha ride (LPS) which acts as barrier (Epand et al. 2016). This made the bioactive compounds that have no ability to dam age the LPS layer, could not able to penetrate the inner cell of Gramnegative bacteria.
Among nine ethyl acetate extracts of endophytic fungi which had antibacterial activity against Grampositive bacteria (S. aureus), eight of them have activity against resistant bacteria (MRSA). The mechanisms of action of the activity of the extract against MRSA was clearly demonstrated by SEM photograph's observation that there were morphological alteration with collapsed cell, and this could be do to the leak in it's cell wall or there were some changes occurred in the cell's membrane permeability. In this case, the damaged occurred by the extract of endo phytic fungi could be cause by the extract had permeabi lized the bacterial cell membrane that influenced the con dition of bacteria. This probability was in line with study conducted by Taufiq and Darah (2019) who reported the extract of Lasiodiplodia pseudotheobromae IBRL OS64 exhibited antibacterial activity by spoil the cell wall of bac terial cells which led to damaged of the cell. As shown by the SEM photograph, the most possible mechanisms of action involved could be the interaction of compound with membrane of the cell and might be linked with the ability of compound contained in the extract to inhibit the pepti doglycan synthesis process and penetrate the cell wall by after binding with penicillinbinding protein 2a (PBP2a). As known, MRSA has an altered PBP structure that is PBP2a caused by the existence of mecA gene, resulting in affinity reduction of βlactam and being resistance to βlactam antibiotics (Stapleton and Taylor 2002).
The highest antibacterial activity against all test bacte ria was displayed by AK32 ethyl acetate extract. This ex tract had small MIC value (1.56%) indicating that AK32 only need a very small amount of bioactive compound to inhibit the bacteria. Bacterial cell treated with AK32 ex tract showed sign of damage, demonstrated by the shape deformation and inward indention of the cell. Through GCMS analysis, ethyl acetate extract of AK32 showed several major compounds with different re tention times. Dinoctyl phthalate was one of the ma jor compounds with the highest % area (7.75%). This compound belongs to the phthalate compound. Phtha late group of compound have known before as plasti cizer and contaminant. Nevertheless, there are many study that reported phthalate compounds and their deriva tives have isolated from natural sources (Thiemann 2021). Dinoctyl phthalate were reported could be produced by various natural sources such as microbe like Strepto myces parvusand (AbdElnaby et al. 2016), Trichoderma asperellum (Bhardwaj and Kumar 2017), Memnoniella (Hande and Kadu 2015), and plant such as Plumbago zey lanica Linn. (Ira et al. 2015), Pachygone ovata (Poir). Miers (Amalarasi and Jothi 2019) and from Coleus fos khohlii (Takshak and Agrawal 2016). Thiemann (2021) also said that these compound have benefit for human health either. Boudjelal et al. (2011) in his study reported that dinoctyl phthalate was a compound produced by the genus Actinoalloteichus and had antibacterial activity against Bacillus subtilis. Another study was conducted by Amalarasi and Jothi (2019) found that dinoctyl pththa late isolated from Pachygone ovata (Poir). Miers pos sessed a cytostatic activity against breast cancer cell. An other major compounds were benzyl alcohol (7.26%), ben zeneacetonitrile (6.05%), benzotriazole (4.98%), octade canoic acid, methyl ester (3.85%); cyclopentenone (3.48); 2,4decadienal, (E,E) (3.00%); 1,2ethanediol, diacetate (2.87%); and 9,12,15octadecatrienal (2.41%) also have been reported had antibacterial activity (Yano et al. 2016; Wei et al. 2011; Wu et al. 2018; Trombetta et al. 2002; Halstead et al. 2015; Boudjelal et al. 2011. In addition to those compounds, minor compounds found in AK32 extract such as βcaryophyllene and far nesol also reported having antibacterial activity. These both compounds belong to terpenoid group. Dahham et al. (2015) said that βcaryophyllene is a terpene that not only has high antibacterial activity but also has antifungal and antioxidant activities. Whereas, farnesol is a sesquiter pene compound that is known to have antibacterial activ ity against S. aureus (Inoue et al. 2016). Based on these findings, AK32 ability to inhibit test bacteria may be due to the synergetic collaboration between major and minor compounds within the extract.
From morphological and molecular identification, it is known that AK32 isolate is Botryosphaeria rhodina. B. rhodina is a teleomorph of Lasidioplodia theobromae. This fungus has been known as an endophyte and op portunistic pathogen. As endophytic fungi B. rhodina has various pharmaceutical activities, including antibac terial. Rukachaisirikul et al. (2009) in his research found that B. rhodina had antibacterial activity against S. au reus ATCC 25922 and MRSA by producing glactone, one dihydronaphthalene2,6dione, one hexahydro indeno fu ran, one cyclopentanone, and lasiodiplodine. Our study has revealed that B. rhodinia besides having antibacterial against S. aureus and MRSA, it also has the same effect on E. coli by producing different bioactive compounds. The difference between these compounds might be affected by the difference of the host plant. B. rhodina is also known has another activity. Abdou et al. (2010) revealed that B. rhodina isolated from medical plant Bidens pilosa has an tifungal activity and cytotoxic depsidones by producing Botryorhodines AD. The finding in this study provides a basis for further research such as isolation that focuses the target antibacterial compound to be developed as a new drug.

Conclusions
This study concludes that the endophytic fungal extracts isolated from M.casturi, endemic plant of South Kali mantan, Indonesia exhibit antibacterial activity due to the bioactive natural compounds production. AK32 iso late was the highest isolate produced antibacterial com pound such as dinoctyl phthalate, phenol, 2methyl, 4 pentadecyne, 15chloro, benzene acetonitrile, and ben ztriazole which had mechanism of action by damaging cell wall in grampositive bacteria and damaging the outer membrane layer in gramnegative bacteria. AK32's com pounds found by GCMS analysis provided new informa tion for developing new antibacterial agents from endo phytic fungi. These active compounds can then be used as a source of antibacterial substance to support M. casturi conservation endeavor.