Xenomyrothecium tongaense PTS8: a rare endophyte of Polianthes tuberosa with salient antagonism against multidrug-resistant pathogens

Introduction Endophytes refer to microorganisms residing within the endosphere of plants, particularly perennials, without inflicting noticeable injury or inducing obvious morphological variations to their host plant or host organism. Endophytic fungi, although often overlooked microorganisms, have garnered interest due to their significant biological diversity and ability to produce novel pharmacological substances. Methods In this study, fourteen endophytic fungi retrieved were from the stem of the perennial plant Polianthes tuberosa of the Asparagaceae family. These fungal crude metabolites were tested for antagonistic susceptibility to Multi-Drug Resistant (MDR) pathogens using agar well diffusion, Minimum Inhibitory Concentration (MIC), and Minimum Bactericidal Concentration (MBC) assays. The chequerboard test was used to assess the synergistic impact of active extract. Results and discussion In early antibacterial screening using the Agar plug diffusion test, three of fourteen endophytes demonstrated antagonism against Methicillin-resistant Staphylococcus aureus (MRSA) and Vancomycin-resistant Enterococcus (VRE). Three isolates were grown in liquid medium and their secondary metabolites were recovered using various organic solvents. Eight extracts from three endophytic fungi displayed antagonism against one or more human pathogens with diameters ranging from 11 to 24 mm. The highest antagonistic effect was obtained in ethyl acetate extract for PTS8 isolate against two MRSA (ATCC 43300, 700699) with 20 ± 0.27 and 22 ± 0.47 mm zones of inhibition, respectively, among different solvent extracts. The extract had MICs of 3.12 ± 0.05 and 1.56 ± 0.05 μg/mL, and MBCs of 50 ± 0.01 and 12.5 ± 0.04 μg/mL, respectively. Antagonism against VRE was 18 ± 0.23 mm Zone of Inhibition (ZOI) with MIC and MBC of 6.25 ± 0.25 and 25 ± 0.01 μg/mL. When ethyl acetate extract was coupled with antibiotics, the chequerboard assay demonstrated a synergistic impact against MDR bacteria. In an antioxidant test, it had an inhibitory impact of 87 ± 0.5% and 88.5 ± 0.5% in 2,2-Diphenyl-1-Picrylhydrazyl and reducing power assay, respectively, at 150 μg/mL concentration. PTS8 was identified as a Xenomyrothecium tongaense strain by 18S rRNA internal transcribed spacer (ITS) sequencing. To our insight, it is the foremost study to demonstrate the presence of an X. tongaense endophyte in the stem of P. tuberosa and the first report to study the antibacterial efficacy of X. tongaense which might serve as a powerful antibacterial source against antibiotic-resistant human infections.


Background
Antimicrobial resistance is not a new phenomenon, but the number of resistant organisms, the geographic areas where drug resistance occurs, and the range of resistance in individual organisms are all unprecedented and escalating.Tuberculosis has been reemerging since the 1980s, and it is now notably multidrug-resistant (MDR), with infection with the human immunodeficiency virus (HIV) exacerbating the disease.Due to the severity of the challenges in treating Multi-Drug Resistant (MDR) strains, the use of many drugs, typically six to seven different medications, is necessary (Levy and Marshall, 2004).However, due to overuse, many antibiotics have lost most of their potency, resulting in more disease-causing bacteria acquiring resistance to them (Abdulmyanova et al., 2020).Antibioticproducing bacteria have a variety of complicated defense systems to protect themselves from the antibiotics they produce (Peterson and Kaur, 2018).Staphylococcus aureus, the dangerous hospital bacterium MRSA (Methicillin-resistant Staphylococcus aureus), or ESBL (Extended-spectrum lactamases) bacteria that manufacture betalactamases, for example, are resistant to a variety of treatment drugs (Wen et al., 2023).Infectious disorders caused by antibiotic-resistant bacteria are becoming more common, posing a substantial medical problem.Poor aseptic settings, inappropriate treatment usage, late analysis of illnesses, and continual movement of travelers all contributed to the increase in instances (Fuego et al., 2021).S.aureus, which is resistant to methicillin, is one of the leading causes of hospital-acquired disease in many Indian hospitals.According to antibiotic susceptibility testing, around 30-80% of them have been documented from various places (Gowrishankar et al., 2013).Over the previous two decades, there has been a rise in the number of MRSA, Streptococcus pneumoniae resistant to penicillin, and Enterococcus faecium resistant to Vancomycin medicines.Furthermore, recent medicines such as Daptomycin and Linezolid have developed resistance (Deshmukh et al., 2015).As a result, there is an urgent need to prevent antimicrobial resistance by improving antibiotic practice and reducing cross-infection in hospitals.Nonetheless, new antibiotic development must continue since it is critical for sustaining antimicrobial therapy effectiveness (Janeš et al., 2007).
Bioactive chemicals derived from natural sources were regarded as the foundation for high-value product development.Their biological functionality has allowed them to continue to be used in agriculture, medicine, and the food sector (El-Sayed et al., 2022).They were deemed possible sources of active metabolites with a variety of distinct restorative properties, such as steroids, xanthones, phenol, flavonoids, tetralones, alkaloids, benzopyranones, and terpenoids (Soni et al., 2021).Several compounds now in clinical trials were modifications of existing antibiotic classes discovered in recent drug discovery and development programs.They represent only transitory remedies to the growing opposition.As a result, there is a growing interest in identifying chemicals from novel families with distinct structures and action mechanisms to treat drug-resistant infections (Sierra-Zapata et al., 2020).Medicinal plants include a varied range of cultural endophytes capable of producing structurally intriguing and bioactive secondary metabolites (An et al., 2020).Endophytes are microorganisms that live in the plant's endosphere but do not cause disease symptoms (Mishra and Priyanka, 2022).They colonize the plant tissue, either inter or intracellularly, and maintain a harmonic symbiotic relationship in all of the plants studied (Soni et al., 2021).
The host and endophyte interact mutually, with the host providing nutrients and shelter and the endophytes acting as chemical sentries (Ababutain et al., 2021).Interestingly, fungal endophytes and their host plants can produce the same or similar bioactive compounds.As a result, they may be employed to produce a replaceable method for producing beneficial bioactive compounds to safeguard plants and the natural environment (Ye et al., 2021).Fungal endophytes have grabbed the interest of researchers because they provide new sources of antibacterial components, cytotoxic substances such as anticarcinogenic agents, and bio-stimulants for essential oil production (Alam et al., 2021).Many plant species have been examined because of their diverse endophyte variety and potential to induce bioactive secondary metabolites (Munshi et al., 2021).To yet, practically all higher plants investigated have been related to endophytes that live above and below ground (Mishra and Priyanka, 2022).Over 300,000 plant species on the planet that are developing in new zones host one or more endophytes (Sharma et al., 2016).
Polianthes tuberosa L., often referred to as Rajanigandha a plant used for ornamental purposes in the Asparagaceae family.It originated in Mexico and is now grown in a variety of tropical and subtropical climates.Because of aromatic compounds, they are mostly used in the perfume industry.Furthermore, the plant comprises a variety of secondary metabolites, including steroid glycosides and flavonoids, whereas the flower mostly contains terpenoid derivatives and benzoid (Fan et al., 2018;Safeena et al., 2019;Alghuthaymi et al., 2021).As a result, there is an urgent need for novel antibacterial drugs to combat the increasing range of illnesses.The main aim of this study was to evaluate the hostile actions of endophytic fungus from the P. tuberosa plant on multi-drug-resistant bacterial infections.The present study goal will lead to a better understanding of endophytic fungi and their use in antibiotic-resistant disease treatment.

Procurement of chemicals and glassware
Chemicals utilized in the experiment were procured from HiMedia Ltd.Mumbai, India.The glassware was from Borosil and the solvents used were acquired from SRL.

Sampling of plant materials for the study of endophytic organisms
To isolate endophytic fungi, a healthy stem of Polianthes tuberosa L. has been collected in sterile polyethylene bags from the Vellore district (12°56′44.8"N79°13′58.2″E),Tamil Nadu, India.Within 12 h after collection, the samples were processed.The plant was certified by Professor Dr. Jayaraman P, PARC (Plant Anatomy Research Centre), West Tambaram, Chennai-600045 (Basha et al., 2012).

Surface sterilization and endophytic fungi isolation
The stem materials were carefully removed from the healthy P. tuberosa and washed for 5 min with distilled (d.H 2 O) water to The stem was then split into small parts 0.5 cm 2 /1 cm long.The explants were transferred and impregnated onto the PDA dish and incubated at 27°C with a daily monitor for 7-10 days till the fungus appeared.Using the single hyphal tip approach, the developing fungus was transferred to a fresh medium.
The pure strain was evaluated based on colony morphology and stored in slants at 4°C (Liu et al., 2019).

Endophytic fungal growth rate assessment
The endophytic fungal isolate PTS8 was cultured on a PDA to probe the growth rate of mycelia.The inoculated fungal plate was incubated at 27 ± 2°C for a week.The mycelial growth rate was documented every 24 h by assessing the diameter of the growing mycelium (Techaoei et al., 2020).

Agar plug diffusion technique
The agar plug diffusion method (primary antibacterial screening) was conducted for all the fungal endophytes isolated.A sterile cotton swab was used to swab the test microorganisms on the Muller Hinton agar (M173 HiMedia) medium.Using a sterile cork borer, mycelium agar discs (5 mm diameter) were collected from seven days of actively developing pure fungal cultures on PDA media.The plugs were put on MHA plates seeded with the test microorganisms and refrigerated overnight at 4°C to allow metabolite diffusion (in triplicates).The plates were then placed in an incubator for 12 h at 37°C to allow microbial development, and growth inhibition was observed (Marcellano et al., 2017).

Antibacterial bioassay by agar well diffusion method
The dried-out crude was reconstituted with DMSO for antibacterial testing.On Muller Hinton agar (MHA) plates, the bacterial lawn culture was evenly dispersed with a sterile cotton swab.Wells of about 2 mm in diameter have been created and filled with extracts of various solvents.The well diffusion technique was used to study dilutions of fungal extract ranging from 25 to 100 μg/ mL against the test pathogens.The system was incubated at 37°C for 24 h.The impact of fungal extracts on pathogen growth was measured after incubation (mm).The trial was carried out in triplicate.Oxacillin (1 mcg) and Vancomycin (30 mcg) commercial antibiotic discs were used as positive controls, while Dimethyl sulfoxide (5%) was used as a negative control.For large-scale fermentation, fungal isolates with promising antibiotic action were chosen (Anitha and Mythili, 2017).

Morphology and microscopic analysis
The fungal isolate was initially identified using scanning electron microscopy (EVO/18 Research, Carl Zeiss) to examine morphological parameters, spore structure, and surface morphology.The culture development profile, spore colors, and morphologies were examined using standard guides.The fungal strain was characterized by the Lacto phenol cotton blue method and detected in the microscope.

Molecular genomic recognition of endophytic fungus
The species-level identification of the potential isolate was accomplished by utilizing 18S rRNA ITS sequencing.Fungal isolate genomic DNA was attained by NucleoSpin ® Tissue Kit.Using ITS1 (5'TCCGTAGGTGAACCTGCGG3') and ITS4 (5' TCCTCCGCTTATTGATATGC 3′) universal primers, the 18S rRNA gene was amplified.Initially, denaturation was carried out for 2 min at 95°C, followed by 25 cycles of 30 s of denaturation at 95°C, primer annealing at 55°C for 30 s, first extension for 1 min at 72°C and last extension for 5 min at 72°C were the PCR thermal cycling conditions.
The sequencing outcomes were achieved using ABI PRISM ® BigDyeTM Terminator Cycle Sequencing Kits with AmpliTaq ® DNA polymerase (Applied Biosystems).Using the Blast tool, the attained sequence of isolate PTS8 was matched through the NCBI database.By the neighbor-joining tree technique, the phylogenetic tree was built.The consensus sequences were deposited in GenBank (Gautam et al., 2016(Gautam et al., ). 10.3389/fmicb.2024.1327190 .1327190Frontiers in Microbiology 04 frontiersin.org

Minimum inhibitory and minimum bactericidal concentration
The NCCLS-recommended broth microdilution susceptibility assay was used to determine the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal concentration (MBC) of crude with the greatest inhibitory effect.The entire experiment was conducted in Muller Hinton broth (MHB).To dissolve the fungal extracts, DMSO (10%) was employed, and a 96-well plate dilution was generated from (50 μg/mL-0.39μg/mL) concentration.Following this, each well received 100 μL of Muller Hinton broth (M391 HiMedia) and 10 μL of preprepared bacterial suspension.The plates were incubated for 24 h at 37°C.The MIC was later determined to be the well with the lowest concentration and no observable growth of microbes.The drugs Oxacillin and Vancomycin were utilized as positive controls for MRSA and VRE, respectively, whereas MHB was used as a negative control.The MIC findings identified the dilutions that had no obvious bacterial growth.50 μL of broth was transferred from each of these wells to the MHA medium and incubated according to the preceding limitations.MBC was the final concentration that inhibited total bacterial growth.The experiment was performed in triplicates (dos Santos et al., 2015).

MIC index of fungal extract
To confirm the efficacy of ethyl acetate extract whether it is bactericidal or bacteriostatic.By dividing MBC by MIC value, the MIC index was assessed.The MIC index was calculated by dividing MBC by the MIC value.If the index value is less than or equal to 4, it is called bactericidal, and if it is larger than 4, it is considered bacteriostatic (Sadrati et al., 2020).

Synergistic study of crude extract
The checkerboard experiment was used to appraise the fungal extract's synergistic interaction with antibiotics such as Oxacillin and Vancomycin.Synergistic combinations of the extract and antibiotics were created at the MIC value to which the bacterial strains were resistant, and then serially diluted in two steps.The FICI (Fractional inhibitory concentration index) was calculated using the method below to determine the optimal interaction combination.

( )
Fractional inhibitory concentration FIC of fungal crude extract Extract MIC in combination with antibiotic / Extract MIC alone.

= ( )
Fractional inhibitory concentration FIC of antibiotic Antibiotic MIC in combination with extract / Antibiotics MIC alone.

Qualitative phytochemical screening
The screening was performed for ethyl acetate solvent crude extracts of PTS8 isolate as per standard procedures described by Devi et al. (2012).This method aims to check the presence or absence of Flavonoids, Tannins, Alkaloids, Saponins, Steroids, Cardiac glycosides, and Phenolic compounds in the fungal crude.

Quantitative phytochemical screening
Based on the qualitative examination, a quantitative investigation was carried out for Tannins, Flavonoids, Phenols, and saponins by following (Gokilavani and Banu, 2021).

Gas chromatography-mass spectrometry sample preparation
The obtained active endophytic fungal ethyl acetate dry extract was liquefied with the equivalent solvent and dissolved thoroughly to make a concentration of 1 mg/mL and subjected to GC-MS analysis.

GC-MS analysis of PTS8 fungal crude extract
GC-MS analysis was carried out in Perkin Elmer Clarus-680 equipped with a Clarus 600 mass spectrometer and a capillary column (30 m, 0.25 mm ID, 250 m df).The temperature in the early oven was kept at 60°C for 2 min, then ramped to 300°C at 10°C/min for 6 min.Helium was supplied at a constant flow rate of 1 mL/min, and the mass transfer line and source temperature were set at 240°C.The entire procedure takes 25 min to complete.Turbo mass software (5.4.2 version) was used for the spectrum analysis.The configurations of the chemical were compared to the mass spectral outlines in the NIST collection (2008) (Gautam et al., 2016).

DPPH radical scavenger assay
The anti-oxidative property of the fungal crude extracts was determined in vitro using DPPH (2,2-Diphenyl-1-Picrylhydrazyl).For the investigation, fungal extracts were dissolved in DMSO.The extract and standard (Ascorbic acid) were diluted to 50, 100, and 150 μg/mL in a test tube, and 2 mL of DPPH was supplied.The tubes were incubated for 30 min at room temperature in the dark, and absorbance was recorded using a UV-Vis spectrophotometer at 517 nm.The fraction of inhibitory free radicals was calculated using (Soni et al., 2021).

[ ]
Inhibition percentage of free radicals Abs of control Abs of sample / Abs of control 100.= − ×

Reducing power assay
The radical scavenging activity of fungal extracts was determined using the reducing power method.Various extract concentrations (50,100,150 μg/mL) were mixed with 2.5 mL of 0.2 M phosphate buffer (pH 6.6) and 2.5 mL of potassium ferricyanide (1%).At 50°C, the mixtures were incubated for 20 min and 2.5 mL of 10% trichloroacetic acid was added.Then it is centrifuged for 10 min at rpm of 1,000.Post centrifugation, the supernatant 2.5 mL was added to 2.5 mL d.H 2 O and 0.5 mL ferric chloride (0.1%) and vortexed.The absorbance was read at 700 nm.Ascorbic acid is used as the reference drug.The test was done in triplicates (Zeng et al., 2011).

Percentage inhibition
Where ' A' is the absorbance.

Statistical analysis
Each of the in vitro experiments was run in triplicates and the outcomes were estimated by Version 9.5.1 (733) GraphPad Prism software.The data were provided concerning average ± standard deviation.The results were analyzed with one-way ANOVA.

Isolation and screening of potential endophytic fungi
According to morphological characteristics, 14 isolates of endophytic fungi from P. tuberosa were obtained from the stem (Figure 1) with no bacterial or fungal growth in the control plate.According to their tissue of origin, the isolates obtained were coded as PTS1-PTS14 from stems.All 14 fungal endophytes were exposed to determine their antibacterial efficacy by the agar plug diffusion technique (primary screening).Three fungal endophytes that showed positive antibacterial activity in initial screening (Table 1) were cultivated independently for 21 days at 27 ± 2°C in potato dextrose broth and extracted with various polarity solvents.Each resulting extract was individually tested for antagonistic ability using the agar well diffusion technique (secondary screening).PTS8 isolate crude extracts were the most active against all of the pathogenic microorganisms tested.The highest antagonism was seen in ethyl acetate extract with inhibitory zones of 20 ± 0.25 mm and 24 ± 0.14 mm against MRSA ATCC 43300 and ATCC 700699, respectively, which was 3 to 4 times greater than the positive control.Inhibition was seen against S.aureus ATCC 25923 and MTCC 3160 at 20 ± 0.27 mm and 22 ± 0.47 mm, respectively.At 100 μg/mL concentration, the extract inhibited VRE with an inhibition zone of 18 ± 0.23 mm and E.faecalis with an inhibition zone of 21 ± 0.11 mm.The dichloromethane crude had a ZOI of 16-20 mm in diameter against the pathogens tested.Bacterial growth was disrupted when ethyl acetate extract concentrations increased, resulting in bacterial growth inhibition.In contrast, no zone was found for the negative control (DMSO).Table 2 validates the results as the diameter of inhibition.Based on the findings, PTS8 isolate has the potential to be a spectacular antibacterial medication that may be studied further.The results of the ANOVA indicated that there is a significant difference between the means of antibacterial results.The antagonistic activity of the PTS8 isolates in primary and secondary screening is shown in Figures 2, 3.

Identification of potential inhibitory isolate
The fungal isolate PTS8 showed strong antagonistic activity against the tested resistant pathogens and was further characterized morphologically and molecularly.The fungal isolate PTS8 is a fastgrowing white fungus that turns purple at the center after 72 h.The potent fungal endophyte PTS8 was identified as Xenomyrothecium tongaense by 18 s rRNA ITS sequencing with 98% identity.The resulting sequence was deposited in GenBank with the accession number ON678071.The nucleotide BLAST sequence result was used to construct the phylogenetic tree using the neighbor-joining technique (Figure 4C). Figure 5B depicts the colony form of Xenomyrothecium tongaense PTS8 cultivated on PDA.Figures 5C,  4A,B show the microscopic appearance of LCB staining and spore morphology.According to our knowledge, no research has been conducted on the X. tongaense endophyte, which inhibits MDR bacterial development.

MIC and MBC of the fungal extracts
The endophytic fungus X. tongaense that showed the highest inhibitory effect in the agar well diffusion method was analyzed for its minimum inhibitory and bactericidal effect by a two-fold serial dilution protocol.The MIC values varied from 0.78 to 6.25 μg/mL.The ethyl acetate fungal extract of X. tongaense has the highest MIC of 3.12 ± 0.05 and 1.56 ± 0.05 μg/mL against MRSA ATCC 43300 and ATCC 700699, whereas the MIC value was 6.25 ± 0.25 μg/mL against VRE.The antagonistic activity of the extracts seems to be directly proportional to their concentration.Therefore, higher antibacterial substance concentrations yielded a more significant bacterial growth suppression.The MBC of the ethyl acetate extracts from the two dilution units evaluated was above the MIC of the extract.The extract showed a bactericidal effect in the 50-12.5 μg/mL range.The lowest MBC of the extract exhibited complete bacterial growth inhibition was 12.5 ± 0.04 μg/mL for MRSA ATCC 700699 and 25 ± 0.01 μg/mL for VRE.The results of the crude extract against tested pathogens are presented in Table 3.

Synergistic testing of ethyl acetate extracts
The combination of ethyl acetate fungal crude extract with Oxacillin and Vancomycin was evaluated against test pathogens.Table 4 shows the findings of the chequerboard assay.The MIC value of the fungal extract was 3.12 ± 0.05 and 1.56 ± 0.05 μg/mL against MRSA ATCC 43300, and ATCC 700699, respectively.Whereas, the combination of fungal extract and Oxacillin performed synergistically,drastically lowering the MIC value to1.56 ± 0.05 and 0.19 ± 0.21 μg/mL, respectively.Vancomycin combinations with the extract decreased MIC from 12.5 ± 0.05 to 1.56 ± 0.2 μg/mL with FICI of 0.24 against MRSA (ATCC 43300) indicating a strong synergistic impact (FICI 0.24).Furthermore, the fungal extract showed MIC of 6.25 μg/mL whereas thecombination of fungal extract and Vancomycin acted synergistically against VRE.It decreases the MIC level to 1.56 μg/mL with an FICI of 0.4, demonstrating synergism against VRE (ATCC 56299).Thus, the findings indicate that the fungal extracts exhibit significant synergistic impact when it is combined with the antibiotics than it is used alone may aid in the treatment of infections caused by drugresistant bacteria.

Mycelium growth rate
The radial mycelial diameter was measured after 7 days of incubation of the colony plates to evaluate the growth rate of the vegetative mycelium.The results showed that the colony's radial expansion was faster on days 4 and 6 (Figure 5D).According to the

Qualitative secondary metabolite screening
According to the qualitative secondary metabolite analysis, fungal ethyl acetate extracts include phenol, alkaloids, tannins, saponins, and flavonoids.As a result, the existence of these active metabolites serves as a marker that may be used as a pioneer in synthetic drug creation and progress.

Quantitative estimation
Quantitatively the active metabolites present in the ethyl acetate extract of X. tongaense PTS8 were screened.The active chemicals estimated quantitatively are represented in Table 5.The results revealed that there is a significant difference between the mean value p < 0.05.

Antioxidant analysis of fungal extract
The DPPH and reducing power method was used to determine the antioxidant capacity of all 14 endophytic fungi.The results showed that fungal extracts inhibited free radicals significantly.Furthermore, the percentage of inhibition rise as the crude extract concentration increased (Figure 7).At 150 μg/mL concentration, X. tongaense PTS8 ethyl acetate extract has a maximum inhibitory activity of 87 ± 0.5%, whereas ascorbic acid has a maximum inhibitory action of 96 ± 0.5%.The PTS5 isolate had the second-greatest scavenging property, with 75 ± 0.5%.The highest free radical scavenging activity was observed in PTS8 ethyl acetate extract of 88.5 ± 0.5% in reducing power assay.The results of the ANOVA indicated that there is a significant difference between the means of antioxidant results (p < 0.001).

Discussion
The antibacterial activity of the Xenomyrothecium tongaense PTS8 isolate isolated by the plug agar technique might be related to the generation of diffusible extracellular metabolites in the agar medium, with these compounds having a specific antibacterial function.This is a straightforward and widely used method for identifying non-volatile compounds produced by bacteria.The fungal endophyte X. tongaense 10.3389/fmicb.2024.1327190Frontiers in Microbiology 09 frontiersin.orgPTS8 ethyl acetate extract proved considerably active against the tested MDR human infections.Ethyl acetate was shown to be the most effective organic solvent for extracting fungal bioactive components.
According to these findings, the antibacterial bioactive chemicals are semi-polar and may be extracted using ethyl acetate (Sadrati et al., 2020).Endophytic fungi are taxonomically diverse.These fungi can control the morphological and physiological activity of host plants in a variety of ways.They are found in almost every type of tissue studied and include potential biologically active chemicals.Despite these characteristics, endophytic fungi receive little attention; hence, different ecological functions and biological assets should be extended globally (Du et al., 2022).The ability of endophytic microorganisms, mostly fungi, to create a diverse variety of active compounds that might protect the plant against disease is one of their most important characteristics (dos Santos et al., 2015).
Metabolites obtained from Zingiber officinale rhizome endophytic fungus GFV1 fungal sp.(KX247125) isolated from layanikkara variety exhibit antibacterial activity against S. aureus (MTCC 96), B. subtilis   (MTCC 121) and Salmonella enterica Typhimurium (MTCC 1251) with inhibition of 20, 10, and 10 mm, respectively.It also demonstrated antifungal activity against Pythium myriotylum on dual culture method with inhibition of 53.3%.Their fungal extracts also produced bioactive compounds such as benzene acetic acid, dehydromevalonic lactone and n-hexadecanoic acid which was identified by GC-MS.The fungal endophyte Chaetomium fusiforme also reported the presence of a Benzene acetic acid molecule (Anisha and Radhakrishnan, 2017).
A study reported that purified Benzyl Benzoate compound from the toluene fraction obtained from Emericiella quadrilineata-derived fern Pteris pellucida showed significant antagonism against Aeromonas hydrophilla and S. aureus at a zone of inhibition of 22 ± 0.8 15 ± 0.8 in disc diffusion assay.GC-MS analysis of toluene fraction disclosed the presence of benzyl benzoate (27.3%).Medically benzyl benzoate is an active ingredient of Ascabiol that is generally used as an ointment to treat scabies and various skin diseases (Goutam et al., 2016).Propenoic acid, tridecyl ester, a bioactive molecule produced by Paecilomyces sp.(JN227071.1)endophytic fungus reported to exhibit antifungal action against Rhizoctonia solani (Hawar et al., 2023).As a result, the current study focuses on the antibacterial properties of Xenomyrothecium tongaense to battle multi-drug-resistant bacterial infections.

Conclusion
Our study reports highly novel findings of (1) endophytic fungi from the plant P.tuberosa; (2) the endophytic nature of the fungi Xenomyrothecium tongaense PTS8; and (3) the antibacterial efficacy of the isolated endophytic fungus X. tongaense PTS8 against MDR pathogens.This is the first report on the endophytic fungus association in the stem of the P. tuberosa plant and the first report on the endophytic nature of Xenomyrothecium tongaense fungus.Also, this is the first report to study the antibacterial activity of X. tongaense to battle multi-drug-resistant bacterial human pathogens.Moreover, the X. tongaense fungal extract was significantly inhibiting the MDR pathogens at its minimal extract concentration and it is more potent than the positive control used.The present research will contribute to the application of endophytic fungus as a potent antibacterial agent for enhanced human infectious disease management.Further, the limitations of the study are analytical chemistry investigation on purification and identification of the bioactive secondary metabolite and its mechanistic involvement in suppressing the growth of drugresistant infections are yet to be done.In vitro and in vivo toxicity and efficacy of the crude extract are yet to be validated in future studies.In addition to laying the groundwork for future research, this study examines the possibility of producing effective antibacterial drugs to tackle drug-resistant bacterial illnesses.

FIGURE 1
FIGURE 1Endophytic fungal isolates derived from the stem of P. tuberosa.
FIGURE 5 (A) Polianthes tuberosa plant, (B) Mycelial growth of PTS8 isolate from the stem on the PDA medium, (C) PTS8 was stained with Lactophenol cotton blue and observed under a light microscope at 100x magnification, Scale bar (0.2 μm), and (D) Mycelial growth rate of PTS8.

TABLE 1
Inhibition of pathogens by active isolates isolate of P. tuberosa.

TABLE 2 Antibacterial
Susceptibility of the X. tongaense secondary metabolites from ethyl acetate crude extract.

TABLE 3 MIC
and MBC of fungal ethyl acetate crude extract.

TABLE 4
Synergistic interaction between fungal crude extract with antibiotics.

TABLE 5
Quantitative phytochemical estimation of the X. tongaense PTS8 ethyl acetate extract.