BIODIVERSITAS ‘Voinme 21, Number 5 Pages: 1953-1960 ISSN: 1412-033 ESN: 2085-4722 ‘DOK: 10,13057/biediv/d2 10523 May 2020 Fungal isolates from marine sponge Chelonaplysilla sp.: Diversity, antimicrobial and cytotoxic activities DIAN HANDAYANT™, MUH. ADE ARTASASTA™, NILDA SAFIRNA!, DIANA FITRI AYU ‘TRINA EKAWATI TALLEP, TRIANA HERTIANT "suman Biota Laboratory, Faclty of Pharmacy. Universtas Andala. J Utand, Kampus Lima Mais, Padang 25163, West Sumatra Indonesia Depantnant of Biomedeal Faculty of Medics, Uaiverates Andlas Tl Und Kanipas Lara Manis Padang 25165, Wst Samat, indonesia Tel “+62-751-31746. tema: dnhandayans@phar wnand acid Deparment of Biology, Faculty of Mathematis and Natural Scienos, Universes Sem Ratlang. Kempus UNSRAT Klsak, Manado, 95115, Noth Slaves Indonesia “Faclty of Pharmacy, Universitas Gadjah Mada J, Kaluran, Sakip Utara, Sloman $5281, Yopyskarta Indonesia ‘Manuscript received: 3 February 2020, Revision aospted: 13 pil 2020. Abstract. Hondayani D, Artasasta MA, Safira N, Ayuni DF, Tallei TE, Hertiani T. 2020. Fungal isolates from marine sponge ‘Chelonaplysilla sp.: Diversity, antimicrobial and cytotoxic activities. Blodiversitas 21; 1954-1960, The purpose of this veseasch Was to study the diversity of fngi associated with marine sponges Chelonaplysilla sp. and their bioactivties. Fungal isolation was carried out by the multilevel dilution method in Saboraud Dewrose agar (SDA). Twelve fungal isolates were successfully purified, then cultivated Using rice for 4-8 weeks at room temperature and subsequently extracted using ethyl acetate, Antimicrobial activities of the fhngal extracts were tested against Staphylococcus aureus, Escherichia coli and Candida albicans by using the agae diffusion method, The extracts of isolates ChOS and Chi? showed a significant antagonistic effect against S. aureus and E.coli with the diameter that ranged from 15 to 17 mm. Using the brine shrimp lethality test (BSLT), six fungal extcacts revealed cytotoxic activity with LCs <100 gm, Ikolate Ciht0 was the most potential fungus with the strong cytotoxic aetivity of LCs of 0.90 jig/mL. The 3-(4,5-dimethylthiazol->-y!. -diphenyltetrazolium bromide (MTT) assay was conducted also for six potential fungal extracts against breast cancer cell (T47D), ‘The isolate Ch0S showed moderate cytotonie activity with ICs of $3.69 ygimL. The molecular identification was castied out for potential fangi using the ITS marker. The results showed that ChO2 was spergillus oryzae, Ch0S was Phomompsis sp... Ch06 was Penicilium simplicissimum, Ch10 was & bassiana and Chi2 was Aspergillus mellimus. This stay concluded that fungal isolates from ‘Chelonaplvsila sp. can be explored further for new sources of aatimicrobial and aatieancer compounds Keywords: Antimicrobial Chelonaplyslla, cytotoxic, marine-derived fungi INTRODUCTION Bioactive compounds produced by fungi associated ‘with marine sponges have shown potential pharmacological activities and have similar metabolites produced by their hosts (Indraningrat etal. 2016; Youssef etal. 2019). These types of fungi are thought to be the original producer of bioactive compounds in sponges (Proksch et al, 2002), Several new bicactive compounds from marine associated fungi have shown potential new bioactivities, for example, secondary metabolites produced by Asperaillus similanensis contained similanpytone C, similanamide, aud yripyropene (Thomas et al. 2010; Prompanya et al. 2015), Averantin isolated fiom A. versicolor associated with ‘marine sponge Neopetrosia sp. was proven to have antibacterial and cytotexic activities (Lee etal. 2010). ‘Marine sponges which were collected from Mandeh island in west Sumatra-Indonesia were known to be associated with potential fungi which produced antimicrobial and cytotoxic activities (Artasasta etal, 2017 Handayeni and Aminah 2017; Handayani and Artasasta 2017; Amina etal, 2019; Handayani etal. 2019a, 20190) This study focused to discover the diversity of Chelonaplsitia sp. associated fungi and their biological activities against pathogens and breast cancer cells (T47D), MATERIALS AND METHODS ‘The extraction process of the pure fungal isolate from marine sponge Chelonapiysilia sp. ‘Marine sponge Chelonapivsilla sp. (Fig. 1) from ‘Mandeh island, west Sumatra-Indonesia (1o 6’-10 13°S, 1000 19°-1000 25°E) (Fig. 2) was used as a fungal source. Identification of the sponge was conducted by Dr. Nicole J De Vooad, at the Natural Biodiversity Center, Netherland ‘The fungal isolation was conducted following our previous study using a multi dilution method (Handayani and Artasasta 2017). Pure isolates were cultivated om rice ‘media at room temperature for 4-6 weeks. The extraction Process was conducted after the fungal isolates overgrew on the media, Ethyl acetate was used for extracting ‘nonpolar and semi-polar compounds of the fungal isolates with the ratio of 200 mL solvent to 100 mg rice. To complete the extraction process, the fungi were immersed in the ethyl acetate for 3 days. The solvent was evaporated to obtain the dry extracts that were used for antimicrobial and cytotoxic activity as well as phytochemical tests.HANDAYANT etal. ~ Fingal isolates from marine sponge Chelonaplysilla sp. 1955 = Figure 2. Location of sample taking of marine sponge Chelonapiysila sp. in Mande Island, West Sumatra Indonesia (1° 6-1 13°S, 100° 19°-100° 25°) Figure 1. Marine spoage Chelonaplysillasp. Bar = em Screening of antimicrobial activity S. aureus ATCC 2592, E, coli ATCC 25922, and C. albicans were used as the pathogens for antimicrobial screening. This study followed the procedure provided by Balouiri ct al (2016) using the agar diffusion method. Sterile paper disk (6 mm) was soaked in each of $ % fungal extract, Nutrient agar (NA) was used for an antibacterial test, while SDA was used as media for an antifungal test AS positive controls, 30 yg/dise chloramphenicol (antibacterial) and nystatin (antifungal) were used. After incubation for 24 h at room temperature, the diameter of inhibition zones (mm) was measured as an indication of antagonistic effect, Screening of cytotoxic activity BLT BSLT was used for the evaluation of cytotoxic activity. “The eggs of Artemia salina were hatched in seawater for 2 days at 37°C. After hatching, 10 active naplit were put into a test tube containing the fungal extracts which were dissolved in DMSO (dimethyi sulfoxide) up to a concentration of 1000, 100 and 10 yom. Probit analysis ‘was used for determining the value of LCSO (Meyer et al 1982). Chrotoricity assay MIT assay was carried out to study the cytotoxic activity of fungal extracts against breast cancer cell T47D. ‘These cells were seeded at | = 10° cells/mL in 96 well iicrotiter plates containing DMEM (Dulbecco's modified Eagle's medium), a modification of Basal Medium Eagle1956 (BME), The cells could attach on the well overnight, Once the cells were confluent, 20 yL of the fangal extract was added to the well with various concentrations (1000, 500, 125, 62.5, and 31.25 ygimL) then the plates were placed in 5% CO2 incubator at 370C for 24 h. Thereafter, the media were replaced with PBS. The cells were subsequently treated with $ mg/mL 3-[4,5-dimethylthiazol- 2-ylJ-2,5-diphenyltratrazolium bromide (MTT) (Sigma Chemical Co., St. Louis, MO) and incubated for 4 hours. DMSO was used to dissolve the remaining formazan crystals. Using ELISA reader, the absorbance was measured at $70 nm. The percentage of viability of the cells was determined then converted into the ICS0 value (Arasasta etal. 2017). Fungal identification Macroscopic and microscopic identification Macroscopic appearance of the fungi such as color. the diameter of the colony, and colony reverse was observed Microscopic observation was conducted by using a Jactophenol cotton blue solution as a mounting medium and staining agent. The hyphae were put on a slide then mixed with fungal coloring. Conidiophores, vesicles, and conidia were observed under @ light microscope (Charya and Garg 2019) Molecular identification Molecular identification was performed using ITS1 primer (FS'-ICC GTA GGT GAA CCT GCG G-3") aud ITS4 primer (RS’-TCC TCC GCT TAT TGA TAT GC-3") with the two-step procedure, DNA extraction, and PCR amplification. DNA extraction was conducted by following Saitoh et al., (2016), The PCR process was conducted by 34 cycles included denaturation at 9$0C for 5 min, annealing at $50C for 1 min, and extension at 72°C for 1 min (Ferrer ct al. 2001). The PCR products were sequenced in First Base Malaysia then were tinmed and assembled by using BioEdit V.7.0.5. Furthermore, the sequences were subjected to identification using the BLAST program on NCBI The Neighbor-Foining (NJ) phylogenetic tree was generated by MEGA 7.0 sofiware (Kumar et al. 2016), using Kimura-2-Parameter with 1,000 bootstrap replications Secondary metabolites examination The standard protocol of Harborne (1984) was followed in this step. Dragendorft reagent for alkaloid, Lieberman Bourchard’s reagent for terpenoid and steroid, FeCl reagent for phenolic, and Citroborat reagent for flavonoid were used. The fungal extract was spotted on the G60 F254 silica plate then eluted with n-hexane: ethyl acetate eluent (i: 4), Each of the reagents was swapped to each different silica. Each of the secondary metabolites produced different colors based on different reagents. The orange color indicated the presence of alkaloid, pink color for terpenoid, blue or green color for the steroid, purple, red, oF pink colors for phenolic, and green color for flavonoid (Harborne 1984). BIODIVERSITAS 21 (5): 1954-1960, May 2020 RESULTS AND DISCUSSION Antimicrobial activity of marine sponge derived fungi Twelve fungal isolates were purified from marine sponge Chelonapirsilla sp. This sponge was known to ‘exhibit antimicrobial and eytoroxic compounds (Bobain ‘and Faulkuer 19912, 19916). Presumably, the bioactive compounds were produced by its associated fangi ‘Although the active compound produced by the fangus associated with Chelonaphysitia sp. has never been reported before, some studies stated the family of this genus was associated with the fungus Streptomyces sp. which has broad-spectrum antimicrobial activity (Selvin ct al. 2004: Selvin 2009; Selvin et al. 2009). Antimicrobial activity from twelve fungal extracts showed different results (Table 1), Among all isolates, only three isolates (ChOS, Ch06, and Ch12) performed strong antagonistic activity against both S: aureus and E, coli. These fungal extracts could be categorized as broad-spectrum because of their abilities to inhibit the growth of both Gram-negative and Gram- positive pathogenic bacteria, Isolate Ch12 was the most potential isolate with a diameter inhibition zone of 16.69 = 0.51 and 7.96 = 0.73 mm, respectively. However there was zno fungal extract that showed a strong antagonistic effect against C. albicans. ‘Table 1. Antimicrobial activity result of fungal extracts from ‘marine sponge Chelonapiysttasp. ‘Diameter inhibition zone (mm) = Standard Fungat Deviation (SD) {solates colle 5 aureus E.coli G albicans Chor hod 8831.01 hos 8250.35 chor = 8134076 hos 15925052 16332051 hos 11962079 12292072 cho? - : chos 8.754087 ccho9 - . 867=036 . ress076 76: : 172102 8. 1692051 16882057 7 ‘Note: The value is expressed as the mean = standard deviation; 3 ‘Table 2. MTT assay of the extracts of fing isolated from marine sponge Chelanoplsitia sp. against breast cancer cell T47D ‘Fungal iolates code 1Ceo(ugmk) chor 2 choz 743.42 cnos 83.96 choo cho 70.75 cud 637.24HANDAYANT etal. ~ Fingal isolates from marine sponge Chelonaplysilla sp. * swf oe ee oe Frngal isolates code Figure 3, Cytotoxic activity results of the extracts of fungi isolated fom masine sponge Chelanoplysila sp. by using BSLT method (Cytotoxte activity of marine sponge derived fumgt BSLT is one of the preliminary toxicity tests for the active compound obtained from the fungal extract. This method is comparable to expensive bioassay methods (Pisutthanan er ai,, 2004; Wu, 2014), In this studs Fungal isolates (ChO1, Ch02, Cho5, ChO9, Ch10, and C12) hhad LCs value <100 g/ml. (Fig. 3). These isolates could bbe categorized as having strong cytotoxic activities according to Meyer ef al, (1982), Among these isolates, isolate Ch10 had the lowest LCso value (0,90 ng/mL). MTT assay was also conducted for these six potential fingi against breast cancer cell T47D (Table 2). The isolate ChOS fungal exhibited moderate cytotoxic activity with ICs0 $3.69 ugimL. Other isolates did not provide enough cytotoxic activity on these cell lines. Presumably, every cancer cell has its sensitivity against each anticancer agent Macroscopic, microscopic, and molecular identification of marine sponge derived fungi Antimicrobial and cytotoxic assays revealed that isolates Ch02, C05, Ch06, Chi0, and Ch12 strains were the most potential to be studied further. Fungal identification of these isolates was conducted macroscopically, microscopically (Fig. 4), and molecularly (Fig. and Tab. 3), From the phylogenetic tre, it was seen the grouping of cach isolate following the results of identification. The surface observation of isolate ChO2 exhibited a white cotton colony and showed a white color in the reverse colony. The diameter of the colony after 5 days was 7 cm (AL). Conidiophore and asexual spore ofthis isolate were similar to these of genus Aspergillus. The surface of the colony of isolate Ch0S was observed to have white threads appearance. The colony diameter after $ days was 10 cm (BL). This isolate had light blue conidiophore, and asexual spores of this fungus were similar to genus Phomompsis (B2). Isolate ChO6 had a dark green surface colony. Its reverse colony was gray color with diameter after § day-growth was $6 em (Cl). This isolate had gray conidiophore and asexual spores which were similar to szenus Peniciliu. Isolate Chi2 had a white colony with a rough surface after 5 days, the diameter of the colony was 1957 around 3-5 cm (El), This fungus had blue light conidiophore and striated vesicle. These characters place this fungus similar to gems Aspergiilus (E2). Isolate Ch10 hhad a white surface colony with a diameter of §-10 em after 10 days. This fangus had gray conidiophore (D1). Asexual spores of this fungal were similar to genus Becuneria (D2) The results of molecular identification for isolate ‘ChO2, Ch0S, Ch06, C10, and Ch? are presented in Table 3 and Figure 4. The conclusions drawn based on the results of identification using the ITS gene indicated that ChO2 had a 100% identity with Aspergillus oryzae and Aspergillus, flavus. The 100% identity between these (0 ‘fungal species resulted in uncertainty regarding the identification method. According to Frisvad et al (2019). 4 ‘oryzae was the result of domestication of 4. flavus, thus ‘giving confusion to the naming of those unfamiliar with the morphological characteristics of these two fungi. To sistinguish the two, a suggestion will be placed suck as the ‘examination of aflatoxins, since A. oryzae lost its ability to produce aflatoxin, although phylogenetically it is very Closely related to A. flavus. Aspergillus oryzae produced 4- hydroxy-4-methylpent-2-enyl moiety which was assumed to inhibit the growth of E coli while 4. flavus produced a compound that showed a strong inhibition activity against S aureus (Xu etal. 2015). Three portals used to identify fungi molecularly gave different results based on the ITS gene sequence of isolate ‘Ch05. The GenBank identified this isolate as Phomapsis sp. (99.8%), Mycobank revealed identity 97.51% with P. perseae, while BOLD identified this isolate as Diaporthe hhongkangensis (99.2%). Phomopsis actually is the asextal stage of Diaporthe (Gomes et al. 2013). Kobayashi et al (2003), was able to isolate phomopsidin produced by Phomopsis sp., @ mavine-derived fungus, Using GenBank and BOLD, Ch06 was confirmed molecularly as, Penicilium simplicissimnmm. Zn et al (2016), reported the presence of three new dihydroisocoumarins prostuced by P. simplictssionom MA-332, a fungus derived from marine ‘mangrove, which showed toxicity activity against brine shrimp and broad-spectrum antimicrobial activities. Isolate Ch10 was identified by GenBank as Becnrveria dassiana, and this result was in line with the results obtained from MycoBank and BOLD. Yamazaki et al (2012) reported that they were able to isolate chrysazin and globosuxanthone A from 8 bassiana TPU942, a marine dcrived fungus. Chrysazin and globosuxanthone A were able to inhibit the growth of C. albicans, and ¢globosuxanthone A showed cytotoxic activity against two ‘human cancer cell lines, HCT-15 (colon) and Jurkat (T-cell Iymphoma), Isolate Chi2 was verified molecularly as Aspergillus mellinus. Aspergillus meltimus RSPG_204 showed antimicrobial activities against S aureus, P. aeruginosa and C. albicans, and high cytotoxic activity against MCF7 (breast) cell line (El-Hady et al, 2014). The results from secondary metabolites analysis showed that ‘Chi2 contained terpenoid, while Ch 10 contained saponin. Terpenoid compounds have been widely isolated from Aspergillus gests. Most of the terpenoid compounds also hhave antimicrobial and cytotoxic activities. Aspergilloxide fa terpenoid compound from the marine-derived fungus1958 Aspergillus sp. bas potent cytotoxic activity toward HCT 116 colon carcinoma (Cueto et al. 2002). Another terpenoid. compound from Aspergillus sp. asterpenols A and B, was known also as a potent antimicrobial (Xiao et al. 2013) Less information provided on saponin compounds from B. bassiana, Nevertheless, B. bassiana bas been explored by several researchers. Beauvericin, a major compound from B. bassiana, is a potent cytotoxic. This compound also has another bioactivity such as antimicrobial, antiviral and insecticidal (Wang and Xu 2012). Pyridovericin and pyridomacrolidin, other compounds ftom B. basstana, have been reported to have antimicrobial and cytotoxic activities, (Takahashi et al. 1998), ED BIODIVERSITAS 21 (5): 1954-1960, May 2020 In summary, fungal isolate Ch12 and Ch10, from marine sponge Chelanoplysilla sp... showed potential ‘antimicrobial and cytotoxic activities, respectively. Molecular identification revealed that Ch 12 was 4. ‘ellirus and Ch 10 was B, Bassiana, Isolate Chi0 and Ch 12 have already submitted to NCBI with accession number ‘of MT000965 and MT003976, respectively. Further study is important to conduct due to the presence of secondary ‘metabolites which can be investigated further for the possibility of being developed as antimicrobial and anticancer agents. Figure 4, Macroscopic and microscopic identification of nga isolates Ch02, ChOS, Ch06, Ch10, and Chi? ble 3. The results of Molecular identification of fungal isolates Ch02, Ch05, Ch06, Ch1O, and Ch12 Fungal toate GenBank Identity (0) MyeoBank Identity (6) BOLD Ldentity (0) Cho? dspergillus onzae 100% flaws 100% 4 one A flaris TOP hos Phomapsis sp. 99.30 Piperseae 97.51 Diaporthe hongkongensis 99.2 Choe. simplicissimum 100 Profi 97.461 Psiplicissimam 100 ChlO—-Reauveria basstana 100 B bassiana 99.184 B barsiona 100 Chi. meltinus 100 A.meltisus 100 Aspergillus meltinus 100HANDAYANT etal. ~ Fingal isolates from marine sponge Chelonaplysilla sp. 1959 KaN1045571 Pencil spear cmos 44684901 Peictum simpletsimum seo) ne 119812.1Peictiom wth HIN1045781 Peneitum spleen /AF203084 1 Pencilum senlcissimum uvs25703.1 Asperilis rye unsast27 1 Aspergihis yzae G87e097 1 Acpergtus ryzae Oi] MHOASS02 4 Ampergiis eyzae 0346200.1 Aspens oryzae 11K229158.1 Phomopsis eucommi i017795.1Aspergus metus 965118.1Aspergaus mets i685256.1 Aspergius metinus Tecate Aspergiss moins len 12 MF 800868 1 Phomapsis sp. KFSE9097 4 Phomampsis 3p. JF726516.1 Phomopss sp. 3171941 Phamopsis sp. on 05 1uns247061 Beawverta bassin KU523254 1 Beawerabessann onto 7] 006825661 Seauvena basstane uxz23322 1 Beawvera beste KCA10951 Beasseriatassione as Figure §, The phylogenetic wee of ITS sequences of fhngal isolate ChO2, ChOS, Ch06, ChiO, and Chi Joining method ACKNOWLEDGEMENTS: This research was supported by BOPTN of Andalas University, Padang, Indonesia, in the project of “Penenelitian Dasar Unggulan Klaster Riset-Publikasi Guru Besar Universitas Andalas (PDU-KRP1GB-Unand)", No, T/32/UN 1617/PP.OK-KRPIGBLPPM 2019 We ate also_gratefil to Dr. Nicole J. De Voogd, Natural Biodiversity Center, the Netherlands for the identification of the marine sponge. REFERENCES ‘Amimh 1, Puta AE, Avbein D, Handayani D. 2018. Screning of ‘oto activites foward WiDr and Vero call lines of es] acetate SStisets of fangrdervad fom the imanine sponge Acanthostongylophora ingens. Appl Pharm Sci 9:15 Anasada MA Dyzniaan A. Handayans D- 2017. Cytotoxic activity sereering of ethyl acetate fingal exacts derived om the marine ‘sponge Neaperosia chaliormic AR-OD. 3 Appl Phar Set 7-174- tr. Bobzin SC, Faulkner DI. 19912. Aromatics alkaloids fom the marine ‘sponge Chlongpisila sp. Org Chem 56: 4403-1407, inferred using the Neighbor- [Bobrin SC, Falkner DJ 19918 Diterpenoide fom the Pohapsian marie sponge Cheloapvsilla ep. Nat Prod 54; 225-232 Balourt M, Sadi M, Tbnsouda SK. Methods for in vito evaluating stimicroba activity: A review. J Phan Anal 6: 71-75, ‘Chaya LS, Garg $2019 Advances sn methods and practioes of Sstonayconzal research 2TH a Adv Biol Sei Res 2019 303-525 DOr: 10.10168975-0-12-817497-500019- Cueto M. Jensen PR. Ferical W. 2002 Aspersilloxde, 2 novel ‘estaterpane eponude Som 2 manine-derved fungus af the gens Aspergillus Org Lat 227: 2001-2008 errr C, Colom F, Frais §, Must E, Abad IL, Ali IL, 2001. Detection ‘an6 identification of fuse putogens by PCR and by ITS? and 85, Dbosomal DNA ‘ping in cular infections J Cha Microbiol 38 2573-2879, Frisyad 3C, Hubka V, Ezekiel CN, Hong SB. Novikov A, Chen AJ ‘Hovbraken J. 2019. Texouomy of Asperglve section Flvi and their ‘production of aliosin, ocbratosins and other mysotoni. Stud Niveal 98" 1-63) (Gomes RR. Gienke C, Videra SIR, Lombard L, Grosnensld JZ, Crous PW. 2013. Diaporthe: 2 gemns of endophytic, saprobic and plant pataogenis ings Persona 1 1-42 Hanied AA, Abdel-Azz MS, Abd I Hedy FK. 2018. Aatimcrobial and ftioudaat settee of diteent exacts dom deperclueungust SPMD-EGY grow on diferent media. Boll Nal Res Centre 42.29, DOI: 10.1186 912269-018.0027-0 Handayan: D, Amina 1 2017 Antbactoral and cyotono activities of iu] acetate exract of symbiotic fing fom West Sumatra marine sponge icanthrongylophora ingen. 3 App Pharm S27: 2372401960 Handayani D, Asands N, Arasasta MA, Ruslan R, Fadrivanti O, Tall TE, 20184, Aatiaicbial acivty screening of endophytic fangs stats saat Som Senin lane Paina sp. 3 Appl Phan Sei 99 5 Honan D, Artasasta MA_2017. Antitacerial and estotowie activities ‘sesoring of symbiotic fing extracts isolated fFom manne sponse Neoperosia caliniformis ARI. J AppI Pharm Sei 7: 665 Handayens D, Sondkavet: N, Akbar §, Syafu N, Pama D. 2019b. ‘Tyrosinse itty activity of eh avetateeitaes fom marine sponge derived Fungi. Bios Ree 16° 2369-2373 Harterne JB. 1984, Methods of Plant Analysis Chapman and Hall, London Indraningrst AAG, Swigt H,Sipems D. 2016, Riopeospecting sponge. associated rcrobes for antimicrobial compounds. Mar Drugs 14° 1- 66 Kobayeshi H, Meguro S, Youhmoto T and Namikochi M2003. Absolute irdture, biosynthes, and ant-serotbule acti of phomopeidn, ‘nolated Som a marine danved fungus Phonapss op Tetahedro <9 455-459, Kumar , Sttchor G, Tamura K. 2016. MEGAT: Molecalar evolutionary ‘enetics analysis version 70 for bigger detases. Mal Biol Evol 33: 1- i {Leo YM, Li H, Hong J, Cho HY, Boo KS, Kim MA, Kim DK, fang J Bioactive metabolites fom the sponge-derived fungus Asperefis ‘verstealor- Asch Pharma Res 33:231-235, Meyer BN, Ferigni NR, Putnam JE, Jacobsen LB, Nichols DE, ‘MeLavghin JL 1980 rine shring: a connie ganar casey fot civ plant consttont, Planta Mad 45-51-34 Pista §, Plantenochang P, Pisuthanon N. 2004. Brine cximp lethality stonty of Tha! medicinal plane sn the famaly Malacens, Naresuen Uni 1213-18 Droksch P.Edrese RA Ebel R 2002 Digs Som the Seas Curent Status ‘and Microbiclogcal Implications Appl Microbiol Biotechnol 59 125.134 Prompenya C, Femandas C, Craw 8, Pinto MMM, Dethoup 7, Sie AMS, Kijon A’ 2015. A new cyclic hewpetide and a new ‘sooumarin dexative fom the manne sponge associated Rangos Asperalue silanes KUFA 0013, Mar Drop 7: 1432-1450. Sibiro ME. gatas ¥, Radjasa OK, Sabdono A. Thanto A, Zlda DS, ‘Wijaya VF. 2019. Sponge associated fing fom e mangrove habitat in Indonesia: species composition, antimicrobial activity. enzyme sovonng and reactive prodlung ail Aquat Ree 11-173 186 BIODIVERSITAS 21 (5): 1954-1960, May 2020 Selvin J Joregh S_ Asha KR, Manjoha WA, Sangeeta VS, Iayassems DM. Antany MC, Vintha AID. 2004. Aatiactenal potential of sstgonisic Soeptonnes ep seat fom marine sponge Devarilla gra FEMS Microbiol Eeal 50-117 Selvin J. 2009 Exploring the anagonssie: producer Srepronces "MSIOS1: implications of polyketide synthase gene type T and a ubigutows defense enzyme phospholipase AZ in the host sponge Denaila nigra, Car Microbicl $8: 459-463 Selvin J Shanmughapriva S, Ganthimathi X, Kiran GS, Raji TR ‘Natasjasenvasan K. Homa TA. Optinization aod prediction of oval aimirobiel agents fom spouge-ascctod marine Actinomyeates Nocandopsis dassomille MADOS. Appl Micrbiat Bictaenaal 83: 135-115 ‘Tohahaskt S. Uchida K, Sakimma N, Hashimoto R, Yamagisnws T, ‘Nakagsva A580" The" stmuomre of pyedavencin and Prrkomaoidin, new muabolites fom the’ solomnpathogenic Fangus, Beauveria BassianaJ Antibiot (Tokyo) $1: 1051-1054 ‘Troms TRA, Kevsiar DP. Loksbaat PA. 2010 Maniae dawg somn ‘sponge-microbe asroiaion —a review, Mar Drugs 8 1417-1468. Wang QS 12012 Basen, «Hoare enpoand praca frag: A short review. Molecules 17 ‘wy C3014 An portant plaer an tine srnp lett oa The solvent J Ad Pharm Technl Res 557-38, ‘Nino Z, Hang H, Sho C, Xia X, Ma L, Huang X. 2013. Aspetepenols "A aad Bow eestertespenoide slated fom a mangrove endopiic flings perans sp 085282. Org Let 15. 2522-2575. 2 L, Mong W. C20, Wang J Shan W & Wana Q.2015, Antibacterial ad Antfingel Compouie fom Mann Fung. Mar Droge 13 (6 3479-3513, ‘Yamazali H, RetasohH, Kansko T, Murakami, Fujara H, Ukat K, ‘Namikoshy M. 2012. A New Dibenbslonspine Dervatve, 1 Hydeoxy-10-methaxy-dibena[heJoxepin 5 11-dioaa, fom Maine Denved Fungus, Beauera basiana TPUB2. Mar Drugs 10 (12) 269.2697 ‘Youse# ¥S, Ashour ML, Singab ANB, Wink M. 2019. compeehenaive review of bleacive peptides ffom manne fing and ther bieloial significance Mar Drags 17-1.24 Xo R Li XM, Wang B-G 2016. Penicsimpins AC, three new dhnydrisccoumanne fom Penicillium sinplicisimume MA332, 8 ‘marine fingus derived ftom the dhizosphere ofthe manarove pant Briguicra sexangula vat. riynchopetala.Phytocheatty Lett 17 aisle