01. Hallis.cdr Vol.15, No.1, March 2021, p 1-7 DOI: 10.5454/mi.15.1.1 Molecular Diversity of Mold Associated with Gatotan STEFFANUS PRANOTO HALLIS , ANASTASIA TATIK HARTANTI , 1 2* AND AGUSTIN WYDIA GUNAWAN 3 1 Program of Biology, Faculty of Biotechnology, Atma Jaya Catholic University of Indonesia. Jalan Jenderal Sudirman 51, Jakarta 12930, Indonesia; 2Department of Food Technology, Faculty of Biotechnology, Atma Jaya Catholic University of Indonesia, Jalan Raya Cisauk-Lapan 10, BSD City, Tangerang 15345, Banten, Indonesia; 3 Department of Biology, Faculty of Biotechnology, Atma Jaya Catholic University of Indonesia, Jalan Raya Cisauk-Lapan 10, BSD City, Tangerang 15345, Banten, Indonesia. Gatotan gatot is a raw material to make , a fermented food made from n Indonesian , especially in Central Java, cassava tubers. Traditionally, the cassava tubers are dried for several days until the black color appears. sun- However, natural fermentation allowed by many types of microorganisms, especially mold in this process could raise concerns about the food safety issues. Previously, the identifications of molds in were based on gatotan morphological observation. Here, we reported the diversity of molds associated with using molecular gatotan identification method. The molecular identification was based on ribosomal DNA internal transcribed spacer (ITS) amplification sequences using combination of ITS4 and ITS5 primers. A total of molds were isolated ten and p ribosomal DNA enceshylogenetic trees were constructed based on sequ . Our results showed that the molds were classified into spp., sp., , and Lasiodiplodia Trichoderma Aspergillus nomius, Fusarium oxysporum Cladosporium sphaerospermum. cassava, fungi, , internal transcribed spacerKey words: gatot makanan fermentasi Indonesia, khususnya di Jawa Gatotan adalah bahan baku dalam pembuatan gatot, Tengah yang terbuat dari umbi singkong. Pembuatan gatot secara tradisional dilakukan dengan mengeringkan umbi singkong ngga muncul warna kehitaman. Fermentasi alami yang langsung dengan paparan sinar matahari hi terjadi pada proses ini melibatkan pertumbuhan berbagai mikroorganisme, terutama kapang, yang tidak terkendali dan dapat menimbulkan permasalahan keamanan pangan. dentifikasi kapang yang terlibat dalam I pembuatan gatotan telah dilakukan secara morfologi. Pada penelitian ini, identifikasi kapang yang berasosiasi dengan gatotan dilakukan secara molekuler. Identifikasi molekuler dilakukan dengan menggunakan amplifikasi situs (ITS) pada DNA ribosom menggunakan pasangan primer ITS4 dan ITS5. internal transcribed spacer Sebanyak sepuluh kapang telah diisolasi dan pohon filogenetik disusun berdasarkan pada sekuen DNA ribosom. Hasil identifikasi yang diperoleh ialah Lasiodiplodia Trichoderma Aspergillus nomius, Fusariumspp., sp., oxysporum Cladosporium sphaerospermum. , dan Kata kunci: cendawan, gatot, , singkonginternal transcribed spacer MICROBIOLOGY INDONESIA Available online at http://jurnal.permi.or.id/index.php/mionline ISSN 1978-3477, eISSN 2087-8575 *Corresponding author: Phone ); : +62-21-80827200 (ext. 1039 Fax: +62-21-5747912 E-mail: anast.hartanti@atmajaya.ac.id; cassava is usually used to make . Traditionally, gatot cassava tubers are peeled off, cut, and washed until clean, then sun-dried for 3-5 days until become . gaplek Gaplek is placed outdoor within approximately two weeks until the tubers colour changed to black, indicating natural fermentation to form which gatotan can be further processed into by steaming gatot (Purwandari 2014a)et al. . flour had 90.33% antioxidant scavenging Gatotan activity 18.92 mg × 100 g equivalent to vitamin E -1 (Purwandari 2014b)et al. . Development of as gatot noodle would not only increase the blood sugar level quicker but also reduce it quicker than white bread , consumption control, suggesting the low glycemic potential of . Furthermore, the starch resistant of gatotan gatotan gatot (22.5%) and (21.85%) were lower than cassava (24.44%) in glucose oxidase assay Fermentation of cassava ( ) has Manihot esculenta been considered as one strategy to improve nutrient quality and reduce its natural cyanide content. Recently, Lactobacillus plantarum, a lactid acid bacteria commonly found in cassava waste, was reported decrease up to 97% of the cyanide content in cassava after 36 hours of fermentation . (Hawashi 2018)et al. Furthermore, together with , several L. plantarum m i c r o o r g a n i s m s c o n s o r t i u m i n c l u d i n g Corynebacterium manihot Saccharomyces exiguus, , and reduced the cyanide content Geotrichum candidum and improved the protein and uality of fatty acid q cassava after 96 hours fermentation (Samson and Akomolafe 2017). In Indonesia, fermentation of (Puspaningtyas 2018)et al. . Additionally, the dietary fiber of (14%) and (17.36%) were found gatotan gatot to be higher than cassava (8.61%), respectively suggesting a prominent as functional food for gatot people with diabetes mellitus . (Sari 2018)et al. Considering the benefits of as fermented food gatot product, identification of microorganisms involving in this process were reported. Mold, such as Aspergillus flavus Rhizopus oryzae Lasiodiplodia theobromae , , and were showed to be associated with from various gatotan regions in Indonesia . In addition, (Purwandari 2000) phenotypic identification showed predominant type of fungi, , , Botryodiplodia theobromae R oligosporus. together with indigenous lactic acid bacteria L. fermentum L manihotivoransand were potential as . starter culture for controlled fermentationgatotan ( Moreover, spontaneous Astriani 2018)et al. . fermentation of cassava to make resulted in gatot growth of and , R. oryzae Acremonium charticola proposed as indigenous fungi which had salt-tolerant properties and potential antioxidant activity (Yudiarti and Sugiharto 2016; Sugiharto 2016)et al. . As fungal consortium in fermentation was broadly gatotan diverse, its clustering needs to be re-confirmed using molecular approaches. However, there was limited report about molecular identification regarding fungal consortium in . gatot Amo ng ma ny DN A ba r c od e fo r f ung al identification, nuclear ribosomal internal transcribed spacer (ITS) is considered as the most accurate region to differentiate the gap between inter- and intraspecific variations . ITS region, comprised (Schoch 2012)et al. of ITS1, ITS2, and small 5.8S ribosomal RNA (rRNA), is located between the large 18S and 28S ribosomal RNA in fungal nuclear cistron. Splitting process in posttranslational modification would remove ITS1 and ITS2 since they act as non-coding region (Schoch et al. 2012). Distinguish properties of this region influenced efficacy for systemic identification, including in diagnostic mycology area . The (Ciardo 2010)et al. universal primer combination of ITS4 and ITS5 anneals to tip part of large rRNA subunits, consequences in amplification of whole ITS region for phylogeny construction . Furthermore, (White 1990)et al. improved ITS primer combination has been evaluated to be capable for food fungal community profiling (Walters 2016)et al. . Thus, ITS would be a promising tool as a key of mold identification in . This gatotan research aimed to identify the diversity of mold associated with using molecular approach of gatotan ITS region. MATERIALS AND METHODS were made from cassava (Gatotan. Gatotan M. esculenta) tubers in Bogor. All the water used was distilled water using GFL Water Distillation Unit 2008 (GFL, Burgwedel, Germany). Potato dextrose base (Oxoid, Hampshire, United Kingdom) was mixed with 15 g L agar base for potato dextrose agar (PDA), while -1 A. flavus A. parasiticus and agar (AFPA), and dichloran 18% glycerol (DG18) agar were made according to the protocols (Hocking and Pitt 1980; Pitt et al. 1983). The DNA extraction reagent DNA Phytopure™ Kit Extraction was provided by GE Healthcare Life Sciences (Amersham, United K i n g d o m ) . P r i m e r p a i r s o f I T S 4 ( 5 ' - TCCTCCGCTTATTGATATGC-3') (5'- dan ITS5 GG AA GTAA A AGT CGTAA CAAG G-3 ') w er e synthesized by Integrated DNA Technologies (Singapore). of m Mold Isolation. The methods old isolation were carried out using direct and dilution method . n s I direct isolation method septically, inside part of , a gatotan were placed on PDA and incubated at 30 . n °C I dilution method AFPA and DG18 medium were used. , First, 25 g were suspended with 250 mL of gatotan aquadest. The suspension was diluted 10 , 10 , and 10 -2 -4 -6 using physiological salt solution. A total of 1 mL diluted suspension was mixed with warm (45 °C) AFPA and DG18 medium prior to solidification. Solid mixtures were incubated at °C. 30 Mold growth was monitored everyday for total four days. Different growing colonies were placed in new PDA for further examination. DNA Isolation, PCR mplification, and A S . equencing Four days old mycelia grown on PDA was ixed with distilled water ntrifuged at m , then ce 10,000 rpm for 10 Pellet were mixed with glass min. beads (Oxoid, Hampshire, United Kingdom) and vortexed to disrupt the cell wall. DNA were extracted using DNA Phytopure™ Kit Extraction (GE Healthcare Life Sciences, Amersham, United Kingdom) according to manufacturer's protocol. A total of 200 ng DNA was amplified using ITS4 and ITS5 primer pairs to obtain ITS rDNA sites (White et al. 1990). The PCR procedures were carried out using Go Taq Green Master Mix PCR 2× (Promega) with ® reaction as follows: pre-denaturation at 94 °C for 2 min, 35 cycles of denaturation at 94 °C for 15 s, annealing at 55 °C for 30 s, and elongation at 72 °C for 1 min with final elongation at 72 °C for 5 min (Abe et al. 2007). The visualization was done on 1% agarose gel 2 HALLIS ET AL . Microbiol Indones Volume 15, 2021 Microbiol Indones 3 electrophoresis ( ) at 60 Mini-Sub Cell GT Cell BioRad ® V for 90 min followed by ethidium bromide staining and imaging using Gel Doc™ XR System (BIO-RAD, Germany). PCR products were sent to First BASE (Malaysia) for sequencing. Phylogenetic Analysis. The nucleotide sequence were analyzed using basic local alignment search tool ( ht tp : // www. bla s t.n c b i.n lm. nih . go v/ Bla s t.c g i) (Altschul 1990)et al. to acquire the mold species. The sequences were aligned using SeqTrace software version 0.9.0 . The phylogenetic trees (Stucky 2012) were obtained using maximum likelihood (ML) parameter on molecular evolutionary genetics analysis 6 (MEGA6). Kimura 2 model was chosen to represent the phylogenetic tree. Strength of the internal branch of phylogenetic tree was analyzed with 1,000 bootstraps (BS) . The reference and outgroup (Felsenstein 1985) s e q u e n c e w e r e o b t a i n e d f r o m G e n B a n k (www.ncbi.nlm.nih.gov/genbank) (Benson et al. 2005). RESULTS A total of ten mold strains were isolated and identified, including strains Lasiodiplodia (4 ), Trichoderma Aspergillus Fusarium, , (3 ), strains and Cladosporium . Identification (Table 1) based on ITS sequence on showed four strains of spp. Lasiodiplodia that these particular strains were found on the same clade with with low BS valueBotryosphaeria rhodina (0.47) . (Fig 1) The ITS region sequence resolution on phylogenetic tree could not describe Lasiodiplodia spp. . to species level There were two species types in one same cluster, namely with its B. rhodina anamorphic name and L. theobromae L. parva. T4 sp. Strain S of was found on theTrichoderma clade Trichoderma reesei, T. consisting of longibrachiatum, T. orientale, Hypocrea orientalisand with BS value Fig ITS region sequence 0.80 ( 2). resolution on phylogenetic tree based on analysis ML could not differentiate the intraspecies variations Two . specimens, and were in T. reesei T. longibrachiatum , the same cluster he comparison result from . However, t the NCBI database showed 95% similarity of strain ST4 with respectively (data not T. longibrachiatum, shown). One strain was identified asAspergillus A. nomius (Fig 3). According to the ITS tree produced from ML analysis, is included in the clade with STP5 A. nomius high BS value In addition, three of0.99. Fusarium strains were identified as StrainsF. oxysporum (Fig 4). STA2, STA4, STA5 were foundand in the F. oxysporum clade 0.63. Meanwhile, with BS value s STD1 train was identified as Cladosporium sphaerospermum 0.86 ( 5). The with BS value Fig phylogenetic tree formed specific clade of C. sphaerospermum with STD1 isolate included. DISCUSSION Cassava fermentation into involves several gatotan molds to produce unique and distinct taste. Several previous studies showed the involvement of this particular mold genera in fermentation gatotan including A. flavus R. oryzae R. oligosporus L. , , and theobromae (Astriani 2018 Purwandari 2000)et al. ; . Our results showed similar mold genera were identified using molecular identification (Fig 1 and 3). These include Lasiodiplodia A. nomius spp. and . Importantly, spp. was isolated and Lasiodiplodia characterized in every investigation. However, gatotan the mor pho log ic al c h ar ac te riz at io n s h owe d inconsistent identification, while Purwandari (2000) identified this mold as and Astriani L. theobromae (2018) as . In addition, based on B. theobromae the ref eren ce and In de x Fungor um Myc oba nk , Table 1 BogorMolecular identity of mold strains associated with fromgatotan Isolation medium Strain code Molecular identity Potato dextrose agar ST2 Lasiodiplodia sp. ST3 Lasiodiplodia sp. ST4 Trichoderma sp. STP5 Aspergillus nomius STP6 Lasiodiplodia sp. Aspergillus flavus and parasiticus agar STA1 Lasiodiplodia sp. STA2 Fusarium oxysporum STA4 Fusarium oxysporum STA5 Fusarium oxysporum Dichloran glycerol 18% agar base STD1 Cladosporium sphaerospermum 4 HALLIS ET AL . Microbiol Indones Fig 1 Phylogenetic tree and morphological characteristics of spp. was an Lasiodiplodia Diplodia seriata used as outgroup. Phylogenetic tree was Kimura2 made based on maximum likelihood analysis with model (1,000X bootstraps). Fig 2 Phylogenetic tree of sp. was an outgroup. Phylogenetic tree wasTrichoderma Penicillium citrinum used as made based on maximum likelihood analysis with model Kimura2 (1,000X bootstraps). Fig 3 Phylogenetic tree of . was an outgroup. Phylogenetic tree Aspergillus nomius Diaporthe angelicae used as was Kimura2 (1,000X bootstraps) made based on maximum likelihood analysis with model . Volume 15, 2021 Microbiol Indones 5 Lasiodiplodia is an anamorphic form from Botryosphaeria and the usage of the name Lasiodiplodia refers to the determination of one fungus having one name (Hawksworth 2011 Robert et al. et al.; 2013). Importantly, since had been L. theobromae isolated in every studies, the role of this mold gatotan would be crucial in fermentation of gatotan (Purwandari 2000; Astriani 2018)et al. . Other mold genera also reported to be associated with despite of their controversial contribution gatotan and safety for food fermentation. Trichoderma sp. isolated from had been classified as unwanted gatotan mold since its potency to produce toxin ''(Astriani et al. 2018). In addition, Fusarium Cladosporium and were isolated and identified as the major contaminants in gaplek, dried cassava tubers before fermented to gatotan . (Susanti 2010) However, , T. longibrachiatum in particular, was reported to inhibit the growth of F. oxysporum Penicillium oxalicum Rhizoctonia solani, , , and that caused post-harvest disease Sclerotium rolfsii in yam . Nonetheless, (Dania 2016)et al. Trichoderma spp. also has an important role in solid substrate fermentation, particularly in cassava to reduce the cyanide content and improve the nutritional value (Hawashi 2019)et al. . Latterly, , one of generally recognized as A. niger safe microorganism, isolated from was observed gatot to produce extracellular starch degrading enzyme, α- amylase . Both crude and partially (Angelia 2019)et al. purified α-amylase enzyme showed similar degrading activity of starch to glucose and maltose with commercial enzyme. Importantly, black colonies of A. niger gatotan were also isolated in samples from various region in Indonesia, suggesting their presence Fig 4 Phylogenetic tree of . sp. was an outgroup. Phylogenetic tree wasFusarium oxysporum Eurotium used as made based on maximum likelihood analysis with model Kimura2 (1,000X bootstraps). Fig 5 Phylogenetic tree of . was an outgroup. Cladosporium sphaerospermum Paecilomyces variotii used as Phylogenetic tree was Kimura2 (1,000X made based on maximum likelihood analysis with model bootstraps). as unique characteristic of fermentation gatotan (Astriani 2018 Purwandari 2000)et al. ; . Beside L. theobromae Rhizopus, spp. was reported to have an important role in fermentation. , gatotan R. oligosporus together with , , and B. theobromae A. niger Trichoderma sp., was isolated as one of indigenous fungi in fermentation . gatot (Astriani 2018)et al. However, in this study, we didn't identify spp. Rhizopus using molecular technique, despite of their presence during isolation. In conclusion, this study identified Lasiodiplodia s p p . , s p . , , Tr i c h o d e r m a F o x y s p o r u m C. . sphaerospermum, A nomius and as . molds associated with gatotan using ITS identification. Lasiodiplodia spp. and spp., were always found in Aspergillus gatotan, thus they may provide essential contribution in fermentation.gatotan ACKNOWLEDGEMENTS This research was supported by Faculty of Biotechnology, Atma Jaya Catholic University of Indonesia through Faculty Research Grant. REFERENCES Abe A, Oda Y, Asano K, Sone T. 2007. is Rhizopus delemar the proper name for fumaric-malic Rhizopus oryzae acid producers. Mycologia. 99:714-722. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. J Mol Biol. 215:403-410. Angelia C, Sanjaya A, Tanudjaja E, Victor H, Cahyani AD, Tan TJ, Pinontoan R. 2019. 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