Genetic Diversity of Amylomyces rouxii from Ragi tapai in Java Island Based on Ribosomal Regions ITS1/ITS2 and D1/D2

다국어 초록 (Multilingual Abstract)

Amylomyces rouxii is commonly found as amylolytic fungi in tapai fermentation. However, its diversity is rarely reported despite being often used for food production in Southeast Asia. This research aims to analyze the genetic diversity and the distribution pattern of A. rouxii from Ragi tapai in Java Island, Indonesia. We isolated the fungus from samples obtained from Ragi tapai producing centers in Bandung, Sumedang, Muntilan, Blora, Yogyakarta, and Bondowoso. The obtained isolates were molecularly identified based on the ribosomal regions ITS1/ITS2 and D1/D2, then analyzed for phylogenetic tree reconstruction, genetic distance, genetic variation, and haplotype networking. Six isolates showed specific morphological traits of A. rouxii. However, phylogenetic tree reconstruction on the ribosomal genes showed that the isolates were grouped into two different clades related to two species. Clade A included BDG, SMD, and MTL isolates related to A. rouxii, whereas clade B included YOG, BLR, and BDS isolates related to Mucor indicus. The genetic distances between clades for ITS1/ITS2 and D1/D2 were 0.6145 and 0.1556, respectively. In conclusion, we confirmed the genetic diversity of molds from Ragi tapai in Java Island and showed that the isolates are not only related to A. rouxii as reported before.

참고문헌 (Reference)

1 Abe A, "rDNA ITS sequence of Rhizopus oryzae: its application to classification and identification of lactic acid producers" 67 (67): 1725-1731, 2003

2 Abe A, "The molecular phylogeny of the genus Rhizopus based on rDNA sequences" 70 (70): 2387-2393, 2006

3 Ardhana MM, "The microbial ecology of tape ketan fermentation" 9 (9): 157-165, 1989

4 Ellis JJ, "The genus Amylomyces" 68 (68): 131-143, 1976

5 Ko SD, "Tape fermentation" 23 (23): 976-978, 1972

6 Djien KS, "Tape fermentation" 23 : 976-978, 1972

7 Gandjar I, "Tapai from Cassava and Cereals" Kasetsart University 13-17, 2003

8 Siebenhandl S, "Studies on tape Ketan–an Indonesian fermented rice food" 52 (52): 347-357, 2001

9 Pino-Bodas R, "Species delimitation in Cladonia (Ascomycota): a challenge to the DNA barcoding philosophy" 13 (13): 1058-1068, 2013

10 Azmi AS, "Ragi tapai and Saccharomyces cerevisiae as potential culture in viscous fermentation medium for ethanol production" 9 (9): 7122-7127, 2010

11 White TJ, "PCR protocols, a guide to methods and applications" Academic Press 315-322, 1990

12 "Nucleotide. Accession No. EF583634, Rhizomucor sp. Rm-33 small subunit ribosomal RNA gene, partial sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence; and large subunit ribosomal RNA gene, partial sequence" National Library of Medicine, National Center for Biotechnology Information

13 "Nucleotide. Accession No. AY238888, Amylomyces rouxii 18S ribosomal RNA gene, partial sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene and internal transcribed spacer 2, complete sequence; and 28S ribosomal RNA gene, partial sequence" National Library of Medicine, National Center for Biotechnology Information

14 "Nucleotide. Accession No. AB181331, Amylomyces rouxii genes for 18S rRNA, ITS1, 5.8S rRNA, ITS2, 28S rRNA, partial and complete sequence, strain: ATCC 22962" National Library of Medicine, National Center for Biotechnology Information

15 Schoch CL, "Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for fungi" 109 (109): 6241-6246, 2012

16 Karimi K, "Mucor indicus: biology and industrial application perspectives: a review" 31 (31): 466-481, 2013

17 de Souza CF, "Mucor indicus isolated from the semiarid region of Brazil:a first record for South America" 131 (131): 897-906, 2016

18 TajAldeen SJ, "Mucor indicus from Qatar" CBS-KNAW Fungal Biodiversity Centre 2015

19 Kito Hideki Abe A, "Molecular characterization of the relationships among Amylomyces rouxii, Rhizopus oryzae, and rhizopus delemar" 73 (73): 861-864, 2009

20 Kasmidjo RB, "Mikrobiologi ragi" PAU Pangan dan Gizi Universitas Gadjah Mada 1983

21 Saono S, "Microflora of ragi: its composition and as source of industrial yeast" Indonesian Institute of Science 241-249, 1982

22 Maddison WP, "Mesquite: a modular system for’ ’evolutionary analysis (3.61)"

23 Vu D, "Largescale generation and analysis of filamentous fungal DNA barcodes boosts coverage for kingdom fungi and reveals thresholds for fungal species and higher taxon delimitation" 92 : 135-154, 2019

24 Kartika AN, "Isolasi amylomyces dari berbagai jenis ragi untuk pembuatan tape rendah alkohol"

25 Fajarningsih ND, "Internal transcribed spacer (ITS)as dna barcoding to identify fungal species: a review" 11 (11): 37-, 2016

26 Cronk TC, "Indonesian Tapé Ketan fermentation" 33 (33): 1067-1073, 1977

27 Saono S, "Indonesia ragi. Symposium on indigenous foods" 1978

28 Ying L, "Identification of mold isolated from Rice Wine Start" 2012

29 Komagata K, "Identification of lactic acid bacteria isolated from a Chinese starter, ragi, in Indonesia" LIPI 1988

30 Lv X-C, "Identification and characterization of filamentous fungi isolated from fermentation starters for Hong Qu glutinous rice wine brewing" 58 (58): 33-42, 2012

31 Steinkraus K, "Handbook of indigenous fermented foods" CRC Press 1995

32 Peakall R, "GenAlEx 6.5: genetic analysis in excel. Population genetic software for teaching and research-an update" 28 (28): 2537-2539, 2012

33 Raja HA, "Fungal identification using molecular tools: a primer for the natural products research community" 80 (80): 756-770, 2017

34 Dung NTP, "Functionality of selected strains of moulds and yeasts from Vietnamese rice wine starters" 23 (23): 331-340, 2006

35 Dijksterhuis J, "Food mycology:a multifaceted approach to fungi and food" CRC Press 428-, 2007

36 Anupma A, "Filamentous moulds associated with some traditionally prepared mixed starter cultures of North East India" 11 (11): 1-16, 2019

37 O’Donnell K, "Evolutionary relationships among mucoralean fungi (Zygomycota): evidence for family polyphyly on a large scale" 93 (93): 286-, 2001

38 Kwiatkowski NP, "Evaluation of nucleic acid sequencing of the D1/D2 region of the large subunit of the 28S rDNA and the internal transcribed spacer region using SmartGene IDNS [corrected] software for identification of filamentous fungi in a clinical laboratory" 14 (14): 393-401, 2012

39 Sharifyazd S, "Effects of fermentation conditions on valuable products of ethanolic fungus Mucor indicus" 30 : 77-82, 2017

40 Librado P, "DnaSP v5: a software for comprehensive analysis of DNA polymorphism data" 25 (25): 1451-1452, 2009

41 Zein M, "DNA barcode fauna Indonesia" Prenadamedia Group 2013

42 Hebert PDN, "Biological identifications through DNA barcodes" 270 (270): 313-321, 2003

43 Rahayu ES, "Bahan pangan hasil fermentasi"

44 Vitale RG, "Antifungal susceptibility and phylogeny of opportunistic members of the order Mucorales" 50 (50): 66-75, 2012

45 Zheng R, "A monograph of Rhizopus" 59 (59): 273-372, 2007