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Aspergillus caninus (Syn: Phialosimplex caninus): a New Isolate from Field Soils in Korea

  • Adhikari, Mahesh (Division of Biological Resources Sciences, Kangwon National University) ;
  • Gurung, Sun Kumar (Division of Biological Resources Sciences, Kangwon National University) ;
  • Kim, Sang Woo (Division of Biological Resources Sciences, Kangwon National University) ;
  • Lee, Hyun Goo (Division of Biological Resources Sciences, Kangwon National University) ;
  • Ju, Han Jun (Division of Biological Resources Sciences, Kangwon National University) ;
  • Gwon, Byeong Heon (Division of Biological Resources Sciences, Kangwon National University) ;
  • Kosol, San (Division of Biological Resources Sciences, Kangwon National University) ;
  • Bazie, Setu (Division of Biological Resources Sciences, Kangwon National University) ;
  • Lee, Hyang Burm (Division of Food Technology, Biotechnology and Agrochemistry, Chonnam National University) ;
  • Lee, Youn Su (Division of Biological Resources Sciences, Kangwon National University)
  • Received : 2018.07.13
  • Accepted : 2018.08.23
  • Published : 2018.12.01

Abstract

During the study of indigenous fungal communities in soil samples collected from various field soils in Sancheong, Gyeongsangnam-do, Korea in 2017, several species of Aspergillus were discovered. Aspergillus caninus (KNU17-7) was isolated, identified, and described based on the results from macro and micro morphological characteristics and molecular characterization. Morphologically, it was identified using five different growth media: potato dextrose agar, oatmeal agar, yeast extract sucrose agar, czapek yeast extract agar, and malt extract agar. For the molecular identification, sequencing of internal transcribed spacer, ${\beta}-tubulin$, and calmodulin genes was performed. Based on this characterization, our study isolate was identified as Aspergillus caninus. This fungal species has not been officially reported in Korea before, and we report here with its morphological and molecular phylogenetic characterization.

Keywords

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Fig. 1. Morphological features of the Aspergillus caninus KNU17-7 colony grown for 7 days on potato dextrose agar (PDA), oatmeal agar (OMA), yeast extract sucrosea gar (YESA), malt extract agar (MEA), and Czapek yeast extract agar (CYEA) at 25°C. A, F, front and back side of the colony on PDA; B, G, front and back side on OMA; C, H, front and back side oYn ESA; D, I, front and back side on MEA; E, J, front and back side on CYEA.

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Fig. 2. Microstructures of the isolate Aspergillus caninus KNU17-7. Front colony from left to right. A, B, microscopy of conidiophores with phialides; C, microscopy of conidia; D, E, scanning electron microscopy of the conidia; F~H, scanning electron microscopy o fconidiophores-bearing conidia.

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Fig. 3. Neighbor-joining phylogenetic analysis of the partial 18S-ITS1-5.8S-ITS2-28S rDNA sequences of Aspergillus caninus KNU17-7. Tree was constructed using the MEGA ver. 6 program. Sequences obtained in the study are shown in boldface. The mark (T) indicates the type strain. Numerical values (> 50) on branches are the bootstrap values as percentage of bootstrap replication from a 1,000 replicate analysis. Scale bar represents the number of substitutions pers ite.

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Fig. 4. Neighbor-joining phylogenetic analysis of β-tubulin (tub2/BenA) gene sequences of Aspergillus caninus KNU17-7. A phylogenetic tree was constructed using MEGA ver. 6. Sequences obtained in the study are shown in boldface. Numerical values (> 50) on branches are the bootstrap values as percentage of bootstrap replication from a 1,000 replicate analysis. Scale bar represents the number of substitutions per site.

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Fig. 5. Neighbor-joining phylogenetic analysis of Calmodulin (CaM) gene sequences of Aspergillus caninus KNU17-7. Tree was constructed using MEGA ver. 6. Sequences obtained in the study are shown in boldface. Numerical values (> 50) on branches are the bootstrap values as percentage of bootstrap replication from a 1,000 replicate analysis. Scale bar represents the number of substitutions per site.

Table 1. Primers used for amplifcation and sequencing.

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Table 2. Thermal cycle programs used for amplifcation.

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Table 3. Morphological comparison of the study isolate KNU17-7 with the previously reported Aspergillus caninus.

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References

  1. Kirk PM, Cannon PF, David JC, Stalpers JA. Dictionary of the fungi. 9th ed. Wallingford: CABI Publishing; 2001.
  2. Bennett JW. An overview of the genus Aspergillus . In: Machida M, Gomi K, editors. Aspergillus: molecular biology and genomics. Poole: Caister Academic Press; 2010. p. 1-17.
  3. Raper KB, Fennell DI. The genus Aspergillus. Baltimore: Williams and Wilkins; 1965.
  4. Gams W, Christensen M, Onions AH, Pitt JI, Samson RA. Infrageneric taxa of Aspergillus. In: Samson RA, Pitt JI, editors. Advances in Penicillium and Aspergillus systematics. Boston: Springer; 1985. p. 55-61.
  5. Goldman GH, Osmani SA. The aspergilli: genomics, medical aspects, biotechnology, and research methods. Boca Raton: CRC Press; 2008.
  6. Samson RA, Varga J. What is a species in Aspergillus? Med Mycol 2009;47:S13-20. https://doi.org/10.1080/13693780802354011
  7. Diba K, Kordbacheh P, Mirhendi SH, Rezaie S, Mahmoudi M. Identification of Aspergillus species using morphological characteristics. Pak J Med Sci 2007;23: 867-72.
  8. International Mycological Association. MYCOBANKDATABASE [Internet]. Utrecht: International Mycological Association; 2018 [cited 2018 September 10]. Available from: http://www.mycobank.org/.
  9. Davet P, Rouxel F. Detection and isolation of soil fungi. Enfield: Science Publishers; 2000.
  10. Samson RA, Houbraken J, Thrane U, Frisvad JC, Andersen B. Food and indoor fungi. Utrecht: CBS-KNAW Fungal Biodiversity Centre; 2010.
  11. Kornerup A, Wanscher JH. Methuen handbook of color. 2nd ed. London: Methuen; 1967.
  12. White TJ, Bruns T, Lee S, Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, editors. PCR protocols: a guide to methods and applications. San Diego: Academic Press; 1990. p. 315-22.
  13. Glass NL, Donaldson GC. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Appl Environ Microbiol 1995;61:1323-30.
  14. Hong SB, Go SJ, Shin HD, Frisvad JC, Samson RA. Polyphasic taxonomy of Aspergillus fumigatus and related species. Mycologia 2005;97:1316-29. https://doi.org/10.1080/15572536.2006.11832738
  15. National Center for Biotechnology Information. GenBank overview [Internet]. Bethesda (MD): National Center for Biotechnology Information; 2015 [cited 2018 September 10]. Available from: https://www.ncbi.nlm.nih.gov/genbank/.
  16. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol 2013;30:2725-9. https://doi.org/10.1093/molbev/mst197
  17. Kimura M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 1980;16:111-20. https://doi.org/10.1007/BF01731581
  18. Hyde KD, Abd-Elsalam K, Cai L. Morphology: still essential in a molecular world. Mycotaxon 2010;114:439-51.
  19. Raja HA, Miller AN, Pearce CJ, Oberlies NH. Fungal identification using molecular tools: a primer for the natural products research community. J Nat Prod 2017;80:756-70. https://doi.org/10.1021/acs.jnatprod.6b01085
  20. Silva DM, Batista LR, Renzende EF, Fungaro MH, Sartori D, Alves E. Identification of fungi of the genus Aspergillus section nigri using polyphasic taxonomy. Braz J Microbiol 2011;42:761-73. https://doi.org/10.1590/S1517-83822011000200044
  21. Sigler L, Sutton DA, Gibas CF, Summerbell RC, Noels RK, Iwens PC. Phialosimplex, a new anamorphic genus associated with infections in dogs and having phylogenetic affinity to the Trichocomaceae. Med Mycol 2010;48:335-45. https://doi.org/10.3109/13693780903225805
  22. Hinrikson HP, Hurst SF, De Aguirre L, Morrison CJ. Molecular methods for the identification of Aspergillus species. Med Mycol 2005;43:S129-37. https://doi.org/10.1080/13693780500064722