Mycotoxin Research

, Volume 34, Issue 2, pp 151–157 | Cite as

First report of an atypical new Aspergillus parasiticus isolates with nucleotide insertion in aflR gene resembling to A. sojae

  • Sui Sheng T. Hua
  • Dan E. Parfitt
  • Siov Bouy L. Sarreal
  • Bertram G. Lee
  • Delilah F. Wood
Original Article


Aflatoxins are toxic and carcinogenic secondary metabolites produced primarily by the filamentous fungi Aspergillus flavus and Aspergillus parasiticus and cause toxin contamination in food chain worldwide. Aspergillus oryzae and Aspergillus sojae are highly valued as koji molds in the traditional preparation of fermented foods, such as miso, sake, and shoyu. Koji mold species are generally perceived of as being nontoxigenic and are generally recognized as safe (GRAS). Fungal isolates were collected from a California orchard and a few were initially identified to be A. sojae using β-tubulin gene sequences blasted against NCBI data base. These new isolates all produced aflatoxins B1, B2, G1, and G2 and were named as Pistachio Winter Experiment (PWE) strains. Thus, it is very important to further characterize these strains for food safety purposes. The full length of aflR gene of these new isolates was sequenced. Comparison of aflR DNA sequences of PWE, A. parasiticus and A. sojae, showed that the aflatoxigenic PWE strains had the six base insertion (CTCATG) similar to domesticated A. sojae, but a pre-termination codon TGA at nucleotide positions 1153–1155 was absent due to a nucleotide codon change from T to C. Colony morphology and scanning microscopic imaging of spore surfaces showed similarity of PWE strains to both A. parasiticus and A. sojae. Concordance analysis of multi locus DNA sequences indicated that PWE strains were closely linked between A. parasiticus and A. sojae. The finding documented the first report that such unique strains have been found in North America and in the world.


Aflatoxins Aspergillus sojae Aspergillus parasiticus aflR Phylogenetics 



This work is supported by in-house research program funds from US Department of Agriculture, Agricultural Research Services (Project Numbers: 5325-42000-038 and 5325-42000-039). We thank Perng Kuang Chang for valuable discussion; Debbie L Chingcuanco for comments; Tina Williams, John Pool, and Curtis Tsai for technical assistance.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.


  1. Amaike S, Keller NP (2011) Aspergillus flavus. Annu Rev Phytopathol 49:107–133CrossRefPubMedGoogle Scholar
  2. Bennett JW, Klich M (2003) Mycotoxins. Clin Microbiol Rev 16:497–516CrossRefPubMedPubMedCentralGoogle Scholar
  3. Chang P (2003) The Aspergillus parasiticus protein AFLJ interacts with the aflatoxin pathway-specific regulator AFLR. Mol Gen Genomics 268:711–719Google Scholar
  4. Chang PK (2004) Lack of interaction between AFLR and AFLJ contributes to nonaflatoxigenicity of Aspergillus sojae. J Biotechnol 107:245–253CrossRefPubMedGoogle Scholar
  5. Chang PK, Matsushima K, Takahashi T, Yu J, Abe K, Bhatnagar D, Yuan GF, Koyama Y, Cleveland TE (2007) Understanding nonaflatoxigenicity of Aspergillus sojae: a windfall of aflatoxin biosynthesis research. Appl Microbiol Biotechnol 76:977–984CrossRefPubMedGoogle Scholar
  6. Dorner JW, Cole RJ, Diener UL (1984) The relationship of Aspergillus flavus and Aspergillus parasiticus with reference to production of aflatoxins and cyclopiazonic acid. Mycopathologia 87:13–15CrossRefPubMedGoogle Scholar
  7. Ehrlich KC, Yu J, Cotty PJ (2005) Aflatoxin biosynthesis gene clusters and flanking regions. J Appl Microbiol 99:518–527CrossRefPubMedGoogle Scholar
  8. Hua SST, Baker JL, Flores-Espiritu M (1999) Interactions of saprophytic yeasts with a nor mutant of Aspergillus flavus. Appl Environ Microbiol 65:2738–2740PubMedPubMedCentralGoogle Scholar
  9. Hua SST, McAlpin CE, Chang PK, Sarreal SBL (2012) Characterization of aflatoxigenic and non-aflatoxigenic Aspergillus flavus isolates from pistachio. Mycotoxin Res 28:67–75CrossRefPubMedGoogle Scholar
  10. Jørgensen TR (2007) Identification and toxigenic potential of the industrially important fungi, Aspergillus oryzae and Aspergillus sojae. J Food Prot 70:2916–2934CrossRefPubMedGoogle Scholar
  11. Klich MA (2002) Identification of common Aspergillus species. Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands, pp 1–116Google Scholar
  12. Kurtzman CP, Smiley MJ, Robnett CJ, Wicklow DT (1986) DNA relatedness among wild and domesticated species in Aspergillus flavus group. Mycologia 78:955–959CrossRefGoogle Scholar
  13. Kurtzman CP, Robnett CJ (1998) Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences. Antonie Van Leeuwenhoek 73:331–371CrossRefPubMedGoogle Scholar
  14. Lee CZ, Liou GY, Yuan GF (2006) Comparison of the aflR gene sequences of strains in Aspergillus section Flavi. Microbiology 152:161–170CrossRefPubMedGoogle Scholar
  15. Matsushima K, Chang PK, Yu J, Abe K, Bhatnagar D, Cleveland TE (2001a) Pre-termination in aflR of Aspergillus sojae inhibits aflatoxin biosynthesis. Appl Microbiol Biotechnol 55:585–589CrossRefPubMedGoogle Scholar
  16. Matsushima K, Yashiro K, Hanya Y, Abe K, Yabe K, Hamasaki T (2001b) Absence of aflatoxin biosynthesis in koji mold (Aspergillus sojae). Appl Microbiol Biotechnol 55:771–776CrossRefPubMedGoogle Scholar
  17. Peterson SW (2008) Phylogenetic analysis of Aspergillus species using DNA sequences from four loci. Mycologia 100:205–226CrossRefPubMedGoogle Scholar
  18. Pitt JI, Hocking AD, Glenn DR (1983) An improved medium for the detection of Aspergillus flavus and A. parasiticus. J Appl Bacteriol 54:109–114CrossRefPubMedGoogle Scholar
  19. Roze LV, Hong S-Y, Linz JE (2013) Aflatoxin biosynthesis: current frontiers. Annu Rev Food Sci Technol 4:293–311CrossRefPubMedGoogle Scholar
  20. Soares C, Rodrigues P, Peterson SW, Lima N, Venancio A (2012) Three new species of Aspergillus section Flavi isolated from almonds and maize in Portugal. Mycologia 104:682–697CrossRefPubMedGoogle Scholar
  21. Varga J, Frisvad JC, Samson RA (2011) Two new aflatoxin producing species, and an overview of Aspergillus section Flavi. Stud Mycol 69:57–80CrossRefPubMedPubMedCentralGoogle Scholar
  22. Watson AJ, Fuller LJ, Jeenes DJ, Archer DB (1999) Homologs of aflatoxin biosynthesis genes and sequence of aflR in Aspergillus oryzae and Aspergillus sojae. Appl Environ Microbiol 65:307–310PubMedPubMedCentralGoogle Scholar
  23. Wicklow DT (1984) Adaptation in wild and domesticated yellow-green aspergilli. In: Kurata H, Ueno Y (eds) Toxigenic fungi: their toxins and health hazards. Elsevier, Amsterdam, The Netherlands, pp 78–86Google Scholar
  24. Yu J, Chang PK, Ehrlich KC, Cary JW, Bhatnagar D, Cleveland TE, Payne GA, Linz JE, Woloshuk CP, Bennett JW (2004) Clustered pathway genes in aflatoxin biosynthesis. Appl Environ Microbiol 70:1253–1262CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2018

Authors and Affiliations

  • Sui Sheng T. Hua
    • 1
  • Dan E. Parfitt
    • 2
  • Siov Bouy L. Sarreal
    • 1
  • Bertram G. Lee
    • 1
  • Delilah F. Wood
    • 1
  1. 1.US Department of Agriculture, Agricultural Research ServiceWestern Regional Research CenterAlbanyUSA
  2. 2.Department of Plant SciencesUniversity of California, DavisDavisUSA

Personalised recommendations