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Non-Aflatoxigenicity of Commercial Aspergillus oryzae Strains Due to Genetic Defects Compared to Aflatoxigenic Aspergillus flavus
Non-Aflatoxigenicity of Commercial Aspergillus oryzae Strains Due to Genetic Defects Compared to Aflatoxigenic Aspergillus flavus
Journal of Microbiology and Biotechnology. 2014. Aug, 24(8): 1081-1087
Copyright © 2014, The Korean Society For Microbiology And Biotechnology
  • Received : November 06, 2013
  • Accepted : April 18, 2014
  • Published : August 28, 2014
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About the Authors
Lin Tao
Soo Hyun Chung
chungs59@korea.ac.kr

Abstract
Aspergillus oryzae is generally recognized as safe, but it is closely related to A. flavus in morphology and genetic characteristics. In this study, we tested the aflatoxigenicity and genetic analysis of nine commercial A. oryzae strains that were used in Korean soybean fermented products. Cultural and HPLC analyses showed that none of the commercial strains produced detectable amount of aflatoxins. According to the molecular analysis of 17 genes in the aflatoxin (AF) biosynthetic pathway, the commercial strains could be classified into three groups. The group I strains contained all the 17 AF biosynthetic genes tested in this study; the group II strains deleted nine AF biosynthetic genes and possessed eight genes, including aflG , aflI , aflK , aflL , aflM , aflO , aflP , and aflQ ; the group III strains only had six AF biosynthetic genes, including aflG , aflI , aflK , aflO , aflP , and aflQ . With the reverse transcription polymerase chain reaction, the group I A. oryzae strains showed no expression of aflG , aflQ and/or aflM genes, which resulted in the lack of AF-producing ability. Group II and group III strains could not produce AF owing to the deletion of more than half of the AF biosynthetic genes. In addition, the sequence data of polyketide synthase A ( pksA ) of group I strains of A. oryzae showed that there were three point mutations (two silent mutations and one missense mutation) compared with aflatoxigenic A. flavus used as the positive control in this study.
Keywords
Introduction
Aflatoxin (AF), a group of polyketide-derived furanocoumarins, was characterized after the acute hepatotoxic disease in turkeys (turkey X disease) was traced to the consumption of a mold-contaminated peanut meal. The four major aflatoxins are called B 1 , B 2 , G 1 , and G 2 , based on their fluorescence (blue or green) under UV light and relative chromatographic mobility during thin-layer chromatography. AF B 1 is the most potent natural carcinogen known and is usually the major AF produced by toxigenic strains. The data on AF as a human carcinogen are far more dreadful than the data implicating it in acute human toxicities. Exposure to aflatoxins with the diet is considered as an important risk factor for the development of primary hepatocellular carcinoma, particularly in individuals already exposed to hepatitis B [2] .
A. oryzae is an important fungus that has been used for centuries in the oriental food fermentation industry. By contrast, A. flavus is notorious for its production of aflatoxins, for causing aspergillosis in humans and animals, and as an opportunistic pathogen of animals and plants [22] . So far, A. oryzae is generally recognized as safe (GRAS). However, previous studies reported that A. oryzae is closely related to A. flavus in morphology and genetic characteristics [23] . A. oryzae and A. flavus belong to Aspergillus section Flavi . These two fungi are very similar in genome size and number of predicted genes: 36.7 Mb and 12,197 genes for A. flavus , and 36.7 Mb and 12,079 genes for A. oryzae . The high degree of sequence similarity in genes between the two fungi suggests that they may be ecotypes of the same species and that A. oryzae has resulted from the domestication of A. flavus [21] .
The molecular biological elucidation of AF non-productivity in A. oryzae strains has important implications in terms of verifying the safety of the fermented products employing those strains [12 - 14 , 17 , 20] . The molecular biological techniques, including analysis of rDNA internal transcribed spacer regions, restriction fragment length polymorphism, and hybridization with aflatoxin biosynthetic genes, are able to distinguish the A. flavus/oryzae group from the A. parasiticus/sojae group but cannot differentiate A. oryzae from A. flavus as a distinct species [5] . The recent studies of whole genomic sequencing and analysis of the aflatoxin biosynthetic cluster of A. oryzae and A. flavus can provide us with the difference between these two fungi [4 , 11 , 17 , 21 , 26] .
AF biosynthesis is almost fully characterized and involves the coordinated expression of approximately 25 genes clustered in a 70 kb DNA region [28] . The genes involved in AF biosynthesis are clustered together on the fungal gene. AFLR, a translation product of the aflR gene in this cluster, is a positive transcriptional regulator that promotes transcription of AF biosynthetic genes [16] . aflJ (translation product is AFLJ), another transcriptional regulator, is also needed for AF biosynthesis [3 , 18] . A. oryzae possesses the AF biosynthesis gene homolog cluster but does not produce aflatoxin [1 , 11] . Recently, to reduce AF contamination on crops, a biocontrol strategy was applied to use non-aflatoxigenic A. flavus strains to competitively exclude field toxigenic Aspergillus species [7 , 8 , 19] . After molecular analysis of those strains, researchers found that they lacked the producing of aflatoxins owing to the single nucleotide mutations in the pksA gene [9] .
In this study, we collected nine commercial strains of A. oryzae currently used in the soybean fermentation industry in Korea, and molecular analyses were conducted to provide the evidence of non-aflatoxigenicity of the strains for commercial use. For this, two regulatory genes ( aflR and aflJ ) and 15 structural genes in the AF biosynthetic pathway were analyzed to distinguish A. oryzae from aflatoxigenic A. flavus , based on the presence or absence of aflatoxin-specific DNA or mRNAs and pksA gene sequencing as well.
Materials and Methods
- Fungal Strains
Commercial strains of A. oryzae M3, M5, M6, M7, M9, M11, M15, M17, and M18 were obtained from soybean fermentation companies in Korea. A. flavus KACC 2030 used as a positive control for AF production was supplied by Korean Agricultural Culture Collection (KACC) (Suwon, Korea). All the strains were inoculated onto Czapek Agar (MBcell, Seoul, Korea) slants and incubated at 25℃ for 7 days, and then conidia were collected for the following experiments.
- Determination of Aflatoxin Production
The AF production by fungal strains was determined using high-performance liquid chromatography (HPLC) (Dionex Ultimate 3000, Sunnyvale, CA, USA) [10] . A 0.1 ml volume of A. oryzae spore suspension (1 × 10 6 spores/ml) was inoculated into 50 ml of Potato Dextrose Broth (PDB) (MBcell) in a 250 ml Erlenmeyer flask at 25℃. After 14 days, the fungal culture broth was filtered through Whatman No. 1 filter paper (GE Healthcare Co., Buckinghamshire, UK), and 15 ml of the filtrate was then diluted with phosphate-buffered saline (pH 7.5) to 90 ml. The mixture was passed through a Whatman GF/A glass filter (GE Healthcare Co.), and 50 ml of the filtrate was loaded onto an immunoaffinity column (AflaTest, Vicam Co., Milford, MA, USA) at a flow rate of approximately 1 drop per second for clean-up. After washing the column with 10 ml of water at the same flow rate, aflatoxin was eluted with 2 ml of methanol. The eluate was evaporated at 40℃ under a stream of N 2 until dry. The dry residue was derivatized by adding 200 μl of trifluoroacetic acid, and the mixture was left to stand for 30 min before it was diluted with 800 μl of acetonitrile-water (10:90 (v/v)). This derivatized sample was filtered through a 0.22 μm membrane filter, and the filtrate was used for HPLC analysis. Separation of AF B 1 , B 2 , G 1 , and G 2 from the injected 50 μl of samples was carried out using a Waters Nova-Pack C18 column (150 × 3.9 mm, 5 μm; Milford, MA, USA). The mobile phase was acetonitrile-methanol-water (17:17:66 (v/v/v)) pumped at a constant flow rate of 0.5 ml/min. The quantitative determination of each AF was carried out using a fluorescence detector (excitation: 360 nm; emission: 440 nm).
- Preparation of Genomic DNA for Polymerized Chain Reaction
Approximately 10 5 conidia were inoculated into 8 ml of PDB in a test tube. The tube was incubated at 25℃ for 3 days with 120 rpm shaking, and then fungal genomic DNA was extracted from the lyophilized mycelia. Briefly, the ground lyophilized mycelia was mixed with lysis buffer (50 mM EDTA, 0.2% SDS, pH 8.5) and incubated at 65℃ for 1 h, and then purification with phenol-chloroform-isoamyl alcohol (25:24:1) and precipitation with isopropanol were conducted. Finally, the precipitation was washed with 70% ethanol and resuspended in 50 μl of TE solution (10 mM Tris-HCl, 0.1 mM EDTA).
Seventeen primer pairs previously designed for the specific amplification of AF biosynthetic genes [4 , 6 , 11 , 24 - 26] were applied ( Table 1 ). The PCR procedure was performed with 50 μl of reaction mixture containing 5.0 μl of 10× Taq buffer, 3.0 μl of 2.5 mM dNTP, 0.3 μl of Taq polymerse, 0.4 μl of each primer (SolGent, Deajeon, Korea), 39.9 μl of distilled water, and 1.0 μl of the DNA as template. Cycling parameters were 5 min at 94℃, 55℃ (primer 3, 4, 5, 8, 10, 11, 14, 15, 16, and 18) or 60℃ (primer 6, 7, 9, and 17) or 65℃ (primer 1, 2, 12, and 13) for 35 cycles, 30 sec at 94℃, 60 sec at 55℃, 90 sec at 72℃, with a final extension at 72℃ for 7 min in a DNA thermal cycler (Astec PC-708 Program Temp Control System, Tokyo, Japan). DNA fragments were separated in a 1.5% agarose gel electrophoresis.
Primers, target genes, primer sequences, and expected PCR and RT-PCR product sizes.
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Primers, target genes, primer sequences, and expected PCR and RT-PCR product sizes.
- RNA Preparation for Reverse Transcriptional PCR
The tested strains were inoculated in the same conditions for DNA extraction. Total RNA was prepared using an RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA). Fifteen AF biosynthetic genes applied for the above conventional PCR (except aflH and aflI ) were also used in the RT-PCR ( Table 1 ). The housekeeping gene tub1 coding β-tubulin was chosen and used as a system control for reverse transcription. Reverse transcription was performed using an Ominiscript Kit (Qiagen), and the PCR procedure was carried out as described above.
- Sequencing of pksA Gene
A part of the pksA gene encoding the polyketide synthase for aflatoxin biosynthesis was amplified from group I A. oryzae strains and A. flavus KACC 2030 by PCR. The 399 bp amplicons were purified using an EZWay PCR Clean-up Kit (Komabiotech, Seoul, Korea) and sequenced by Solgent Co. Ltd (Daejeon, Korea).
Results
- Aflatoxin Production
After extraction of PDB-culture filtrates with an immunoaffinity column, the eluate was analyzed by fluorescence detection with HPLC, which allowed the separation and identification of AF B 1 , B 2 , G 1 , and G 2 . None of the commercial A. oryzae strains (M3, M5, M6, M7, M9, M11, M15, M17, and M18) produced detectable aflatoxins, whereas A. flavus KACC 2030 strain produced AF B 1 (12.5 μg/ml) and AF B 2 (0.9 μg/ml) after 14 days of incubation ( Fig. 1 ).
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Chromatogram of aflatoxin analysis of commercial A. oryzae strains and A. flavus KACC 2030: 1, A. oryzae M3; 2, A. oryzae M5; 3, A. oryzae M6; 4, A. oryzae M7; 5, A. oryzae M9; 6, A. oryzae M11; 7, A. oryzae M15; 8, A. oryzae M17; 9, A. oryzae M18; and 10, A. flavus KACC 2030. Potato Dextrose Broth was used for the production of aflatoxin by each fungal strain. After 14 days of incubation at 25℃, the fungal culture filtrates were used for aflatoxin analysis.
- AF Biosynthetic Gene Profiles of Commercial A. oryzae
After screening 17 genes ( aflR , aflJ , aflA , aflD , aflE , aflF-aflU , aflG , aflH , aflI , aflK , aflL , aflM , aflO , aflP , aflQ , aflT , and pksA ) through PCR-gel electrophoresis, nine strains of A. oryzae were divided into three groups ( Table 2 ). Group I, strains of M3, M6, M7, M9 and M11, contained all the 17 AF biosynthetic genes, including the regulatory genes aflR and aflJ ; Group II, strains of M15, M17, and M18, had eight genes, including aflG , aflI , aflK , aflL , aflM , aflO , aflP and aflQ ; Group III, only M5, possessed six genes, including aflG , aflI , aflK , aflO , aflP and aflQ . It was clear that group II and group III strains were unable to produce AF owing to the lack of more than half of the AF biosynthetic genes tested in this study. However, based on the PCR results, we could not prove the non-AF production by group I strains at the molecular level.
Classification of commercialA. oryzaestrains based on PCR amplification patterns of aflatoxin biosynthetic genes.
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Classification of commercial A. oryzae strains based on PCR amplification patterns of aflatoxin biosynthetic genes.
- AF Biosynthetic Gene Expression of Group I A. oryzae
RT-PCR analysis was carried out to evaluate the AF biosynthetic gene expression of group I A. oryzae using the 15 pairs of primers that were used for the AF biosynthetic gene profiles mentioned above, except two primers for aflH and aflI . The primer pairs for the amplification of aflH and aflI gene expression could not generate their positive signals with aflatoxigenic A. flavus KACC 2030 in preliminary tests (data not shown).
The results showed that after the expression analysis of the 15 genes, M3, M6, and M7 strains generated 12 positive signals ( aflR , aflJ , aflA , aflD , aflE , aflF-aflU , aflK , aflL , aflO , aflP , aflT , and pksA ) ( Table 3 ). Strains of M9 and M11 generated 14 positive signals ( aflR , aflJ , aflA , aflD , aflE , aflF aflU , aflG , aflK , aflL , aflO , aflP , aflQ , aflT , and pksA ). Consequently, M3, M6 and M7 strains were incapable of producing aflatoxin owing to the lack of expression of aflG , aflM , and aflQ ; M9 and M11 strains were unable to produce AF owing to the lack of expression of the aflM gene. It was reported that the aflG gene represented one of the early steps in the AF biosynthetic pathway, encoding a cytochrome P450 monooxygenase that converted averantin to 5P-hydroxy-averantin [27] . The research also showed that aflM and aflQ lay at the end of the biosynthetic pathway of aflatoxins: aflM was required for the conversion of versicolorin A to demethylsterigmatocystin; aflQ was involved in the conversion of O -methylsterigmatocystin to AF B 1 (and AF G 1 ) and of dihydrodemethylsterigmatocystin to AF B 2 (and AF G 2 ).
Expression of aflatoxin biosynthetic genes by commercialA. oryzaeandA. flavusKACC 2030.
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In aflatoxin production, +: afltoxin production; §—: no aflatoxin production by HPLC. In gene expression, +: amplification signal present; §—: amplification signal absent by RT-PCR.
- Determination of Mutations of pksA Gene of Group I A. oryzae
A gene sequencing for the 0.4 kb partial pksA gene region of group I A. oryzae and A. flavus KACC 2030 strains was performed. The sequence data showed that group I strains had three point mutations compared with A. flavus KACC 2030 ( Fig. 2 ): T to C point mutation at nucleotide position 303, C to T point mutation at position 315, and G to A point mutation at position 325, respectively. The first two point mutations at positions 303 and 315 were silent mutations, which respectively induced the asparagine to asparagine codon and the arginine to arginine codon. The point mutation at nucleotide position 325, referring to a missense mutation, changed from GAG to AAG, resulting in an amino acid change within the polypeptide from glutamic acid to lysine.
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Comparison of the pksA gene of group I A. oryzae strains (M3, M5, M6, M7, M9, and M11) with aflatoxigenic A. flavus KACC 2030. The base sequences of 1-158 and 335-359 were omitted.
Discussion
Kusumoto et al . [15] studied 39 strains of A. oryzae and found those strains could be classified into three groups according to the existence of five genes ( aflR , aflA , aflK , aflM , and pksA ,): In group I, almost all of the AF biosynthetic genes were preserved, containing 24 strains (61.5% of examined strains); in group II (28.2%), more than half of the AF biosynthetic genes (including aflR ) were missed; in group III (10.2%), the AF biosynthetic genes were almost lost. Similarly, Tominaga et al . [26] analyzed the existence of seven AF biosynthetic genes ( aflR , aflD , aflE , aflG , aflK , aflL , and aflT ) and found that the 210 strains of A. oryzae tested were still classified into the three groups (58.1%, 36.7%, and 4.3%, respectively). We studied the existence of 17 AF biosynthetic genes with nine commercial A. oryzae strains and got a consistent result with Kusumoto et al . and Tominaga et al .; the nine A. oryzae strains in this study were also divided into three groups and similar proportions (55.5%, 33.3%, and 11.1%, respectively). Group II and group III strains evidently could not produce AF owing to their gene deletions in the AF biosynthetic pathway. The group I strains, however, possessed intact AF biosynthetic genes, but still needs molecular evidence to explain their non-AF production. It is very important to have molecular evidence to prove that group I A. oryzae strains are incapable of producing aflatoxins and safe for commercial use.
Scherm et al . [24] suggested that RT-PCR could be efficiently used to distinguish between AF-producing and non-producing strains of A. flavus and A. parasiticus by measuring the presence of aflD , aflO , and aflP transcripts. In this paper, we studied the expression of two regulatory genes ( aflR and aflJ ) and 13 structural genes ( aflA , aflD , aflE , aflF-aflU , aflG , aflK , aflL , aflM , aflO , aflP , aflQ , aflT , and pksA ), and found that group I A. oryzae strains were not capable of AF production owing to the lack of expression of one gene ( aflM ) or three genes ( aflG , aflM , and aflQ ). The results proved the safety of group I A. oryzae strains in the molecular level of AF production. Furthermore, it also supported that RT-PCR was an effective method to distinguish A. oryzae from aflatoxigenic A. flavus .
Ehrlich and Cotty [9] reported that the pksA gene from an isolate of non-aflatoxigenic A. flavus had a G to A point mutation (position 192) compared with a strain of aflatoxigenic A. flavus , which induced a TGA stop codon that caused truncation of the resulting polyketide synthase. In our result, there was no stop codon present in the pksA gene of the A. oryzae strains, but depending on the open reading frame of the pksA gene, we found three positions of point mutations: two silent mutations and one missense mutation. The missense mutation was found to result in an amino acid change from glutamic acid to lysine. Whether the pksA -translated protein structure change of this missense mutation affects the production of AF is still not clear and needs to be studied in the future.
In summary, molecular approaches based on the presence or absence of AF-specific DNA or mRNA were employed in an attempt to distinguish nine commercial strains of A. oryzae and aflatoxigenic A. flavus KACC 2030 in this study. PCR experiments using 17 genes in the AF biosynthetic pathway differentiated four of the nine strains of A. oryzae from aflatoxigenic A. flavus , and the other five strains needed RT-PCR for the expression of the aflatoxigenic genes. One ( aflM ) or three genes ( aflG , aflM , and aflQ ) were not expressed in the five A. oryzae strains, which had all the 17 genes tested. Our results show the molecular evidence of non-aflatoxigenicity of the A. oryzae strains used commercially in Korea.
Acknowledgements
This research was supported by a grant from Korea University.
References
An LH , Hu JY , Zhang ZB , Yang M 2006 Quantitative realtime RT-PCR for determination of vitellogenin mRNA in soiuy mullet (Mugil soiuy). Anal. Bioanal. Chem. 386 1995 - 2001    DOI : 10.1007/s00216-006-0846-y
Bennett JW , Klich M 2003 Mycotoxins. Clin. Microbiol. Rev. 16 497 - 516    DOI : 10.1128/CMR.16.3.497-516.2003
Chang PK 2003 The Aspergillus parasiticus protein AFLJ interacts with the aflatoxin pathway-specific regulator AFLR. Mol. Genet. Genomics 268 711 - 719
Chang PK , Abbas HK , Weaver MA , Ehrlich KC , Scharfenstein LL , Cotty PJ 2012 Identification of genetic defects in the atoxigenic biocontrol strain Aspergillus flavus K49 reveals the presence of a competitive recombinant group in field populations. Int. J. Food Microbiol. 154 192 - 196    DOI : 10.1016/j.ijfoodmicro.2012.01.005
Chang PK , Ehrlich KC 2010 What does genetic diversity of Aspergillus flavus tell us about Aspergillus oryzae? Int. J. Food Microbiol. 138 189 - 199    DOI : 10.1016/j.ijfoodmicro.2010.01.033
Chang PK , Scharfenstein LL , Ehrlich KC , Wei QJ , Bhatnagar D , Ingber BF 2012 Effects of laeA deletion on Aspergillus flavus conidial development and hydrophobicity may contribute to loss of aflatoxin production. Fungal Biol. 116 298 - 307    DOI : 10.1016/j.funbio.2011.12.003
Das MK , Ehrlich KC , Cotty PJ 2008 Use of pyrosequencing to quantify incidence of a specific Aspergillus flavus strain within complex fungal communities associated with commercial cotton crops. Phytopathology 98 282 - 288    DOI : 10.1094/PHYTO-98-3-0282
Dorner JW 2004 Biological control of aflatoxin contamination of crops. J. Toxicol. Toxin Rev. 23 425 - 450    DOI : 10.1081/TXR-200027877
Ehrlich KC , Cotty PJ 2004 An isolate of Aspergillus flavus used to reduce aflatoxin contamination in cottonseed has a defective polyketide synthase gene. Appl. Microbiol. Biotechnol. 65 473 - 478    DOI : 10.1007/s00253-004-1670-y
Jung YJ , Chung SH , Lee HK , Chun HS , Hong SB 2012 Isolation and identification of fungi from a meju contaminated with aflatoxins. J. Microbiol. Biotechnol. 22 1740 - 1748    DOI : 10.4014/jmb.1207.07048
Kiyota T , Hamada R , Sakamoto K , Iwashita K , Yamada O , Mikami S 2011 Aflatoxin non-productivity of Aspergillus oryzae caused by loss of function in the aflJ gene product. J. Biosci. Bioeng. 111 512 - 517    DOI : 10.1016/j.jbiosc.2010.12.022
Klich MA , Mullaney EJ 1987 DNA restriction enzyme fragment polymorphism as a tool for rapid differentiation of Aspergillus flavus from Aspergillus oryzae. Exp. Mycol. 11 170 - 175    DOI : 10.1016/0147-5975(87)90002-8
Klich MA , Yu J , Chang PK , Mullaney EJ , Bhatnagar D , Cleveland TE 1995 Hybridization of genes involved in aflatoxin biosynthesis to DNA of aflatoxigenic and non-aflatoxigenic aspergilli. Appl. Microbiol. Biotechnol. 44 439 - 443    DOI : 10.1007/BF00169941
Kumeda Y , Asao T 2001 Heteroduplex panel analysis, a novel method for genetic identification of Aspergillus section Flavi strains. Appl. Environ. Microbiol. 67 4084 - 4090    DOI : 10.1128/AEM.67.9.4084-4090.2001
Kusumoto K , Nogata Y , Ohta H 2000 Directed deletions in the aflatoxin biosynthesis gene homolog cluster of Aspergillus oryzae. Curr. Genet. 37 104 - 111    DOI : 10.1007/s002940050016
Kusumoto KI , Yabe K , Nogata Y , Ohta H 1998 Transcript of a homolog of aflR, a regulatory gene for aflatoxin synthesis in Aspergillus parasiticus, was not detected in Aspergillus oryzae strains. FEMS Microbiol. Lett. 169 303 - 307    DOI : 10.1111/j.1574-6968.1998.tb13333.x
Lee CZ , Liou GY , Yuan GF 2006 Comparison of the aflR gene sequences of strains in Aspergillus section Flavi. Microbiology 152 161 - 170    DOI : 10.1099/mic.0.27618-0
Meyers DM , Obrian G , Du WL , Bhatnagar D , Payne GA 1998 Characterization of aflJ, a gene required for conversion of pathway intermediates to aflatoxin. Appl. Environ. Microbiol. 64 3713 - 3717
Mishra HN , Das C 2003 A review on biological control and metabolism of aflatoxin. Crit. Rev. Food Sci. Nutr. 43 245 - 264    DOI : 10.1080/10408690390826518
Montiel D , Dickinson MJ , Lee HA , Dyer PS , Jeenes DJ , Roberts IN 2003 Genetic differentiation of the Aspergillus section Flavi complex using AFLP fingerprints. Mycol. Res. 107 1427 - 1434    DOI : 10.1017/S0953756203008797
Payne GA , Nierman WC , Wortman JR , Pritchard BL , Brown D , Dean RA 2006 Whole genome comparison of Aspergillus flavus and A. oryzae. Med. Mycol. 44 S9 - S11    DOI : 10.1080/13693780600835716
Ramirez-Camejo LA , Zuluaga-Montero A , Lazaro-Escudero M , Hernandez-Kendall V , Bayman P 2012 Phylogeography of the cosmopolitan fungus Aspergillus flavus: is everything everywhere? Fungal Biol. 116 452 - 463    DOI : 10.1016/j.funbio.2012.01.006
Scheidegger KA , Payne GA 2003 Unlocking the secrets behind secondary metabolism: a review of Aspergillus flavus from pathogenicity to functional genomics. J. Toxicol. Toxin Rev. 22 423 - 459    DOI : 10.1081/TXR-120024100
Scherm B , Palomba M , Serra D , Marcello A , Migheli Q 2005 Detection of transcripts of the aflatoxin genes aflD, af1O, and aflP by reverse transcription-polymerase chain reaction allows differentiation of aflatoxin-producing and non-producing isolates of Aspergillus flavus and Aspergillus parasiticus. Int. J. Food Microbiol. 98 201 - 210    DOI : 10.1016/j.ijfoodmicro.2004.06.004
Shapira R , Paster N , Eyal O , Menasherov M , Mett A , Salomon R 1996 Detection of aflatoxigenic molds in grains by PCR. Appl. Environ. Microbiol. 62 3270 - 3273
Tominaga M , Lee YH , Hayashi R , Suzuki Y , Yamada O , Sakamoto K 2006 Molecular analysis of an inactive aflatoxin biosynthesis gene cluster in Aspergillus oryzae RIB strains. Appl. Environ. Microbiol. 72 484 - 490    DOI : 10.1128/AEM.72.1.484-490.2006
Yu JJ , Bhatnagar D , Cleveland TE 2004 Completed sequence of aflatoxin pathway gene cluster in Aspergillus parasiticus. FEBS Lett. 564 126 - 130    DOI : 10.1016/S0014-5793(04)00327-8
Yu JJ , Chang PK , Ehrlich KC , Cary JW , Bhatnagar D , Cleveland TE 2004 Clustered pathway genes in aflatoxin biosynthesis. Appl. Environ. Microbiol. 70 1253 - 1262    DOI : 10.1128/AEM.70.3.1253-1262.2004