Characterization of Aspergillus species on Brazil nut from the Brazilian Amazonian region and development of a PCR assay for identification at the genus level

BMC Microbiology

Robert NG Miller³  Daniela MC Bittencourt⁶  Virginia S Alvares²  Rivadalve C Gonçalves²  Renata MLC Mesquita⁴  Rogerio E Hanada⁴  Luis IB Kanzaki³  Jurema do Socorro A Dias¹  Otniel Freitas Silva⁵  Maria de Lourdes M de Sousa⁵  Glaucia EO Midorikawa³ 

[1] EMBRAPA Amapa, Rodovia Juscelino Kubitschek, Km 5, No. 2600, CEP 68.903-419 Caixa Postal 10, Macapa, AP, Brazil;EMBRAPA Acre, BR 364, Km 14, Zona Rural, CEP 69.908.970, Caixa Postal 321, Rio Branco, AC, Brazil;Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Campus Universitário Darcy Ribeiro, CEP 70.910-900 Brasília D.F., Brazil;Instituto Nacional de Pesquisas da Amazônia, Avenida André Araújo, 2936, CEP 69.067-375, Caixa Postal 2223, Manaus, AM, Brazil;EMBRAPA Centro Nacional de Pesquisa de Tecnologia Agroindustrial de Alimentos, Avenida das Americas, 29501, Guaratiba, CEP 23.020-470 Rio de Janeiro, RJ, Brazil;EMBRAPA Amazônia Ocidental, Rodovia AM-010, Km 29, CEP 69.011-970, Caixa Postal 319, Manaus, AM, Brazil

关键词: PCR-RFLP;    Mitochondrial small subunit ribosomal RNA gene;    Mycotoxin;    Aspergillus section Flavi;   

Others  :  1140994
DOI  :  10.1186/1471-2180-14-138

 received in 2013-12-20, accepted in 2014-05-23,  发布年份 2014

【 摘 要 】

Background

Brazil nut is a protein-rich extractivist tree crop in the Amazon region. Fungal contamination of shells and kernel material frequently includes the presence of aflatoxigenic Aspergillus species from the section Flavi. Aflatoxins are polyketide secondary metabolites, which are hepatotoxic carcinogens in mammals. The objectives of this study were to identify Aspergillus species occurring on Brazil nut grown in different states in the Brazilian Amazon region and develop a specific PCR method for collective identification of member species of the genus Aspergillus.

Results

Polyphasic identification of 137 Aspergillus strains isolated from Brazil nut shell material from cooperatives across the Brazilian Amazon states of Acre, Amapá and Amazonas revealed five species, with Aspergillus section Flavi species A. nomius and A. flavus the most abundant. PCR primers ASP_GEN_MTSSU_F1 and ASP_GEN_MTSSU_R1 were designed for the genus Aspergillus, targeting a portion of the mitochondrial small subunit ribosomal RNA gene. Primer specificity was validated through both electronic PCR against target gene sequences at Genbank and in PCR reactions against DNA from Aspergillus species and other fungal genera common on Brazil nut. Collective differentiation of the observed section Flavi species A. flavus, A. nomius and A. tamarii from other Aspergillus species was possible on the basis of RFLP polymorphism.

Conclusions

Given the abundance of Aspergillus section Flavi species A. nomius and A. flavus observed on Brazil nut, and associated risk of mycotoxin accumulation, simple identification methods for such mycotoxigenic species are of importance for Hazard Analysis Critical Control Point system implementation. The assay for the genus Aspergillus represents progress towards specific PCR identification and detection of mycotoxigenic species.

【 授权许可】

   
2014 Midorikawa et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 9.

【 参考文献 】

• [1]JECFA. Joint Expert Committee on Food Additives: Evaluation of certain food additives and contaminants. Forty-sixth Report of the Joint FAO/WHO Expert Committee on Food Additives; 1996. WHO Technical Report Series 868. Geneva: World Health Organization; 1997.
• [2]Council For Agriculture Science And Technology (Cast: Mycotoxins: Risks in Plant, Animal and Human Systems. Ames, Iowa: Council for Agricultural Science and Technology; 2003.
• [3]Holzapfel CW: The isolation and structure of cyclopiazonic acid, a toxic metabolite of Penicillium cyclopium Westling. Tetrahedron 1968, 24:2101-2119.
• [4]Rao BL, Husain A: Presence of cyclopiazonic acid in kodo millet (Paspalum scrobiculation) causing “kodua poisoning” in man and its production by associated fungi. Mycopathologia 1985, 89:177-180.
• [5]Rodrigues P, Venâncio A, Kozakiewicz Z, Lima N: A polyphasic approach to the identification of aflatoxigenic and non-aflatoxigenic strains of Aspergillus section Flavi isolated from Portuguese almonds. Int J Food Micro 2009, 129:187-193.
• [6]Samson RA, Varga J: What is a species in Aspergillus? Med Mycol 2009, 47(Suppl 1):13-20.
• [7]Varga J, Frisvad JC, Samson RA: Two new aflatoxin producing species, and an overview of Aspergillus section Flavi. Stud Mycol 2011, 69:57-80.
• [8]Gonçalves SS, Stchigel AM, Cano JF, Godoy-Martinez PC, Colombo AL, Guarro J: Aspergillus novoparasiticus: a new clinical species of the section Flavi. Med Mycol 2012, 50:152-160.
• [9]Soares C, Rodrigue P, Peterson SW, Lima N, Venâncio A: Three new species of Aspergillus section Flavi isolated from almonds and maize in Portugal. Mycologia 2012, 104:682-697.
• [10]Taniwaki MH, Pitt JI, Iamanaka BT, Sartori D, Copetti MV, Balajee A, Fungaro MH, Frisvad JC: Aspergillus bertholletius sp. nov. from Brazil nuts. PLoS One 2012, 7(8):e42480.
• [11]Freitas-Silva O, Venancio A: Brazil nuts: Benefits and risks associated with the contamination by fungi and mycotoxins. Food Res Int 2011, 44:1434-1440.
• [12]Reis TA, Oliveira TD, Baquião AC, Gonçalves SS, Zorzete P, Corrêa B: Mycobiota and mycotoxins in Brazil nut samples from different states of the Brazilian Amazon region. Int J Food Microbiol 2012, 159:61-68.
• [13]Olsen M, Johnson P, Moller T, Paladino R, Lindblad M: Aspergillus nomius, an important aflatoxin producer in Brazil nuts? World Mycotoxin J 2008, 1:123-126.
• [14]Baquião AC, Zorzete P, Reis TA, Assunção E, Vergueiro S, Correa B: Mycoflora and mycotoxins in field samples of Brazil nuts. Food Control 2012, 28:224-229.
• [15]Gonçalves JS, Ferracin LM, Vieira MLC, Iamanaka BT, Taniwaki MH, Fungaro MHP: Molecular analysis of Aspergillus section Flavi isolated from Brazil nuts. World J Microb Biot 2012, 28:1817-1825.
• [16]Rodrigues P, Soares C, Kozakiewicz Z, Paterson R, Lima N, Venancio A: Identification and characterization of Aspergillus flavus and aflatoxins. In Communicating Current Research and Educational Topics and Trends in Applied Microbiology. Edited by Méndez-Villas A. Badajoz: Formatex; 2007:527-534.
• [17]El Khoury A, Atoui A, Rizk T, Lteif R, Kallassy M, Lebrihi A: Differentiation between Aspergillus flavus and Aspergillus parasiticus from Pure Culture and Aflatoxin-Contaminated Grapes Using PCR-RFLP Analysis of aflR-aflJ Intergenic Spacer. J Food Sci 2011, 76:M247-M253.
• [18]Montiel D, Dickinson MJ, Lee HA, Dyer PS, Jeenes DJ, Roberts IN, James S, Fuller LJ, Matsuchima K, Archer DB: Genetic differentiation of the Aspergillus section Flavi complex using AFLP fingerprints. Mycol Res 2003, 107:1427-1434.
• [19]Lee CZ, Liou GY, Yuan GF: Comparison of the aflR gene sequences of strains in Aspergillus section Flavi. Microbiology 2006, 152:161-170.
• [20]Hinrikson HP, Hurst SF, Lott TJ, Warnock DW, Morrison CJ: Assessment of ribosomal large-subunit D1-D2, internal transcribed spacer 1, and internal transcribed spacer 2 regions as targets for molecular identification of medically important Aspergillus species. J Clin Microbiol 2005, 43:2092-2103.
• [21]Samson RA, Hong SB, Frisvad JC: Old and new concepts of species differentiation in Aspergillus. Med Mycol 2006, 44:133-148.
• [22]Carbone I, Kohn LM: A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia 1999, 91:553-556.
• [23]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, 61:1323-1330.
• [24]Pildain MB, Frisvad JC, Vaamonde G, Cabral D, Varga J, Samson RA: Two novel aflatoxin-producing Aspergillus species from Argentinean peanuts. Int J Syst Evol Micr 2008, 58:725-735.
• [25]Godet M, Munaut F: Molecular strategy for identification in Aspergillus section Flavi. FEMS Microbiol Lett 2010, 304:157-168.
• [26]Shapira R, Paster N, Eyal O, Menasherov M, Mett A, Salomon R: Detection of aflatoxigenic molds in grains by PCR. Appl Environ Microbiol 1996, 62:3270-3273.
• [27]Luo J, Taniwaki MH, Iamanaka BT, Vogel RF, Niessen L: Application of loop-mediated isothermal amplification assays for direct identification of pure cultures of Aspergillus flavus, A. nomius, and A. caelatus and for their rapid detection in shelled Brazil nuts. Int J Food Microbiol 2014, 172:5-12.
• [28]Codex: Hazard analysis and critical control point (HACCP) system and guidelines for its application. ANNEX to Recommended International Code of Practice/General Principles of Food Hygiene. CAC/RCP 1–1969, Rev 4. FAO/WHO Codex Alimentarius Commission. Rome: Food and Agriculture Organization of the United Nations, World Health Organization; 2003.
• [29]Costa AKF, Freire FCO, Icaro GPV, Andrade JA, Mendes FNP: Fungos associados a Castanha-do-Brasil (Bertholleti excels Humb. & Bompl) e o amendoim (Arachis hypogaea L.) comercializados em Fortaleza (Ceara). Rev Ciênc Agron 2009, 40:455-460.
• [30]Radstrom P, Lofstrom C, Lovenklev M, Knutsson R, Wolffs P: 2003. Strategies for overcoming PCR inhibition. In PCR Primer: A Laboratory Manual. 2nd edition. Edited by Diefenbach CW, Dveksler GS. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press; 2003:149-161.
• [31]Ito Y, Peterson SW, Wicklow D, Goto T: Aspergillus pseudotamarii, a new aflatoxin producing species in Aspergillus section Flavi. Mycol Res 2001, 105:233-239.
• [32]Calderari TO, Lamanaka BT, Frisvad JC, Pitt JI, Sartori D, Pereira JL, Fungaro MH, Taniwaki MH: The biodiversity of Aspergillus section Flavi in brazil nuts: from rainforest to consumer. Int J Food Microbiol 2013, 160:267-272.
• [33]Dorner JW, Cole RJ, Diener UL: The relationship of Aspergillus flavus and Aspergillus parasiticus with reference to production of aflatoxins and cyclopiazonic acid. Mycopathologia 1984, 87:13-15.
• [34]Dorner JW: Production of cyclopiazonic acid by Aspergillus tamarii Kita. Appl Environ Microbiol 1983, 46:1435-1437.
• [35]Vinokurova NG, Ivanushkina NE, Khmel’nitskaia II, Arinbasarov MU: Synthesis of alpha-cyclopiazonic acid by fungi of the genus Aspergillus. Prikl Biokhim Mikrobiol 2007, 43:486-489.
• [36]FAO: Manual on the application of the HACCP system in mycotoxin prevention and control. FAO Food and Nutrition Paper 73; 2003. http://www.fao.org/docrep/005/y1390e/y1390e00.htm webcite [18/12/13]
• [37]Lima AM, Gonçalves EC, Andrade SS, Barbosa MSR, Barroso KFP, de Sousa MB, Borges L, Vieira JLF, Teixeira FM: Critical points of Brazil nuts: a beginning for food safety, quality control and Amazon sustainability. J Sci Food Agric 2012, 93:736-740.
• [38]Bruns TD, White TJ, Taylor JW: Fungal Molecular Systematics. Annu Rev Ecol Syst 1991, 22:525-564.
• [39]Xu J, Singh RS: The inheritance of organelle genes and genomes: patterns and mechanisms. Genome 2005, 48:951-958.
• [40]Quirk JT, Kupinski JM: Interspecific mitochondrial DNA restriction fragment length polymorphisms in Aspergillus section Flavi. Mycologia 2002, 94:1078-1086.
• [41]Juhász Á, Engi H, Pfeiffer I, Kucsera J, Vágvolgyi C, Hamari Z: Interpretation of mtDNA RFLP variability among Aspergillus tubingensis isolates. Antonie Van Leeuwenhoek 2007, 91:209-216.
• [42]Klich MA, Mullaney EJ: DNA restriction enzyme fragment polymorphism as a tool for rapid differentiation of Aspergillus flavus from Aspergillus oryzae. Exp Mycol 1987, 11:170-175.
• [43]Tominaga M, Lee Y-H, Hayashi R, Suzuki Y, Yamada O, Sakamoto K, Gotoh K, Akita O: Molecular analysis of an inactive aflatoxin biosynthesis gene cluster in Aspergillus oryzae RIB strains. Appl Environ Microbiol 2006, 72:484-490.
• [44]Yuan GF, Liu CS, Chen CC: Differentiation of Aspergillus parasiticus from Aspergillus sojae by random amplification of polymorphic DNA. Appl Environ Microbiol 1995, 61:2384-2387.
• [45]Freire FC, Kozakiewicz Z, Paterson RRM: Mycoflora and mycotoxins in Brazilian black pepper, white pepper and Brazil nuts. Mycopathologia 2000, 149:13-19.
• [46]Pitt JI, Hocking AD: Fungi and Food Spoilage. 3rd edition. New York: Springer; 2009.
• [47]Schmidt-Heydt M, Abdel-Hadi A, Magan N, Geisen R: Complex regulation of the aflatoxin biosynthesis gene cluster of Aspergillus flavus in relation to various combinations of water activity and temperature. Int J Food Microbiol 2009, 135:231-237.
• [48]Raeder U, Broda P: Rapid preparation of DNA from filamentous fungi. Lett Appl Microbiol 1985, 1:17-20.
• [49]White TJ, Bruns T, Lee S, Taylor J: Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR protocols: A Guide to Methods and Applications. Edited by Innis MA, Gelgard DH, Sninsky JJ, White TJ. New York: Academic Press; 1990:315-322.
• [50]Hong SB, Cho HS, Shin HD, Frisvad JC, Samson RA: Novel Neosartorya species isolated from soil in Korea. Int J Syst Evol Microbiol 2006, 56:477-486.
• [51]Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997, 25:3389-3402.
• [52]Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Res 1994, 22:4673-4680.
• [53]Rozen S, Skaletsky HJ: Primer3 on the WWW for general users and for biologist programmers. In Bioinformatics Methods and Protocols; Methods in Molecular Biology. Edited by Krawetz S, Misener S. New Jersey: Humana Press; 2000:365-386.
• [54]Joardar V, Abrams NF, Hostetler J, Paukstelis PJ, Pakala S, Pakala SB, Zafar N, Abolude OO, Payne G, Andrianopoulos A, Denning DW, Nierman WC: Sequencing of mitochondrial genomes of nine Aspergillus and Penicillium species identifies mobile introns and accessory genes as main sources of genome size variability. BMC Genomics 2012, 13:698. BioMed Central Full Text