Para alcançar o objetivo geral do projeto, de desenvolvimento de um sistema de diagnose molecular de A. flavus aflatoxigênico, aplicável em HACCP nas cadeias produtivas de grãos no país, estão em andamento análises de seqüências completas dos genes aflR,
aflP e aflQ de amostras de A. flavus isolados de castanha do Brasil, castanha de caju e
amendoim de oito localidades, onde será analisada a diversidade nucleotídica intra- específica e inter-especifica, identificando haplótipos e desequilíbrio de ligação em relação a aflatoxigenicidade, origem geográfica e hospedeiro.
Em conjunto com análises cromatográficas de TLC, todas as amostras serão re- analisadas por meio de HPLC tendo em vista que o HPLC detecta a presença da toxina em níveis mais baixos que o TLC, gerando resultados mais confiáveis em relação à discriminação de isolados aflatoxigênicos e não aflatoxigênicos, já que resultados não detectáveis da toxina por TLC não significam que o organismo seja não produtor, o que pode este estar produzindo a toxina em níveis baixos não detectáveis pelo TLC.
Outros marcadores em potenciais no desenvolvimento de um sistema robusto de PCR específico para A. flavus aflatoxigênico podem ainda incluir RAPD SCAR, ou marcadores derivados de analises por AFLP. Considerando que a via biossintética de aflatoxinas consta hoje com 29 genes diretamente envolvidos na síntese e regulação de produção de aflatoxinas, vários genes, além dos três estudados neste trabalho, podem servir como candidatos. Com a disponibilidade de métodos de seqüenciamento em larga escala como Illumina (Illimina) e SOLID (Applied Biosystems) cada vez mais accessível, onde análises de expressão gênica diferencial entre isolados aflatoxigênicos e não aflatoxigênicos oferecem um caminho novo para identificação de todos os genes envolvidos na síntese e regulação de produção de aflatoxinas.
REFERÊNCIAS BIBLIOGRÁFICAS
ABDULMAWJOOD, A.; ROTH, S.; BUÈ LTE, M. Two methods for construction of internal amplification controls for the detection of Escherichia coli O157 by polymerase chain reaction. Molecular and Cellular Probes, v. 16, p. 335-339. 2002.
ANDERSEN, J.R.; ZEIN, I.; WENZEL, G.; DARNHOFER, B.; EDER, J.; OUZUNOVA, M.; LUBBERSTEDT, T. Characterization of phenylpropanoid pathway genes within European maize (Zea mays L.) inbreds. BioMed Central Plant Biology, v. 8, n. 2, p. 1-14. 2008.
BATISTA, P.P.; SANTOS, J.F.; OLIVEIRA, N.T.; PIRES, A.P.D.; MOTTA, C.M.S.; LIMA, E.A. Genetic characterization of Brazilian strains of Aspergillus flavus using DNA markers.
Genetics and Molecular Research, v. 7, n. 3 p. 706-717. 2008.
BLUHM, B.H.; COUSIN, M.A.; WOLOSHUK, C.P. Multiplex real-time PCR detection of fumonisin-producing and trichothecene-producing groups of Fusarium species. Journal of
Food Protection, v. 67, n. 3, p. 536-543. 2004.
BRADBURY, P.J.; ZHANG, Z.; KROON, D.E.; CASSTEVENS, T.M.; RAMDOSS, Y.; BUCKLER, E.S.; TASSEL: software for association mapping of complex traits in diverse samples. Bioinformatics Applications Note, v. 23, n. 19, p. 2633-2635. 2007.
BRETAGNE, S.; COSTA, J. M.; MARMORAT, K. A.; PORON, F.; CORDONNIER, C.; VIDAUD.; M. & FLEURY, F. J. Detection of Aspergillus species DNA in bronchoalveolar lavage samples by competitive PCR. Journal of Clinical Microbiology, v. 33, n. 5, p. 1164- 1168. 1995.
BRUNS, T.D.; PALMER J.D.; SHUMARD, D.S.; GROSSMAN, L.I.; HUDSPETH, M.E. Mitochondrial DNAs of Suillus: three fold size change in molecules that share a common gene order. Curren. Genetics; v. 13, n. 1, p. 49-56. 1988.
BRUNS, T. D.; VILGALYS, R.; BARNS, S. M.; GONZALEZ, D.; HIBBETT, D. S.; LANE, D. J.; SIMON, L.; STICKEL, S.; SZARO, T. M.; WEISBURG, W. G. & SOGIN, M. L. Evolutionary
ralationships within the fungi: analysis of nuclear small subunit rRNA sequences. Molecular
Phylogenetics and Evolution, v. 1, p. 231-241. 1991.
BRUNS, T. D. & SZARO, T. M. Rate and mode differences between nuclear and mitochondrial small-subunit rRNA genes in mushrooms. Molecular Biology and Evolution, v. 9, p. 836-855. 1992.
BUFFLIER, E.; SUSCA, A.; BAUD, M.; MULÈ, G.; BRENGEL, K.; LOGRIECO, A. Detection of Aspergillus carbonarius and other black aspergilli from grapes by DNA OLISA™ microarray. Food Additives & Contaminants,v. 24, n. 10, p. 1138-1147. 2007.
BULLERMAN, L.B. Significance of mycotoxins to food safety and human health. Journal of
food protection,v. 42, p. 65-86. 1979.
CAI, J.; ZENG, H.; SHIMA, Y.; HATABAYASHI, H.; NAKAGAWA, H.; ITO, Y.; ADACHI, Y.; NAKAJIMA, H.; YABE, K. Involvement of the nadA gene in formation of G-group aflatoxin in
Aspergillus parasiticus. Fungal Genetics and Biology, v. 45, p. 1081-1093. 2008.
CALDAS, E.D.; SILVA, S.C.; OLIVEIRA, J.N. Aflatoxinas e ocratoxinas A em alimentos e riscos para a saúde humana. Revista de Saúde Pública, v. 36, n. 3, p. 319-323. 2002. CALDAS, E.D.; SILVA, A.C. Mycotoxins in corn-based food products consumed in Brazil: an exposure assessment for fumonisins. Journal of Agricultural and Food Chemistry, v. 55, n. 19, p. 7974-7980. 2007.
CARBONE, I.; RAMIREZ-PRADO, J.H.; JAKOBEK, J.L.; HORN, B.W. Gene duplication, modularity and adaptation in the evolution of the aflatoxin gene cluster. BioMed Central
Evolutionary Biology, v. 7, n. 111, p. 1-12. 2007.
CARY, J.W.; EHRLICH, K.C. Aflatoxigenicity in Aspergillus: Molecular genetics, phylogenetic relationships and evolutionary implications. Mycopathologia, v. 162, p. 167–177. 2006.
CARY, J.W.; OBRIAN, G.R.; NIELSEN, D.M.; NIERMAN, W.; COWARD, P.H.; BHATNAGAR, J.Y.; CLEVELAND, T.E.; PAYNE, G.A.; CALVO, A.M. Elucidation of veA-
dependent genes associated with aflatoxina and sclerotial production in Aspergillus flavus by functional genomics. Applied Microbiology Biotechnology, v. 76, p. 1107-1118. 2007. COUNCIL FOR AGRICULTURE SCIENCE AND TECHNOLOGY (CAST). Mycotoxins: Risks in Plant, Animal and Human Systems. Council for Agricultural Science and Technology. 2003.
CAVALCANTI, S.D.B.; LEVI, J.E.; DANTAS, K.C.; MARTINS, J.E.C. Analysis of the genetic polymorphism of paracoccidioides brasiliensis and paracoccidioides cerebriformis “moore” by random amplified polymorphic dna (rapd) and 28s ribosomal dna sequencing - paracoccidioides cerebriformis revisited. Revista do Instituto de Medicina Tropical. São
Paulo, v. 47, n. 3, p. 119-123. 2005.
CENTER FOR INTEGRATED FUNGAL RESEARCH. Disponível em: <http://www.aspergillusflavus.org>. Acesso em: 2008.
CHANG, P. K.; BHATNAGAR, D.; CLEVELAND, T. E & BENNETT, J. W. Sequence variability in homologs of the aflatoxin pathway gene aflR distinguishes species in Aspergillus Section Flavi. Applied and Environmental Microbiology, v. 61, p. 40-43. 1995.
CHANG, P.K.; HORN, B.W.; DORNER, J.W. Sequence breakpoints in the aflatoxina biosynthesis gene cluster and flanking regions in non-aflatoxigenic Aspergillus flavus isolates. Fungal Genetics and Biology, v. 42, p. 914–923. 2005.
CHANG, P.K.; EHRLICH, K.C.; HUA, S.S. Cladal relatedness among Aspergillus oryzae isolates and Aspergillus flavus S and L morphotype isolates. International Journal of Food
Microbiology. v. 108, n. 2, p. 172-177. 2006.
CHAO-ZONG, L.; GUEY-YUH, L.; GWO-FANG, Y. Comparison of the aflR gene sequences of strains in Aspergillus section Flavi. Microbiology, v. 152, p. 161-170. 2006.
CHEN, R.S.; TSAY, J.G.; HUANG, Y.F.; CHIOU, R.Y.Y. Polymerase chain reaction mediated characterization of molds belonging to the Aspergillus flavus group and detection of A.
parasiticus in peanut kernels by multiplex polymerase chain reaction. Journal of Food
CHO, R.J.; MINDRINOS, M.; RICHARDS, D.R.; SAPOLSKY, R.J.; ANDERSON, M.; DRENKARD, E.; DEWDNEY, J.; REUBER, T.; STAMMERS, M.; FEDERSPIEL, N. Genome- wide mapping with biallelic markers in Arabidopsis thaliana. Nature Genetics, v. 23, n. 2, p. 203-201. 1999.
CHOO, B.K.; MOON, B.C.; JI, Y.; KIM, B.B.; CHOI, G.; YOON, T.; KIM, H.K. Development of SCAR Markers for the Discrimination of Three Species of Medicinal Plants, Angelica
decursiva (Peucedanum decursivum), Peucedanum praeruptorum and Anthricus sylvestris,
Based on the Internal Transcribed Spacer (ITS) Sequence and Random Amplified Polymorphic DNA (RAPD). Biological Pharmceutical Bulletin. v. 1, p. 24-30. 2009.
CHU, F.S. Recente Progress on Analytical Techniques for Mycotoxins in Feedstuffs. Journal
of Animal Science, v. 70, p. 3950-3963. 1992.
CHUN, H.S.; KIM, H.J.; OK, H.E.; HWANG, J.; CHUNG, D. Determination of aflatoxin levels in nuts and their products consumed in South Korea. Food Chemistry, v. 102, n. 1, p. 385- 391. 2007.
CLARK-WALKER, G.D.; MCARTHUR, C.R.; SRIPRAKASH, K.S. Order and orientation of genic sequences in circular mitochondrial DNA from Saccharomyces exiguus: implications for evolution of yeast mtDNAs. Journal of Molecular Evolution, v. 19, n. 5, p. 333-41. 1983. COMPANHIA NACIONAL DE ABASTECIMENTO (CONAB). Disponível em: <http://www.conab.gov.br>. Acesso em: jan. 2009.
CORABI-ADELL, C. Biodiversidade do gênero Trichoderma (HYPOCREALES-FUNGI) mediante técnicas moleculares e análises ecofisiográfica. Tese de Doutorado. UNESP. 2004.
CRISEO, G.; BAGNARA, A.; BISIGNANO, G. Differentiation of aflatoxin-producing and nonproducing strains of Aspergillus flavus group. Letters in Applied Microbiology, v. 33, p. 291–295. 2001.
CRISEO, G.; RACCO, C.; ROMEO, O. High genetic variability in non-aflatoxigenic A. flavus strains by using Quadruplex PCR-based assay. International Journal of Food
DA SILVA, J.B.; POZZI, C.R.; MALLOZZI, M.A.; ORTEGA, E.M.; CORRÊA, B. Mycoflora and occurrence of aflatoxin B(1) and fumonisin B(1) during storage of Brazilian sorghum. Journal
of Agricultural and Food Chemistry, v. 48, n. 9, p. 4352-4356. 2000.
DE MELLO, F.R. & SCUSSEL, V.M. Characteristics of in-shell Brazil nuts and their relationship to aflatoxin contamination: criteria for sorting. Journal of Agricultural and Food
Chemistry. v. 55, n. 22, p. 9305-9310. 2007.
DEGOLA, F.; BERNI, E.; DALL’ASTA, C.; SPOTTI, E.; MARCHELLI, R.; FERRERO, I.; RESTIVO, F.M. A multiplex RT-PCR approach to detect aflatoxigenic strains of Aspergillus
flavus. Journal of Applied Microbiology, v. 103, p. 409-417. 2007.
DEGOLA, F.; BERNI, E.; SPOTTI, E.; FERRERO, I.; RESTIVO, F.M. Facing the problem of "false positives": Re-assessment and improvement of a multiplex RT-PCR procedure for the diagnosis of A. flavus mycotoxin producers. International Journal of Food Microbiology, v. 29, n. 3, p. 300-305. 2008.
DING, H.; DING, Z.; LI, H.; CHEN, N. Application of SCAR molecular marker technology in identification of Monascus strains. Zhongguo Zhong Yao Za Zhi, v. 33, n. 4, p. 359-62. 2008.
EDGAR, R.C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research, v. 32, n. 5, p. 1792-1797. 2004.
EDWARDS, S. G.; O’CALLAGHAN, J.; DOBSON, A. D. W. PCR-based detection and quantification of mycotoxigenic fungi. Mycological Research News, v. 106, n. 9, p. 1005-1025. 2002.
EHRLICH, K.C.; MONTALBANO, B.G.; COTTY, P.J. Sequence comparison of aflR from different Aspergillus species provides evidence for variability in regulation of aflatoxin production. Fungal and Genetics Biology. v. 38, n. 1, p. 63-74. 2003.
EHRLICH, K.C.; CHANG, P.; YU, J.; COTTY, P.J. Aflatoxin biosynthesis cluster gene cypA is required for G aflatoxin formation. Applied Environmental Microbiology. v. 70, n. 11, p. 6518-6524. 2004.
EHRLICH, K.C.; MONTALBANO, B.G.; COTTY, P.J. Analysis of single nucleotide polymorphisms in three genes shows evidence for genetic isolation of certain Aspergillus flavus vegetative compatibility groups. FEMS Microbiology Letters, v. 268, n. 2, p. 231-236. 2007.
EMPRESA BRASILEIRA DE PESQUISA AGROPECUÁRIA (EMBRAPA). Disponível em: <www.embrapa.br>. Acesso em: 2008.
FERNS, R.B.; FLETCHER, H.; BRADLEY, S.; MACKINNON, S.; HUNT, C.; TEDDER, R.S. The prospective evaluation of a nested polymerase chain reaction assay for the early detection of Aspergillus infection in patients with leukaemia or undergoing allograft treatment.
British Journal Haematology, v. 119, n. 3, p. 720-725. 2002.
FIGUEIREDO, L.F.A.; CALATAYUD, C.; DUPUITS, C.; BILLOT, C.; RAMI, J.; BRUNEL, D. et al. Phylogeographic evidence of crop neodiversity in sorghum. Genetics, v. 179, n. 2, p. 997-1008. 2008.
FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS (FAO). FAO
Production Year Book, v. 55, p. 118-119. 2001.
FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS (FAO). Disponível em: <http://www.fao.org/inpho/content/compend/text/ch31/ch31_04.htm>. Acesso em: 2003. FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS (FAO). Disponível em: <http://www.fao.org/inpho/content/compend/text/ch31/ch31_04.htm>. Acesso em: 2008. FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS (FAO). Disponível em: <http://www.fao.org/inpho/content/compend/text/ch31/ch31_04.htm>. Acesso em: 2009. FONSECA, H. Micotoxinas. Disponível em:< http://www.micotoxinas.com.br>. 23 janeiro 2008.
FREIRE, F.C.O.; CAVALCANTE, M.J.B.; BEZERRA, J.L. Deterioração fúngica de amêndoas de cajueiro no Nordeste brasileiro. Agrotrópica, v. 8, n. 3, p. 65-68. 1996.
FREIRE, F.C.O.; ZOKAKIEWICZ, Z.; PATERSON, R.R.M. Mycoflora and mycotoxins in Brazilian cashew kernels. Mycophatologia, v. 145, p. 95-103. 1999.
FRISVAD, J.C.; THRANE, U; FILTENBORG, O. Chemical Fungal Taxonomy. International
Mycological Institute. 1998.
FUNGARO, M.P.; VISSOTTO, P.C.; SARTORI, D.; VILAS-BOAS, L. A.; FURLANETO, M.C.; TANIWAKI, M.H. A Molecular Method for Detection of Aspergillus carbonarius in Coffee Beans. Current Microbiology. v. 49, p. 123–127. 2004.
GARDES, M. & BRUNS, T.D. ITS primers with enhanced specificity for basidiomycetes - application to the identification of mycorrhizae and rusts. Molecular Ecology, v. 2, p. 113- 118. 1991.
GARDES, M.; MUELLER, G.M.; FORTIN, J.A.; KROPP, B.R. Mitochondrial DNA polymorphisms in Laccaria bicolor, L. laccata, L. proxima and L. amethystina. Mycological
Research. v. 95, p. 206–216. 1991.
GEISEN, R. Multiplex polymerase chain reaction for the detection of potential aflatoxin and sterigmatocystin producing fungi. Systematic and Applied Microbiology, v. 19, p.388–392. 1996.
GEISEN, R. PCR Methods for the Detection of Mycotoxin – producing Fungi. In: P.D. Bridge, D.K. Arora, C.A. Reddy and R.P. Elander (eds.) Applications of PCR in Mycology, CAB International, p. 243-266. 1998.
GEISEN, R.; CANTOR, M.D.; HANSEN, T.K.; HOLZAPFEL, W.H.; JAKOBSEN, M. Characterization of Penicillium roqueforti strains used as cheese starter cultures by RAPD typing. International Journal of Food Microbiology. v. 65, n. 3, p. 183-191. 2001.
GEISER, D. M.; DORNER, J. W.; HORN, B. W. & TAYLOR, J. W. The phylogenetics of mycotoxin and sclerotium production in Aspergillus flavus and Aspergillus oryzae. Fungal
GEORGIANNA, D.R. & PAYNE, G.A. Genetic regulation of aflatoxina biosynthesis: From gene to genome. Fungal Genetics and Biology, v. 46, p. 113-125. 2008.
GONZALEZ, P.; LABARÈRE, J. Phylogenetic relationships of Pleurotus species according to the sequence and secondary structure of the mitochondrial small-subunit rRNA V4, V6 and V9 domains. Microbiology, v. 146, p. 209-221. 2000.
GONÇALEZ, E.; NOGUEIRA, J.H.; FONSECA, H.; FELICIO, J.D.; PINO, F.A.; CORRÊA, B. Mycobiota and mycotoxins in Brazilian peanut kernels from sowing to harvest. International
Journal of Food MicrobiologyN, v. 123, n. 3, p. 184-190. 2008.
GONZÁLEZ-SALGADO, A.; GONZÁLEZ-JAÉN, T.; VÁZQUEZ, C.; PATIÑO, B. Highly sensitive PCR-based detection method specific for Aspergillus flavus in wheat flour. Food
Additives & Contaminants Part A, Chemistry, Analysis, Control, Exposure & Risk Assessment. v. 25, n. 6, p. 758-764. 2008.
GOPAL, K.; SREENIVASULU, Y.; GOPI, V.; PRASADBABU, G.; KUMAR, T.B.; MADHUSUDHAN, P.; AHEMED, S.K.; PALANIVEL, S.G. Genetic variability and relationships among seventeen Trichoderma isolates to control dry root rot disease using RAPD markers. Z Naturforsch. v. 63, n. 9-10, p. 740-746. 2008.
GRAY, M. W.; CEDERGREN, R.; ABEL, Y. & SANKOFF, D. On the evolutionary origin of the plant mitochondrion and its genome. Proceedings of the National Academy of Sciences.
U. S. A, v. 86, p. 2267-2271. 1989.
GRIFFIN, G.J.; SMITH, E.P. AND ROBINSON, T.J. Population patterns of Aspergillus flavus group and A. niger group in field soil. Soil Biology & Biochemistry, v. 33, p. 253–257. 2001.
GRIMM, C. & GEISEN, R. A PCR-ELISA for the detection of potencial fumonisin producing
Fusarium species. Letters in Applied Microbiology, v. 26, n. 6, p. 456-462. 1998.
GROSSMAN, L.I.; HUDSPETH, M.E.S. Fungal mitochondrial genomes. In: Bennett JW, Lasure LL (eds) Gene manipulations in fungi. Academic, New York London, p 65–103. 1985.
HAMARI, Z.; TÓTH, B.; BEER, Z.; GÁCSER, A.; KUCSERA, J.; PFEIFFER, I. et al. Interpretation of intraspecific variability in mtDNAs of Aspergillus niger strains and rearrangement of their mtDNAs following mitochondrial transmissions. FEMS Microbiology
Letters, v. 221, n. 1, p. 63-71. 2003.
HAUGLAND, R.A.; VESPER, S.J. Method for identifying and quantifying specific fungi and bacteria. US Patent and Trademark Office, Patent #6,387,652, May 14, 2002, 29 pp. 2000. HAYASHI, K.; HASHIMOTO, N.; DAIGEN, M.; ASHIKAWA, I. Development of PCR-based SNP markers for rice blast resistance genes at the Piz locus. Theoretical and Applied
Genetics, v. 108, p. 1212–1220. 2004.
HEGEDUS, D. D.; PFEIFER, T. A.; MULYL, D. S. & KHACHATOURIANS, G. G. Characterization and structure of the mitochondrial small rRNA gene of the entomopathogenic fungus Beauveria bassiana. Genome, v. 41, n. 3, p. 471-476. 1998.
HIRANO, S.; SHIMA, T.; SHIMADA, T. Proportion of aflatoxin B1 contaminated kernels and its concentration in imported peanut samples. Shokuhin Eiseigaku Zasshi, v. 42, n. 4, p. 237-242. 2001.
HOEBEN, P.; CLARK-WALKER, G. D. An approach to yeast classification by mapping mitochondrial DNA from Dekkera/Brettanomyces and Eeniella genera. Current Genetics, v.10, n. 5, p. 371-379. 1986.
HOFSTETTER, V.; CLÉMENÇON, H.; VILGALYS, R. & MONCALVO, J. M. Phylogenetic analyses of the Lyophylleae (Agaricales, Basidiomycota) base on nuclear and mitochondrial rDNA sequences. Mycological Research, v. 106, n. 9, p. 1043-1059. 2002.
HOMECHIN, M. Potencial e emprego de isolados brasileiros de Trichoderma harzianum
Rifai para controle de patógenos de soja (Glycine max (L) Merril). Piracicaba, 1987. 183p.
Tese Doutorado em Agronomia – Departamento. de Fitopatologia, Universidade de São Paulo. 1987.
HOORFAR, J.; MALORNY, B.; ABDULMAWJOOD, A.; COOK, N.; WAGNER, M.; FACH, P. Practical considerations in desing of internal amplification controls for diagnostic PCR assays. Journal of Clinical Microbiology, v. 42, n. 5, p. 1863-1868. 2004.
HUSSEIN, H. S. & BRASEL J. M. Toxicity, metabolism, and impact of mycotoxins on humans and animals. Toxicology, v. 167, n. 2, p. 101-134. 2001. In Arabidopsis thaliana.
Nature Genetics. 1999.
INFORMATIVO SECEX. Castanha de caju: Uma receita para elevar as exportações. Disponível em: <http://www.mdic.gov.br>. Acessado em: 2004.
ISTITUTO NACIONAL DE METROLOGIA, NORMALIZAÇÃO E QUALIDADE INDUSTRIAL (INMETRO). Disponível em:<http://www.inmetro.gov.br>. Acesso em: 2008
INSTITUTO BRASILEIRO DE GEOGRAFIA E ESTATÍSTICA (IBGE). Produção da Extração Vegetal e da Silvicultura 2007. Disponível em: <http://www.ibge.gov.br>. Acesso em: fev. 2009.
INSTITUTO FNP. Agrianual 2008: Anuário da agricultura brasileira, p. 231-234. 2008. INSTITUTO FNP. Agrianual 2009: Anuário da agricultura brasileira, p. 177-180. 2009.
JAIN, N.; SHASANY, A. K.; SINGH, S.; KHANUJA, S. P. S.; KUMAR, S. SCAR markers for correct identification of Phyllanthus amarus, P. fraternus, P. debilis and P. urinaria used in scientific investigations and dry leaf bulk herb trade. Plant Medicine, v. 74, n. 3, p. 296-301. 2008.
JIMENEZ, M.; RODRIGUEZ, S.; MATEO, J. J.; GIL, J. V. & MATEO, R. Characterization of Gibberella fujikuroi complex isolates by fumonisin B1 e B2 analysis and by RAPD and restriction analysis of PCR-amplified internal transcribed spacers of ribosomal DNA.
Systematic and Applied Microbiology, v. 24, n. 1, p. 146. Erratum in Systematic and
Applied Microbiology. 2001. 24(1), p. 146. 2000.
JUHÁSZ, A.; ENGI, H.; PFEIFFER, I.; KUCSERA, J.; VÁGVÖLGYI, C.; HAMARI, Z. Interpretation of mtDNA RFLP variability among Aspergillus tubingensis isolates. Antonie
JURADO, M.; VÁZQUEZ, C.; MARÍN, S.; SANCHIS, V.; TERESA GONZÁLEZ-JAÉN, M. PCR-based strategy to detect contamination with mycotoxigenic Fusarium species in maize.
Systematic and Applied Microbiology, v. 29, n. 8, p. 681-689. 2006.
KAINZ, P. The PCR plateau phase – towards an understanding of its limitations.; Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, v. 1494, n. 1-2, p. 23-27. 2000.
KHOT, P.D.; KO, D.L.; HACKMAN, R.C.; FREDRICKS, D.N. Development and optimization of quantitative PCR for the diagnosis of invasive aspergillosis with bronchoalveolar lavage fluid. BioMed Central Infectious Diseases, v. 8, p. 73. 2008.
KIM, S.; MISRA, A. SNP- Genotyping: Technologies and Biomedical Applications. The
Annual Review of Biomedical Engineering. n. 9, p. 289-320. 2007.
KLICH, M. A. L. H.; TIFFANY, G. KNAPHUS. Ecology of the aspergilli of soils and litter. JW Bennett and MA Klich, eds. Butterworth-Heineman, Boston, 1994.
KRISTENSEN, R.; BERDAL, K.G.; HOLST-JENSEN, A. Simultaneous detection and identification of trichothecene and moniliformin-producing Fusarium species based on multiplex SNP analysis. Journal of Applied Microbiology, v. 102, p. 1071-1081. 2007. KOVACH, O.L. Programa MVSP – version 3.1: A Multivariate Statistical Package for Windows (software). UK., Pentraeth, Waçes: Kovach Computing Services. p. 133. 1999. KULIK, T.; FORDONSKI, G.; PSZCZOLKOWSKA, A.; PLODZIEN, K.; LAPINSKI, M. Development of PCR assay based on ITS2 rDNA polymorphism for the detection and differentiation of Fusarium sporotrichioides. FEMS Microbiology Letters, v. 239, p. 181– 186. 2004.
KUMEDA Y, ASAO T. Single-strand conformation polymorphism analysis of PCR-amplified ribosomal DNA internal transcribed spacers to differentiate species of Aspergillus section Flavi. Applied and Environmental Microbiology, v. 62, n. 8, p. 2947-2952. 1996.
LAHLALI, R.; SERRHINI, M.N.; JIJAKLI, M.H. Studying and modelling the combined effect of temperature and water activity on the growth rate of P. expansum; International Journal of
Food Microbiology, v. 103, n. 3, p. 315-322. 2005.
LEE, C.; LIOU, G.; YUAN, G. Comparison of the aflR gene sequences of strains in
Aspergillussection Flavi. Microbiology, v. 152, p. 161-170. 2006.
LIN, L.; ZHANG, J.; WANG, P.; WANG, Y.; CHEN, J. Thin-layer chromatography of mycotoxins and comparison with other chromatographic methods. Journal of
Chromatography A, v. 815, n. 1, p. 3-20. 1998.
LOURENÇO, A.; DURIGON, E.L.; ZANOTTO, P.; MADEIRA, J.E.G.C.; ALMEIDA, A.P.; CORREA, B. Genetic diversity of environmental Aspergillus flavus strains in the state of São Paula, Brazil by random amplified polymorphic DNA. Mémórias do Instituto Oswaldo Cruz, Rio de Janeiro, v. 102, n. 6, p. 687-692. 2007.
LU, B. C. K. Chromosomes, mitosis and meiosis. In: Fungal genetics: principles and pratices, Bos, C. J., Marcel Dekker, New York, p. 119-176. 1996.
LUBULWA, A.S.G. AND DAVIS, J.S. Estimating the social costs of the impacts of fungi and aflatoxins in maize and peanuts, p. 1017-1042, 1994. In: Stored Product Protection. Proceedings of the 6th International Working Conference on Stored-product Protection. Highley, E, Wright, E J, Banks, H J and Champ, B R (eds). CAB International, Wallingford, UK.
MA, Z. AND MICHAILIDES, T. J. Approaches for eliminating PCR inhibitors and designing PCR primers for the detection of phytopathogenic fungi. Crop Protection, v. 26, n. 2, p. 145- 161. 2006.
MAGAN, N. Mycotoxin contamination of food in Europe: early detection and prevention strategies. Mycopathologia, v. 162, n. 3, p. 245-253. 2006.
MARTIN, J.P. Use of acid rose bengal and streptomycin in the plate method for estimating soil fungi. Soil Science, v.134, p.1528-1529, 1950.
MAYER, Z.; BAGNARA, A.; FARBER, P.; GEISEN, R. Quantification of the copy number of nor-1, a gene of the aflatoxina biosynthetic pathway by real-time PCR, and its correlation to the cfu of Aspergillus flavus in foods. International Journal of Food Microbiology. v. 82, p. 143– 151. 2003.
MEENA, B.; RADHAJEYALAKSHMI, R.; MARIMUTHU, T.; VIDHYASEKARAN, P.; VELAZHAHAN, R. Biological control of groundnut late leaf spot and rust by seed and foliar applications of a power formulation of Pseudomonas fluorescens. Biocontrol Science and
Technology, v. 12, p. 195-204. 2002.
MEGNEGNEAU, B.; DEBETS, F.; HOEKSTRA, R.F. Genetic variability and relatedness in the complex group of black Aspergilli based on random amplification of polymorphic DNA.
Current Genetics, v. 23, n. 4, p. 323-329. 1993.
MIDORIKAWA, G.E.O.; PINHEIRO, M.R.; VIDIGAL, B.S.; ARRUDA, M.C.; COSTA, F.F.; PAPPAS, G.J.Jr.; RIBEIRO, S.G.; FREIRE, F.; MILLER, R.N. Characterization of Aspergillus
flavus strains from Brazilian Brazil nuts and cashew by RAPD and ribosomal DNA analysis.
Letters in Applied Microbiology, v. 47, n. 1, p. 12-18. 2008.
MISHRA, P.K.; FOX, R.T.V.; CULHAM, A. Development of a PCRbased assay for rapid and reliable identification of pathogenic Fusaria. FEMS Microbiology Letters, v. 218, n.329– 332. 2003.
MÖLLER, E.M.; CHELKOWSKI, J.; GEIGER, H.H. Species-specific PCR assays for the fungal pathogens Fusarium moniliforme and Fusarium subglutinans and their application to diagnose maize ear rot disease. Journal of Phytopathology, v. 147, p. 497–508. 1999. MONCALVO, J.M.; DREHMEL, D.; VILGALYS, R. Variation in modes and rates of evolution in nuclear and mitochondrial ribosomal DNA in the mushroom genus Amanita (Agaricales, Basidiomycota): phylogenetic implications. Molecular Phylogenetics Evolution, v. 16, n, 1, p. 48-63. 2000.
MOODY, S.F.; TYLER, B.M. Restriction enzyme analysis of mitochondrial DNA of the
Aspergillus flavus group: A. flavus, A. parasiticus, and A. nomius. Applied and
MORALES-LOREDO-A; GALLARDO-ZAPATA-EA; LEAL-KLEVEZAS-DS; TAMEZ- GUERRA-RS; ROCHA-PENA-MA; MARTINEZ-SORIANO-JPSO. Use of RAPD-PCR for the taxonomic differentiation of Aspergillus spp.OT: Uso de RAPD-PCR para la diferenciacion taxonomica de Aspergillus spp. Revista-Mexicana-de-Fitopatologia, v. 14, n. 2, p. 108-113. 1996.
NATIONAL ADVISORY COMMITTEE ON MICROBIOLOGICAL CRITERIA FOR FOODS. Hazard Analysis and Critical Control Point Principles and Application Guidelines. Journal of
Food Protection, v. 61, n. 9, p. 1246-1259. 1998.
NICOLAISEN, M.; JUSTESEN, A.F.; ULF THRANE, U.; SKOUBOE, P.; HOLMSTRBM, K. An oligonucleotide microarray for the identification and differentiation of trichothecene producing and non-producing Fusarium species occurring on cereal grain. Journal of Microbiological
Methods, v. 62, p. 57– 69. 2005.
NIESSEN, L. PCR-based diagnosis and quantification of mycotoxin-producing fungi.
Advances in Food & Nutrition Research. p. 5481-138. 2008.
O’BRIAN, G.R..; GEORGIANNA, D.R.; WILKINSON, J.R.; YU, J.; ABBAS, H.K. et al. The effect of elevated temperature on gene transcription and aflatoxin biosynthesis. Mycologia, v. 99, p. 232–239. 2007.
O’DONNELL, K. Ribosomal DNA internal transcribed spacers are highly divergente in the phytopathogenic ascomycete Fusarium sambucinum (Gibberella pulicaris).Current
Genetics, v. 22, n. 3, p. 213-220. 1992.
OBRIAN, G.R.; FAKHOURY, A.M.; PAYNE, G.A. Identification of genes differentially expressed during aflatoxin biosynthesis in Aspergillus flavus and Aspergillus parasiticus.
Fungal Genetics and Biology, v. 39, n. 2, p. 118-127. 2003.
PAN, L.; WANG, B.; GUO, Y. RAPD analysis of Aspergilli and its application in brewing industry. Wei Sheng Wu Xue Bao, v. 47, n. 3, p. 533-536. 2007.
PATERSON, R. R. M. & BRIDGE, P. D. Biochemical techniques for filamentous fungi.
PATERSON, R.R.M. Identification and quantification of mycotoxigenic fungi by PCR;
Process Biochemistry, v. 41, p. 1467–1474. 2006.
PATERSON, R.R.M. Internal amplification controls have not been employed in fungal PCR