Characterization of Iranian Nonaflatoxigenic Strains of Aspergillus flavus Based on Microsatellite-primed PCR

Document Type: Original article


1 Plant Protection department faculty of Agriculture Ferdowsi university mashhad Iran

2 Department of Plant Protection, Faculty of Agriculture, Ferdowsi University, Mashhad

3 Department of Agricultural Biotechnology, Faculty of Agriculture, Ferdowsi University, Mashhad, Iran

4 Department of plant Protection faculty of agriculture Ferdowsi University


Out of fifty-two Iranian nonaflatoxigenic strains of Aspergillus flavus, which were collected from various substrates (soil and kernel) and sources (peanut, corn and pistachio), fifteen representatives were selected according to their different geographical origins (six provinces: Guilan and Golestan, Ardebil, Fars, Kerman and Semnan) and vegetative compatibility groups (VCGs, IR1 to IR15) for microsatellite-primed PCR analysis. Two inter-simple sequence repeat (ISSR) primers AFMPP and AFM13 were used to determine the polymorphism and the relationship among strain isolates. The A. flavus isolates were identified by their morphologies and their identities were confirmed by PCR amplification using the specific primer pair ITS1 and ITS4. The results revealed variations in the percentages of polymorphisms. In ISSR analysis, primers AFMPP and AFM13 generated a total of 18 and 23 amplicons among the fungal strains, which 12 (66.7%) and 22 (95.7%) were polymorphic, respectively. Cluster analysis of ISSR data was carried out by using 1 D DNA gel image analysis. The two dendrograms obtained through these markers showed six different clustering of testing nonaflatoxigenic A. flavus L strains, but we noted that some clusters were different in some cases. The microsatellite-primed PCR data revealed that the Iranian nonaflatoxigenic isolates of A. flavus were not clustered based on their origins and sources. This study is the first to characterize Iranian nonaflatoxigenic isolates of A. flavus using ISSR markers.


1. Frisvad JC, Thrane U, Samson R. In: Dijksterhuis J, Samson RA. (Ed.), Food Mycology: A Multifaceted  Approach to Fungi and Food, CRC Press, Taylor and Francis Group, Boca Raton, 2007; pp.135–140.

2. Robens J, Cardwell FK. The cost of mycotoxin managment to the USA: Management of aflatoxin in the United States. J Toxicol 2003;22:139-152.

3. IARC (International Agency for Research on Cancer). World Health Organization, International Agency for Research on Cancer, Lyon, France, 1993; 56:571.

4. Manonmani HK, Anand S, Chandrashekar A, Rati ER. Process Biochem 2005;                 40:2859-2864.

5. Oktay HI, Heperkan D, Yelboga E, Karaguler NG. Aspergillus flavus – primary causative agent of aflatoxins in dried figs. Mycotaxon 2011;115:425-433.

6. Egel DS, Cotty PJ, Elias KS. Relationships among isolates of Aspergillus sect. Flavi that vary in aflatoxin production. Phytopathol 1994;84:906-912.

7. Gams W, Christensen, M, Onions AH, Pitt, JI, Samson RA. Infragenetic taxa of Aspergillus. In Advances in Penicillium and Aspergillus Systematics, ed. by Samson RA, Pitt JI, Plenum Press, New York, 1985; pp. 55-62.

8. Paplomatas EG. Molecular diagnostics for soil-borne fungal pathogens. Phytopathol Mediterr 2004;43:213-220.

9. Bretagne S, Costa JM, Marmorat-Khuong A, Poron F, Cordonnier C, Vidaud M, Fleury-Feith J. Detection of Aspergillus species DNA in bronchoalveolar lavage samples by competitive PCR. J Clin Microbiol 1995;33:1164-1168.

10. Einsele H, Hebart H, Roller G, Loffler J, Rothenhofer I, Muller CA, Bowden RA, van Burik J, Engelhard D, Kanz L, Schumacher U. Detection and identification of fungal pathogens in blood by using molecular probes. J Clin Microbiol 1997;35: 1353-1360.

11. Yamakami Y, Hashimoto A, Tokimatsu I, Nasu M. PCR detection of DNA specific for Aspergillusspecies in serum of patients with invasive Aspergillosis. J Clin Microbiol 1996;34:2464-2468.

12. O´Donnell K. Ribosomal DNA internal transcribed spacers are highly divergent in the phytopathogenic ascomycete Fusarium sambucinum (Gibberella pulicaris). Curr Genet 1992;22:213-220.

13.Bruns TD, White TJ, Talyor JW. Fungal molecular systematics. Annu Rev Ecol Sys 1991;22:525-564.

14. Beckmann JS. Oligonucleotide polymorphisms: A new tool for genomic genetics. Biotechnology 1988;6:161-164. 

15. Frankham R, Ballou JD, Briscoe DA. Introduction to Conservation Genetics. Cambrige University Press, Cambridge, UK. 2002

16. Louis M, Louis L, Simor AE. The role of DNA amplification technology in the diagnosis of infectious diseases. Can Medical Assoc J 2000;163:301-309.

17. Reischl U, Lohmann CP. [Polymerase chain reaction (PCR) and its possible applications in diagnosis of infection in ophthalmology.] Klin Monatsbl Augenheilkd 1997;211:227-234. [in German]

18. Morgante  M, Hanafer M, Powell W. Microsatellites are preferentially associated with nonrepetitive DNA in plant genomes. Nat Genet2002;30:194-200.

19. Kurtzman CP, Smiley MJ, Robnett CJ, Wicklow DT. DNA relatedness among wild and domesticated species in the Aspergillus flavus group. Mycologia 1986;78:955-959.

20. Tautz D, Renz M. Simple sequences are ubiquitous repetitive components of eukaryotic genomes. Nucleic Acids Res 1984;12:4127-4138.

21. Klaassen CH. MLST versus microsatellites for typing Aspergillus fumigates isolates. Med Mycol 2009;47:27-33.

22. BallouxF, Lugon-Moulin N. The estimation of population differentiation with microsatellite markers. Mol Ecol 2002;11:155-165.

23. Bornet B, Muller C, Paulus F, Branchard M. Highly informative nature of inter simple sequence repeat (ISSR) sequence samplified using tri- and tetra-nucleotide primers from DNA of cauliflower( Brassica oleracea  var. botrytis L.) Genome 2002;45:890-896.

24. 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. (Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds). Academic Press, New York, USA: 1990; pp. 315-322.

25. Chen Z-Y, Brown RL, Damann KE, Cleveland TE. Identification of unique relevated levels of kernel proteins in aflatoxin-resistant maize genotypes through proteome analysis. Phytopathology 2002;92:1084-1094.

26. Criseo G, Bagnara A, Bisignano G. Differentiation of aflatoxin-producing and non-producing strains of Aspergillus flavus group. Lett Appl Microbiol 2001;33:                 291-295.

27. Prabha TR, Revathi1 K, Vinod MS, Shanthakumar SP, Bernard P. A simple method for total genomic DNA extraction from water moulds. Curr Sci 2012;104:345-347.

28. Batista PP, Santos JF, Oliveira NT, Pires APD, Motta CMS, Luna-Alves LimaEA. Genetic characterization of Brazilian strains of Aspergillus flavus using DNA markers. Genet Mol Res 2008;7:706-717.

29. Tran-Dinh N, Carter D. Characterization of microsatellite loci in the aflatoxigenic fungi Aspergillus flavus and Aspergillus parasiticus. Mol Ecol 2000;9:2170–2172.

30. Naik AS, Taware SD. Cytogenetic diversity analysis of Coix species using ISSR markers. Biosci.Biotech Res Asia 2009;6:647-652.

31. Farr DF, Castlebury LA, Rossman AY. Morphological and molecular characterization of Phomopsis vaccinii and additional isolates of Phomopsis from blueberry and cranberry in the eastern US. Mycologia 2002;94:494-504.

32. Rehner SA, Uecker FA. Nuclear ribosomal internal transcribed spacer phylogeny and host diversity in the Coelomycete Phomopsis. Can J Bot 1994;72:1666-1674.

33. Yin Y, Lou T, Yan L, Michailides TJ, Ma Z. Molecular characterization of toxigenic and atoxigenic Aspergillus flavus isolates, collected from peanut fields in China. J Appl Microbiol 2009;107:857-865.

34. Gupta M, Chyi YS, Romero-Severson J, Owen JL. Amplification of DNA markers from evolutionary diverse genomes using single primers of simple sequence repeates. Theoretical Appl Genet 1994;89:998-1006.

35. Meyer W, Mitchell TG, Freedman EZ, Vilgalys R. Hybridisation probes for conventional DNA fingerprinting used as single primers in the polymerase chain reaction to distinguish strains of Cryptococcus neoformans. J Clin Microbiol 1993; 31:2274-2280.

36. Wu KS, Jones R, Danneberger L, Scolnik PA. Detection of microsatellite polymorphisms without cloning. Nucleic Acids Res 1994;22:3257-3258.

37. Archibald JK, Crawford DJ, Santos-Guerra A, Mort ME. The utility of automated analysis of inter-simple sequence repeat (ISSR) loci for resolving relationships in the Canary Island species of Tolpis (Asteraceae). Am J Bot 2006;93:1154-1162.

38. Clausing G, Vickers K, Kadereit W. Historical biogeography in a linear system: genetic variation of Sea Rocket (Cakile maritima) and Sea Holly (Eryngium maritimum) along European coasts. Mol Ecol 2001;9:1823-1833.

39. Reddy MP, Sarla N, Siddiq EA. Inter simple sequence repeat (ISSR) polymorphism and its application in plant breeding. Euphytica 2002;128:9-17.

40. Rogers D.What is a genetic marker? Why we care about genetics.  2006; Vol 5.

41. Ziekiewicz E, Rafalski A, Labuda A. Genome fingerprinting by simple sequence repeat (SSR) anchored polymerase chain reaction amplification.Genomics 1994; 20:178-183.

42. Joshi SP, Gupta VS, Agganoal RK, Rangekar PK, Brar DS. Genetic diversity and phylogenetic relationship as revealed by ISSR polymorphism in the genus Oryza. Theo Appl Genet 2000;100:1311-1320.

43. Hatti AD, Taware SD, Taware AS, Pangrikar PP. Genetic diversity of toxigenic and non-toxigenic Aspergillus flavus strains using ISSR markers. Int J Curr Res 2010;        5:61-66.

44. Hadrich I, Makni F, Sellami H, Cheikhrouhou F, Sellami A, Bouaziz H, Hdiji S, Elloumi M, Ayadi A. Invasive aspergillosis: epidemiology and environmental study in haematology patients (Sfax, Tunisia). Mycoses 2010b;53:443-447