Analysis of Molecular Variability Among Isolates of Aspergillus Flavus by PCR-RFLP of the its Regions of rDNA

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Analysis of Molecular Variability Among Isolates of Aspergillus Flavus by PCR-RFLP of the its Regions of rDNA

A total of seventeen isolates of Aspergillus flavus from maize were collected from different agro-ecological zones of Tamil Nadu, India. The isolates were tested for their ability to produce aflatoxin B1 (AFB1) in vitro by indirect competitive enzyme-linked immunosorbent assay (ELISA). The amount of AFBM1 produced by the isolates of A. flavus ranged from 1.9 to 206.6 ng/ml. Among the various isolates of A. flavus, the isolate AFM46 produced the highest amount of AFB1. DNA was extracted from A. flavus isolates and their molecular variability was investigated by using restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified internal transcribed spacer (ITS) regions of ribosomal DNA. PCR amplification with ITS1 and ITS4 primers resulted in the amplification of a product of approximately 600 bp. Digestion of the PCR products with the restriction enzymes EcoRI, HaeIII and TaqI produced fragments of different sizes. Analysis of the genetic coefficient matrix derived from the scores of RFLP profiles showed that minimum and maximum per cent similarities among the tested A. flavus strains ranged from 0 to 88%. Cluster analysis using the unweighted pair-group method with arithmetic average (UPGMA) clearly separated the isolates into five groups (group I-V) confirming the genetic diversity among the A. flavus isolates from maize.

Appiah A. A., Flood J., Archer S. A., Bridge P. D. 2004. Molecular analysis of the major Phytophthora species on cocoa. Plant Pathol. 53: 209-219.

Bayman P., Cotty P. 1993. Genetic diversity in Aspergillus flavus: association with aflatoxin production and morphology. Can. J. Bot. 71 (1): 23-31.

Bennett J. W., Christensen S. B. 1983. New perspective of aflatoxin biosynthesis. Adv. Appl. Microbiol. 29: 53-92.

Bruns T. D., White T. J., Taylor J. W. 1991. Fungal molecular systematics. Annu. Rev. Ecol. Syst. 22: 525-564.

Cooke D. E. L., Duncan J. M. 1997. Phylogenetic analysis of Phytophthora species based on ITS1 and ITS2 sequences of ribosomal DNA. Mycol. Res. 101 (6): 667-677.

Cooke D. E. L., Kennedy D. M., Guy D. C., Russell J., Unkle S. E., Duncan J. M. 1996. Relatedness of group I species of Phytophthora as assessed by random amplified polymorphic DNA (RAPDs) and sequences of ribosomal DNA. Mycol. Res. 100 (3): 297-300.

Crawford A. R., Bassam B. J., Drenth A., MacLean D. J., Irwin J. A. G. 1996. Evolutionary relationships among Phytophthora species deduced from rDNA sequence analysis. Mycol. Res. 100 (4): 437-443.

Egel D. S., Cotty P. J., Elias K. S. 1994. Relationships among isolates of Aspergillus sect. Flavai that vary in aflatoxin production. Phytopathology 84 (9): 906-912.

Fatehi J., Bridge P. D. 1998. Detection of multiple rRNA-ITS regions in isolates of Ascochyta. Mycol. Res. 102 (6): 762-766.

Goodwin S. B., Zismann V. L. 2001. Phylogenetic analyses of the ITS region of ribosomal DNA reveal that Septoria passerinii from barley is closely related to the wheat pathogen Mycosphaerella graminicola. Mycologia 93 (5): 934-946.

Harlton C. E., Levesque C. A., Punja Z. K. 1995. Genetic diversity in Sclerotium (Athelia) rolfsii and related species. Mol. Plant Pathol. 85 (10): 1269-1281.

Henry T., Iwen P. C., Hinrichs S. H. 2000. Identification of Aspergillus species using Internal transcribed spacer regions 1 and 2. J. Clin. Microbiol. 38 (4): 1510-1515.

IARC. 1993. IARC-WHO Monographs on the Evaluation of Carcinogenic Risks to Humans. Vol. 56: "Some Naturally Occurring Substances: Food Items and Constituents, Heterocyclic Aromatic Amines and Mycotoxins. Int. Agency for Research on Cancer World Health Organization, 599 pp.

Karthikeyan M., Sandosskumar R., Mathiyazhagan S., Mohankumar M., Valluvaparidasan V., Kumar S., Velazhahan R. 2009. Genetic variability and aflatoxigenic potential of Aspergillus flavus isolates from maize. Arch. Phytopathol. Plant Protect. 42 (1): 83-91.

Khoodoo M. H. R., Jaufeerally-Fakim Y. 2004. RAPD-PCR fingerprinting and southern analysis of Xanthomonas axonopodis pv. dieffenbachiae strains isolated from different aroid hosts and locations. Plant Dis. 88 (9): 980-988.

Levy L., Castlebury L.A, Carris L. M., Meyer R. J., Pimentel G. 2001. Internal transcribed spacer sequence-based phylogeny and polymerase chain reaction-restriction fragment length polymorphism differentiation of Tilletia walkeri and T. indica. Mycology 91 (10): 935-940.

Liu D., Coloe S., Baird R., Pederson J. 2000. Rapid mini preparation of fungal DNA for PCR. J. Clin. Microbiol. 38, p. 471.

Lourenço A., Durigon E. L., Zanotto P., Madeira J. E. G. C., De Almeida A. P., Correa B. 2007. Genetic diversity of environmental Aspergillus flavus strains in the state of São Paulo, Brazil by random amplified polymorphic DNA. Memorias do Instituto Oswaldo Cruz 102: 687-692.

O'Donnell K., Cigelik E., Nirenberg H. I. 1998. Molecular systematics and phylogeography of the Gibberella fujikuroi species complex. Mycologia 90 (3): 465-493.

Peterson S. W. 1991. Phylogenetic analysis of Fusarium species using ribosomal RNA sequence comparisons. Phytopathology 81 (9): 1051-1054.

Reddy S. V., Mayi D. K., Reddy M. U., Thirumala Devi K., Reddy D. V. 2001. Aflatoxins B1 in different grades of chillies (Capsicum annum L.) in India as determined by indirect competitive ELISA. Food Additiv. Contam. 18 (6): 553-558.

Salazar O., Schneider J. H. M., Julian M. C., Keijer J., Rubio V. 1999. Phylogenetic subgrouping of Rhizoctonia solani AG2 isolates based on ribosomal ITS sequences. Mycologia 91 (3): 459-467.

Seifert K. A., Wingfield B. D., Wingfield M. J. 1995. A critique of DNA sequence analysis in the taxonomy of filamentous ascomycetes and ascomycetous anamorphs. Can. J. Bot. 73 (1): 760-767.

Singh K., Frisvad J. C., Thrane U., Mathur S. B. 1991. An Illustrated Manual on Identification of Some Seed-Borne Aspergilli, Fusaria, Penicillia and Their Mycotoxins. Danish Government Institute of Seed Pathology for Developing Countries, Denmark, 133 pp.

Toth B., Mesterhazy A., Nicholson P., Teren J., Varga J. 2004. Mycotoxin production and molecular variability of European and American isolates of Fusarium culmorum. Eur. J. Plant Pathol. 110 (5-6): 587-599.

Tran-Dinh N., Pitt J. I., Carter D. A. 1999. Molecular genotype analysis of natural toxigenic and nontoxigenic isolates of Aspergillus flavus and Aspergillus parasiticus. Mycol. Res. 103 (11): 1485-1490.

van Egmond H. P. 1995. Mycotoxins: regulations, quality, assurance and reference materials. Food Additiv. Contam. 12 (3): 321-330.

White T. J., Bruns T., Lee S., Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. p. 315-322. In: "PCR Protocols: A Guide to Methods and Applications" (M. A. Innis, D. H. Gelfand, J. J. Sninsky, T. J. White, eds.). San Diego, CA: Academic Press

Zhang Z. G., Zhang J. Y., Zheng X. B., Yang Y. W., Ko W. H. 2004. Molecular distinctions between Phytophthora capsici and P. tropicalis based on ITS sequences of ribosomal DNA. J. Phytopathol. 152 (6): 358-364.

Journal of Plant Protection Research

The Journal of Polish Society of Plant Protection, Committee of Plant Protection; Polish Academy of Sciences, Institute of Plant Protection – National Research Institute

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