Adeela HUSSAİN, Awais AFZAL, Muhammad IRFAN, Kauser Abdulla MALİK

Molecular Detection of Aflatoxin Producing Strains of Aspergillus Flavus from Peanut (Arachis Hypogaea)

Aflatoxins are the potential carcinogens produced as secondary metabolites by Aspergillus flavus. They have the ability to contaminate large number of food which ultimately affect the human population. Malt extract agar was selected for the growth of control stains of fungus. The aim of the study was to develop a reliable and quick method for the detection of aflatoxin producing strains in peanuts by using molecular approaches. Total 80 samples of infected peanuts were collected from four different cities of Punjab and checked for their aflatoxin contamination. For aflatoxin detection, three target genes nor1, ver1 and aflR were selected which was involved in the aflatoxin biosynthesis. In all examined cases, 24 out of 80 (30%) samples successfully amplified all three genes indicating aflatoxigenic activity. Discrimination between aflatoxigenic and nonaflatoxigenic strains were also determined on the basis of amplification of these three target DNA fragments. In this study, it was also demonstrated that only specific strains were able to produce the aflatoxin contamination in peanuts.

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Ahmad N, Rahim M. 2007. Evaluation of promising groundnut Arachis hypogaea L. varieties for yield and other characters. J. Agric. Res., 45:185-189.

Bankole SA, Ogunsanwo BM, Mabekoje OO. 2004. Natural occurrence of moulds and aflatoxin B1 in melon seeds from markets in Nigeria. Food and Chem. Toxicol., 42:1309-14.

Bhattacharya K, Raha S. 2002. Deteriorative changes of maize, groundnut and soybean seeds by fungi in storage. Mycopathol., 155:135141.

Bokhari F M, Aly M. 2009. Trials towards reduction of fungal growth and aflatoxin G1 production in Arabic coffee using different additives. Afr. J. Food Sci., 3:068-076.

Bradburn N, Coker RD, Blunden G. 1994. The etiology of turkey x disease. Phytochem., 35: 817-817.

Chen RS, Tsay JG, Huang YF, Chiou RYY. 2002. Polymerase chain reaction-mediated characterization of molds belonging to Aspergillus flavus group and detection of Aspergillus parasitictus in peanut kernels by a multiplex polymerase chain reaction. J. Food Prot., 65: 840-844.

Cotty PJ, Jaime-Garcia R. 2007. Influences of climate on aflatoxin producing fungi and aflatoxin contamination. Intl. J. Food Microbiol., 119:109-115.

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

Degola F, Berni E, DallAsta C, Spotti E, Marchelli R, Ferrero I, Restivo FM. 2007. A multiplex RT-PCR approach to detect aflatoxigenic strains of Aspergillus flavus. J. Appl. Microbiol., 103: 409-417.

Ehrlich KC, Montalbano VG, Cotty PJ. 2003. Sequence comparison of aflR from different Aspergillus species provides evidence for variability in regulation of aflatoxin production. Fung. Genet. Biol., 38: 6374.

El-Maghraby OMO, El-Maraghy M. 1987. Mycoflora and mycotoxins of peanuts (Arachis hypogaea L.) seeds in Egypt. 1-sugar fungi and natural occurrence of mycotoxins. Mycopathol., 98: 165-170.

Färber P, Geisen R, Holzapfel WH. 1997. Detection of aflatoxinogenic fungi in figs by a PCR reaction. Intl. J. Food Microbiol., 36: 215-220.

Geisen R. 1996. Multiplex polymerase chain reaction for the detection of potential aflatoxin and sterigmatocystin producing fungi. Syst. and App. Microbiol., 19: 388-392.

Gonçalez E, Nogueira JHC, Fonseca H, Felicio JD, Pino FA, Corrêa B. 2008. Mycobiota and mycotoxins in Brazilian peanut kernels from sowing to harvest. Intl. J. Food Microbiol., 123:184190.

Hadi AA, Carter D, Magan N. 2011. Discrimination between aflatoxigenic and non-aflatoxigenic Aspergillus section Flavi strains from Egyptian peanuts using molecular and analytical techniques. World Myco. J., 4: 69-77.

Hinrikson HP, Hurst SF, de Aguirre L, Morrison CJ. 2005. Molecular methods for the identification of Aspergillus species. Med. Mycol., 43:129-37.

Horn BW, Dorner JW. 1999. Regional differences in production of aflatoxin b1 and cyclopiazonic acid by soil isolates of Aspergillus flavus along transect within the United States. App. and Environ. Microbiol., 65:1444-1449.

Isabel ML, Alicia R, Andrade MJ, Martín A, Córdoba JJ. 2012. Development of a PCR Protocol to Detect Aflatoxigenic Molds in Food Products. J. Food Prot., 75: 85-94.

Konietzny U, Greiner R. 2003. The application of PCR in detection of mycotoxigenic fungi in food. Braz. J. Microbiol., 34: 283-300.

Liu BH, Chu FS. 1998. Regulation of aflR and its product associated with aflatoxin biosynthesis. App. Environ. Microbiol., 10: 3718-22.

Nakai VK, de Oliveira L, Rocha A, Gonçalez E, Fonseca H, Ortega EMM, Corrêa B. 2008. Distribution of fungi and aflatoxins in a stored peanut variety. Food Chem., 106: 285290.

Paterson RRM. 2007. Aflatoxins contamination in chilli samples from Pakistan. Food Ctrl., 18: 817820.

Pildain BM, Frisvad JC, Vaamonde G, Cabral D, Varga J, Samson RA. 2008. Two novel aflatoxin-producing Aspergillus species from Argentinean peanuts. Intl. J. Syst. and Evol. Microbiol., 58: 725735.

Rashid M, Khalil S, Ayub N, Ahmed W, Khan AG. 2009. Categorization of Aspergillus flavus and Aspergillus parasiticus isolates of stored wheat grains into aflatoxinogenics and non-aflatoxinogenics. Pak. J. Bot., 40: 2177-2192.

Rodrigues P, Venencio A, Kozakiewicz Z, Lima N. 2009. A polyphasic approach to the identification of aflatoxigenic and non-aflatoxigenic strains of Aspergillus section Flavi isolated from Portuguese almonds. Intl. J. Food Microbiol., 129: 187-193.

Rodrigues P, Soares C, Kozakiewicz Z, Paterson RRM, Lima N, Venâncio A. 2007. Identification and characterization of Aspergillus flavus and aflatoxins. IN: Méndez-Villas A. (Editor) Communicating Current Research and Educational Topics and Trends in Applied Microbiology. Formatex., 527-534.

Schaad NW, Cheong SS, Tamaki S, Hatziloukas E, Panopoulos NJ. 1995. A combined biological and enzymatic amplification (BIOPCR) technique to detect Pseudomonas syringae pv. phaseolicola in bean extracts. Phytopath., 85: 243248

Scherm B, Palomba M, Serra D, Marcello A, Migheli Q. 2005. Detection of transcripts of the aflatoxin genes aflD, aflO and aflP by reverse transcriptase-polymerase chain rection allows differentiation of aflatoxin-producing and nonproducing isolates of Aspergillus flavus and Aspergillus parasiticus. Int. J. Food. Microbiol., 98: 201-210.

Shapira R, Paster N, Eyal O, Menasherov M, Mett A, Salomon R. 1996. Detection of aflatoxigenic moulds in grains by PCR. App. Environ. Microbiol., 62:3270-3273.

Sultan Y, Magan N. 2010. Mycotoxigenic fungi in peanuts from different geographic regions of Egypt. Mycotoxin Res., 26: 133-140.

Sweeney MJ, Pamies P, Dobson ADW. 2000. The use of reverse transcription-polymerase chain reaction (RT-PCR) for monitoring aflatoxin production in Aspergillus parasiticus. Intl. J. Food Microbiol., 56: 97-103.

Trucksessy MW, Dombrink-Kurtzman MA, Tournasy VH, White KD. 2002. Occurrence of afatoxins and fumonisins in Incaparina from Guatemala. Food Addit. Contam., 7: 671- 675.

Vaamonde G, Patriarca A, Fernández-Pinto V, Comerio R, Degrossi C. 2003. Variability of aflatoxin and cyclopiazonic acid production by Aspergillus section flavi from different substrates in Argentina. Intl. J. Food Microbiol., 88:79-84.

Williams JH, Phillips TD, Jolly PE, Stiles JK, Jolly CM, Aggarwal D. 2004. Human aflatoxicosis in developing countries: a review of toxicology, exposure, potential health consequences, and interventions. American J. Clin. Nutr., 80: 1106-1122.

Wyss P, Bonfante P. 1993. Amplification of genomic DNA of arbuscular- mycorrhizal (AM) fungi by PCR using short arbitrary primers. Mycol. Res., 97: 13511357.

Youssef MS, El-Maghraby OMO, Ibrahim YM. 2008. Mycobiota and mycotoxins of Egyptian peanut (Arachis hypogeae L.) seeds. Intl. J. Bot., 4: 349-360.

Yu J, Chang PK, Ehrlich KC, Cary JW, Bhatnagar D, Cleveland TE, Payne GA, Linz JE, Woloshuk CP, Bennett W. 2004. Clustered pathway genes in aflatoxin biosynthesis. App. Environ. Microbiol., 70: 12531262.