Development of Molecular Marker and Variability Characterization of Aspergillus flavus Isolates of Chilies (Capsicum frutescens L.) through RAPD-PCR and Estimation of Aflatoxin B1 by Indirect Competitive ELISA in India
Aflatoxin is the most potent toxic substances, these are the group of closely related mycotoxins produced by fungus i.e. Aspergillus flavus. Twenty seven isolates of A. flavus designated as Af-1, Af-2, Af-4, Af-5, Af-7, Af-9, Af-11, Af-12, Af-13, Af-15, Af-25, Af-28, Af-31, Af-33, Af-34, Af-35, Af-36, Af-37, Af-39, Af-40, Af-46, Af-47, Af-54, Af-55, Af-58, Af-59 and Af-60 were collected from different agro climatic conditions from chilies (Capsicum frutescence) samples. Random amplified polymorphic DNA-PCR analysis was used to investigate genetic variation among these isolates. Molecular variability studied was done to find out the best annealing temperature and sixty primers were screened to select for maximum polymorphism of DNA. The best annealing temperature was recorded 34 oC for the pathogen and most efficient amplification and polymorphism of DNA was found with random primer 5’-CAGGCGCACA-3’. This primer sequence had also been identified as a typical A. flavus isolate which produces sclerotia in APA medium. In another set of experiments, occurence of aflatoxin B1 in chilies from India was determined by using indirect competitive ELISA in work undertaken in Indian Institute of Vegetable Research, Varanasi, India. Among twenty chilies samples, the toxin concentration ranged from 15.3-782.3 μg/ kg and all chilies samples were positive for aflatoxin B1. Out of twenty seven chilies samples, nine samples were found to be toxigenic. UAS Dharwad (B) chilies sample was found most toxigenic because it was found to be producing maximum yellow fluorescence on exposure of UV (365 nm) light
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Kumar, V. , Kumar, A. , Kannojia, S. K. and Singh, R. P. (2014). Development of Molecular Marker and Variability Characterization of Aspergillus flavus Isolates of Chilies (Capsicum frutescens L.) through RAPD-PCR and Estimation of Aflatoxin B1 by Indirect Competitive ELISA in India. Open Access Library Journal, 1, e1161. doi: http://dx.doi.org/10.4236/oalib.1101161.
Bhatnagar, D.K.,
Khlich, C. and Cleveland, T.E. (1993) Biochemical Characterization of an Aflatoxin B2 Producing
Mutant of Aspergillus flavus. FASEB
Journal, 7, A1234.
Saba, I. and Rabiya, N. (2012) Molecular Characterization of Seven Different Species of Aspergillus through Random Amplified
Polymorphic DNA (RAPD) and Enzymes Analysis. Journal
of Microbiological Research, 2, 47-50. http://dx.doi.org/10.5923/j.microbiology.20120203.01
Paterson, R.R.M. and Lima, N. (2010) Toxicology of Mycotoxins. In: Lunch, A., Ed., Molecular Clinical
and Environmental Toxicology, Vol. 2, Springer, Basel, 31-63. http://dx.doi.org/10.1007/978-3-7643-8338-1_2
Kirschstein, R.L. and Sidransky, H. (1965) Mycotic Endocarditis of
Tricuspid Vave Due to Aspergillus flavus. Archive of Phtopathology and Plant
Protection, 62, 103-106.
Ito, Y., Peterson, S.W.,
Wiclow, D.T. and Goto, T. (2001) Aspergillus pseudotamari, a New Aflatoxin-Producing
Species in Aspergillus Section Flavi. Mycological Research, 105,
233-239. http://dx.doi.org/10.1017/S0953756200003385
Goto, T. (2005)
Aflatoxigenic and Non Aflatoxigenic Species in Aspergillus Section Flavi. Journalof the Faculty of Agriculture Shinshu University, 41, 1-6.
Deiner, U.L., Cole, R.J.,
Sanders, T.H., Payne, G.A. and Lee, K. (1987) Epidemiology of Alfatoxin Formation of by Aspergillus flavus. Annual Review of Phytopathology, 25,
249-270. http://dx.doi.org/10.1146/annurev.py.25.090187.001341
Leger, R.J., Screen,
S.E. and Shams-Pirzadeh, B. (1999) Lack of Host Specilization in Aspergillus flavus.Applied
and Environmental Microbiology, 66, 320-324.
Reddy, S.V., Mayi, D.K., Reddy, M.U., Thirumala-Devi, K. and Reddy, D.V.R. (2001) Aflatoxin B1 in Different Grades of
Chillies (Capsicum annum L.) in India
as Determined by Indirect Competitive ELISA. Food Additives & Contaminants, 18, 553-558. http://dx.doi.org/10.1080/02652030119491
Manicom, B.Q., Bar-Joseph, M., Rosner, A., Vigodsky-Hans, H. and Kotze, J.M. (1987) Potential Applications of Random DNA Probes, and
Restriction Fragment Length Polymorphism in the Taxonomy of Fusaria. Phytopathology, 77, 669-672. http://dx.doi.org/10.1094/Phyto-77-669
Weir, T.L., Huff, D.R., Christ, B.J. and Romaine, C.P. (1998) RAPD-PCR Analysis of Genetic Variations among Isolates
of Alternaria solani and Alternaria alternata from Potato and
Tomato. Mycologia, 90, 813-821. http://dx.doi.org/10.2307/3761323
Hara, S.,
Fennell, D.I. and Hesseltine, C.W. (1974) Aflatoxin Producing Strain of Aspergillus flavus Detected by Fluorescence of Agar Medium under
U.V. Light. Applied Microbiology, 27, 1118-1123.
Mohankumar, M., Vijayamundeeswari, A., Karthikeyan, M., Mathiyazahagan, S., Paranidhavan, V. and Velzhahan, R. (2010) Analysis of Molecular Variability among Isolates of Aspergillus
flavus by PCR-RFLP of the Its Regions of rDNA. Journal of Plant Protection Research, 50, 446-451.
Midorikawa, G.E.O., Pinheiro, M.R.R., Vidigal, B.S., Arrunda, M.C., Costa, F.F., Pappas
Jr., G.J., Ribeiro, S.G., Freire, F. and Miller, R.N.G. (2008) Characterization of Aspergillus flavus Strains from Brazilian Brazil Nuts and Cashew by
RAPD and Ribosomal DNA Analysis. Society for Applied Microbiology, 11, 1472-1765.
Ajithkumar, K. and Naik, M.K. (2006) Detection of Aflatoxin Producing A. flavus Isolates from Chili and Their
Management by Post Harvest Treatment. Journal
of Food Science and Technology, Mysore, 43, 200-204.
Ravi
Kiran, D., Narayana, K.J.P. and Vijaylakshmi, M. (2005) Aflatoxin B1 Production in Chillies (Capsicum annuum L.) Kept in Cold
Storage. African Journal of Biotechnology, 4, 791-795.
Shweta, S., Kanan, K., Madhawan, S., Adithya, R., Paranidharan, V. and Velazhahan, R. (2013) RAPD Analysis of Genetic Diversity among the Isolates of Aspergillus
flavus from Different Host and Locations. Archives of Phytopathology and Plant Protection.
Tran-Dinh, N., Pitt, J.I. and Carter, D.A. (1999) Molecular Genotype Analysis of Natural Toxigenic and
Nontoxigenic Isolates of Aspergillus
flavus and A. parasiticus. Mycological Research, 103, 1485-1490. http://dx.doi.org/10.1017/S0953756299008710
Gashgari, R.M., Shebany, Y.M. and Ghabacoy, Y.A. (2010) Molecular Characterization of Mycobiota and Aflatoxin
Contamination of Retail Wheat Flour from Jeddah Markets. Foodborne Pathogens and Disease, 7, 1047-1054. http://dx.doi.org/10.1089/fpd.2009.0506
Barros, G.G., Chitta, M.L., Reynoso, M.M., Torres, A.M. and Chulze, S.N. (2007) Molecular Characterization of Aspergillus Section Flavi Isolates Collected from Peanut
Field in Argentina Using AFLPs. Journal
of Applied Microbiology, 103, 900-909. http://dx.doi.org/10.1111/j.1365-2672.2007.03312.x
Singh,
I. and Singh, V.P. (2002) Comparative Studies on Growth Patterns
and Metabolic Status of Aflatoxin, Producing and Non Producing Strains of Aspergillus flavus.Current Science, 82, 1425-1426.
Saito, M. and Machida, S. (1999) A Rapid
Identification Method for Aflatoxin-Producing Strains of Aspergillus flavus and A. parasiticus by Ammonia Vapor.Mycoscience, 40, 205-208. http://dx.doi.org/10.1007/BF02464300
Schearer, C.G., Wright, S., Krosch, J., Tatnell, A. and Kyei (1999) Effect of
Temperature on Grown and Aflatoxin Production by Non-Toxigenic (Strain 4351)
and Toxigenic (Strain 2754). Aspergillus
flavus. ACIAR Food Legume, News Letter No. 29, 4-7.
Abbas, H.K., Weaver, M.A., Zabitowicz, R.M., Horn, B.W. and Shier, W.T. (2005) Relationships between
Aflatoxin Production and Sclerotia Formation among Isolates of Aspergillus Section Flavi from the Mississippi Delta.European Journal of Plant Pathology, 112, 283-287. http://dx.doi.org/10.1007/s10658-004-4888-8
Shier, W.T., Lao, Y.B., Steele, T.W.J. and Abbas, H.K. (2005) Yellow Pigments Used in Rapid Identification of Aflatoxin
Producing Aspergillus Strains Are
Anthraquinones Associated with the Aflatoxin Biosynthesis Pathway. Bioorganic Chemistry, 33, 426-438. http://dx.doi.org/10.1016/j.bioorg.2005.09.002
Fente, C.A., Ordaz, J.J., Vozquez, B.I., Frnaco, C.M. and Cepeda, A. (2001) New Additive for
Culture Media for Rapid Idenitification of Aflatoxin-Producing Aspergillus Strains. Applied and Environmental Microbiology, 87, 4858-4862. http://dx.doi.org/10.1128/AEM.67.10.4858-4862.2001
Maxwell, S.M., Apeagyei, F., De Vries, H.R., Mwanmut, D.D. and Hendrickse, R.G. (1998) Aflatoxin in Breast Milk, Neonatal Cord Blood and Sera of
Pregnant Women. Toxicological Reviews, 8, 12-29.