TITLE:
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
AUTHORS:
Virendra Kumar, Ajay Kumar, Shailesh Kumar Kannojia, Rana Pratap Singh
KEYWORDS:
Chilies, RAPD-PCR, Aspergillus flavus, Aflatoxin B1, ELISA, Toxigenic
JOURNAL NAME:
Open Access Library Journal,
Vol.1 No.9,
December
22,
2014
ABSTRACT: 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 study 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℃ 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 produced sclerotia in APA medium. In another set of experiments, occurrence 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 A. flavus isolates, seven 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 light on exposure of UV (365 nm) light.