Morphological and molecular identification of Aspergillus flavus isolated from rice (Oryza sativa L.) grain samples in Karnataka and Tamil Nadu in Southern India

Abstract

Mycotoxin contamination especially aflatoxin is of grave concern in food safety, due to its carcinogenicity. Rice is a staple food for all sections of people in India. The present investigation thus aimed at enumeration of fungal microflora in ten samples of rice collected from Karnataka and Tamil Nadu in South India. The main genera found were Aspergillus, Cladosporium, Alternaria, Rhizopus and Aspergillus which was found with highest frequency (90%) and relative density (52%) with A. ochraceous and A. flavus comprising ninety and sixty percent respectively. The presence of A. flavus, a principal aflatoxin producer, was confirmed by polymerase chain reaction (PCR) amplification of the specific internal transcribed spacer (ITS) ITS1-5.8S-ITS2 of the ribosomal DNA region of the fungus and its sequencing in selected isolates. This region is also found in the non-aflatoxigenic A. oryzae with 100 % homology. The isolates were hence confirmed as A. flavus by amplification of an important gene in aflatoxin biosynthetic pathway, namely nor-1 (aflD) gene and its expression by reverse transcription PCR (RT-PCR) as this gene is present in both species but expressed only in A. flavus. The co-occurrence of A. ochraceous in most of the samples also raises concern in consumer health which needs to be addressed during the storage and public distribution of rice. The presence and expression of nor-1 gene could also be used as a molecular tool for detection of A. flavus in stored rice and other food grains.