Genetic Diversity of Aspergillus spp. in Stored Grains Using RAPD-PCR Technique

Abstract

Background: Aspergillus species are the dominant storage fungi of cereal grains and include aflatoxigenic and opportunistic human pathogens. Data on the genetic structure of grain-borne Aspergillus populations in Iraq remain limited. Objectives: To isolate and identify Aspergillus spp. from stored wheat, maize and rice in Iraq and to assess their intra- and interspecific genetic diversity using random amplified polymorphic DNA (RAPD)-PCR. Methods: Ninety stored grain samples (30 each of wheat, maize and rice) were collected from warehouses and retail markets in Haditha, Hit, Ramadi and Rutba, Anbar Governorate, Iraq, between January and March 2026. Fungi were isolated by direct and dilution plating, identified morphologically and confirmed by ITS and calmodulin sequencing, and section Flavi isolates were screened culturally for aflatoxigenic potential. Genomic DNA of 36 isolates was amplified with five decamer RAPD primers. Bands were scored as binary data and analysed by polymorphism indices, polymorphic information content (PIC), Jaccard similarity, UPGMA clustering, principal coordinate analysis and analysis of molecular variance (AMOVA). Results: Aspergillus was recovered from 47 of 90 samples (52.2%). A. flavus was the dominant species (36.4% of 55 isolates), and 55.6% of section Flavi isolates were presumptively aflatoxigenic. The five primers generated 58 bands, of which 48 (82.8%) were polymorphic, with PIC values of 0.22–0.44. Jaccard similarity ranged from 0.31 to 0.91. UPGMA (cophenetic r = 0.89) resolved three clusters at 65% similarity that corresponded largely to species. AMOVA attributed 82% of the variation to differences within grain sources and 18% to differences among them (ΦPT = 0.18; P = 0.018). Conclusion: Aspergillus populations in stored Iraqi cereals are genetically diverse and structured mainly by species rather than by grain source. RAPD-PCR is a rapid, low-cost tool for baseline surveillance of storage Aspergillus and should be combined with aflatoxin-gene PCR for risk assessment.