CHARACTERIZATION OF AFLATOXIN-PRODUCING ASPERGILLUS SPECIES IN CASHEW NUTS COLLECTED FROM THE COASTAL REGION OF KENYA

Abstract

Cashew trees (Anacardium occidentale) are among the main cash crops in coastal Kenya grown for their nut and apples. Cashew nuts have a high nutritional value which makes them highly susceptible to fungal growth and mold contamination resulting in biodeterioration of the nutritional value and production of secondary metabolites including aflatoxins. Incidences of contamination of aflatoxins in cashew nuts from coastal Kenya have been reported, leading to great losses and refusal in the international markets. This study is aimed at morphological and molecular identification and characterization of the aflatoxin-producing Aspergillus species isolated from the cashew nuts postharvest. Cashew nut samples were collected from Kilifi, Kwale, and Lamu counties in Kenya’s coast and examined for moisture content and the abundance of aflatoxigenic fungal species. The cashew nut seeds were direct plated in modified Rose Bengal agar (MRBA) medium and pure isolates were obtained from potato dextrose agar (PDA) and malt extract agar (MEA) media. Lactophenol cotton blue (LPCB) stain was used to evaluate the isolates at 40X and 100X for microscopic differences. Sixty-three isolates were used for genomic DNA extraction and ITS and 28S rRNA regions were amplified and sequences for identification of the isolates and genetic diversity estimation using MEGA X and Arlequin. The AflD (Nor1), AflM (Ver1) and AflR genes were amplified to identify the potentially aflatoxigenic from the Aspergillus isolates. The Aflatoxins’ presence on the isolates was screened using UV (365nm) and the ammonia vapour test on coconut milk agar. Moisture content comparison between the counties showed a significant difference (p<0.05) in the cashew nuts from Kwale (7.15 ± 0.25%) compared to both Kilifi (4.87 ± 0.99%) and Lamu (4.73 ±0.74%) counties. Strains from two Aspergillus sections Flavi and Nigri were identified, and Aspergillus flavus and Aspergillus niger were the predominant species recovered from the cashew nuts. The amplification of the aflatoxin biosynthesis pathway genes showed that 39.68% isolates were potentially aflatoxigenic. The aflatoxins rapid tests showed that 22.22% isolates were aflatoxigenic, they belong to Aspergillus section Flavi and Aspergillus flavus was more abundant. Diversity estimation of the isolated species showed no significant difference in the genetic makeup in isolates from Kilifi, Kwale and Lamu counties. The presence of Aspergillus section Flavi species is an indication of the probable aflatoxin production and the recovery Aspergillus section Nigri points to the possibility of the production of other mycotoxins in the cashew nuts. This study establishes a foundation for the management of aflatoxin contamination in cashew nuts in coastal Kenya.