Aspergillus population diversity and its role in aflatoxin contamination of cashew nuts from coastal Kenya

Abstract

Cashew nuts are among the main cash crops in coastal Kenya, due in large part to their high nutritional value. Unfortunately, they also make them highly susceptible to mold contamination, resulting in biodeterioration of the nutritional value and potential contamination with toxic secondary metabolites, such as aflatoxins, that cause them to be rejected for sale at the market. We determined the population diversity of the Aspergillus species and their role in aflatoxin contamination in cashew nuts in selected coastal regions of Kenya. Fifty raw cashew nut samples were collected from post-harvest storage facilities across three counties in Kenya's coastal region and examined for moisture content and the presence of Aspergillus fungi. About 63 presumptive isolates were recovered from the cashew nuts. ITS and 28S rDNA regions were sequenced. The aflD, aflM and aflR genes were amplified to identify the potentially aflatoxigenic from the Aspergillus isolates. The Aflatoxins' presence on the isolates was screened using UV and the ammonia vapour test on coconut milk agar and validated using ELISA assay. A comparison of cashew moisture content between the three counties sampled revealed a significant difference. Sixty-three isolates were recovered and identified to section based on morphological characters and their respective ITS regions were used to obtain species identifications. Three sections from the genus were represented, Flavi and Nigri, and Terrei with isolates from the section Nigri having slightly greater abundance (n = 35). The aflD, aflM and aflR genes were amplified for all isolates to assess the presence of the aflatoxin biosynthesis pathway, indicating the potential for aflatoxin production. Less than half of the Aspergillus isolates (39.68%) contained the aflatoxin pathway genes, while 22.22% isolates were aflatoxigenic, which included only the section Flavi isolates. Section Flavi isolates identification was confirmed by calmodulin gene. The presence of species from Aspergillus section Flavi and section Nigri indicate the potential for aflatoxin or ochratoxin in the cashew nuts. The study established a foundation for future investigations of the fungi and mycotoxins contaminating cashew nuts in Kenya, which necessitates developing strategies to prevent infection by mycotoxigenic fungi, especially during the storage and processing phases.

Fig 1

Box plots comparing the average moisture content of cashew nuts between (A) and within (B, C and D) each of the three sampling counties. Between-county values compared using the Kruskal Wallis test, followed by the Post Hoc test (p < 0.05). Within-county values compared using a one-way ANOVA, while Lamu County values were compared sing T-test (p < 0.05).

Fig 2. Box plots comparing the average moisture content of cashew nuts from each of the 13 sampling locations.

The means were assessed for statistically significant differences by one-way ANOVA (p < 0.05), yielding a test statistic of (F) = 3.423 and p-value of p = 0.002. To confirm the significantly different pairs, the Tukey’s Honest Significant Difference (HSD) test was performed. * p < 0.05, ** p < 0.01, no asterisk indicates lack of significance.

Fig 3. Graph showing the correlation between cashew kernel moisture content with growth rates of Aspergillus isolates.

Data was evaluated using the Pearson correlation method and resulted in r = 0.4582 at 95% confidence interval, R square = 0.2100, p-value = 0.0001. The correlation was Significant at (alpha = 0.05) for the number of XY Pairs = 65. The growth rates were measured in mm/day.

Fig 4. Phylogeny of Aspergillus isolates recovered from cashew nuts using nucleotide sequences from A. 28S rDNA region.

The phylogeny was inferred using maximum likelihood method of newick tree generated using the Fasttree package. A bootstrap replication of 1000 was used.

Fig 5. Phylogeny of Aspergillus isolates recovered from cashew nuts using nucleotide sequences from ITS region.

The phylogeny was inferred using maximum likelihood method of newick tree generated using the Fasttree package. A bootstrap replication of 1000 was used.

Fig 6. Phylogenetic tree of Aspergillus species section Flavi confirmed by use of calmodulin gene.

The tree shows Aspergillus isolates previously detected by ITS and 28S rDNA regions. The phylogeny was inferred using maximum likelihood method of newick tree generated using the Fasttree package. A bootstrap replication of 1000 was used.