Prevalence of Mycotoxigenic Fungi and Efficacy of Multi-spectral Kernel Sorting and Irradiation Techniques in Management of Mycotoxigenic Fungi and Mycotoxins in Maize

Abstract

Maize, the main staple food in Kenya is largely affected by mycotoxin contamination mainly aflatoxin and fumonisins due to attack by toxigenic Aspergillus and Fusarium species, resulting in health and economic burdens. The aim of this study was to assess the prevalence of mycotoxigenic fungi and enhance food safety and human health by reducing mycotoxin contamination in maize using multi-spectral sorting and irradiation techniques. The study was carried out during the short rains cropping season of 2019/2020 in Eastern Kenya: Embu, Meru, Tharaka Nithi, Machakos, Makueni and Kitui Counties. Ninety-nine maize samples were collected from standing crop in farmers’ fields while 97 samples were collected from farmers stores. In order to determine the effects of farmer practice on the population of fungi and mycotoxins, a structured questionnaire in a surveyCTO software was administered to the farmers during harvesting of the maize. Prior to the baseline survey, an ethical approval was obtained through the National commission of Science, Technology and Innovation. During the survey, 20 to 30 maize cobs were sampled in a zigzag manner in each farmer’s field. The cobs were shelled manually by hand, mixed, and subdivided by quartering to obtain 1kg of kernels which was shipped to the Regional Mycotoxin Laboratory in Kenya Agricultural and Livestock Research Organization in Katumani for processing and shipment to the University of Illinois for microbial analysis by plate count and mycotoxin analysis using enzyme-linked immunosorbent assay method. Secondly, to determine the effect of multi-spectral sorting on reduction of fumonisin, toxigenic Fusarium and other microbes, maize kernels were sorted in a calibrated multi-spectral sorter and samples in the accepted and rejected streams were assayed for fumonisin levels by enzyme-linked immunosorbent assay; toxigenic Fusarium by qPCR; and other microbes by sequencing of ITS1F- ITS2 and V3-V5 regions for fungi and bacteria respectively. Lastly to evaluate the efficacy of E-beam irradiation in reducing Aspergillus, Fusarium, aflatoxin and fumonisins, 24 samples out of the 97 samples collected in farmers’ stores and had aflatoxins greater than100 μg/kg and fumonisins greater than 1000 μg/kg were treated with E-beam irradiation dose of 5, 10 and 20 kGy. The Aspergillus and Fusarium in the sample were assayed by culture plating method on potato dextrose agar modified with antibiotics and qPCR while the aflatoxin and fumonisin levels were assayed by enzyme-linked immunosorbent assay. Both microbial populations and mycotoxin levels were compared before and after E-beam treatment. Data were analyzed by an open-source R-software. A higher proportion of the farmers in Eastern Kenya planted improved maize varieties (45.2%); practiced intercropping (83.7%); preferred planting maize using organic manure (45.5%); tilled their fields by hand (52.6%); and after harvesting, majority of the farmers (65.6%) stored their crops on wooden racks in the house. The aflatoxin contamination levels increased with the increased use of tractors (p= 0.03, r= 0.85); a similar trend (p= 0.02, r=0.87) was also seen in use of mixed varieties (combining both improved and local varieties) with fumonisin accumulation. No relation was established between farmers’ practice and fungal population. Single kernel multi-spectral sorting reduced fumonisins by an average of 88.9 % (ranged between 27.6 to 99.8% reduction) with a low median rejection rate of 1.87% (ranged 0% to 48%). The proportion of toxigenic Fusarium infection on the maize kernel was significantly (p= 0.005) lower in the accept stream (1.4%) than in the reject stream (30.1%). Similarly, there was a significant decrease (p=0.002) of 31% and 90 % in the total fungal and bacterial counts in accepted maize kernels respectively. E-beam irradiation at doses greater than 5 kGy reduced fungal loads (average 3.7 log CFU/g) to below limits of detection by culturing method. E-beam dose of 20 kGy caused a significant (p = 0.03) 6.2 ng/g reduction in aflatoxins in the maize slurry. However, the 20 kGy did not reduce fumonisin in the maize slurry. Farmers’ practices influence accumulation of mycotoxins accumulation in the field. Multi-spectral sorting was effective in reducing fumonisins, toxigenic Fusarium and other microbes. Furthermore, E-beam irradiation was effective in reduction of preformed aflatoxins and complete elimination of microbes in maize. Since no relation was seen between farmer practices and population of fungi, there is need to investigate other factors that may affect the abundance of fungi in soil. There is also need to upscale multi-spectral sorting technique to suit large scale farmers. Lastly, emerging technologies like E-beam irradiation should be adopted in Sub-Saharan Africa and high doses should be explored to manage fumonisins.