Fungi belonging to the genus Aspergillus produce various toxins, which are of importance to human health. In particular, Aspergillus flavus and Aspergillus parasiticus mainly produce aflatoxins (Abbas et al., 2005) while other congeneric species: Aspergillus sections Nidulantes, Versicolores, Usti, Circumdati and Nigri produce various metabolites related to aflatoxins which cause food poisoning (Blumenthal, 2004; Do and Choi, 2007). These fungi colonize grain in the field during planting and continue to do so during storage when they may produce toxins (Waliyar et al., 2015). Apart from aflatoxins, these fungi produce other metabolites such as ochratoxins and oxalic acid, which are hazardous to humans (Palencia et al., 2010). The effect of mycotoxin ingestion is dose-dependent, it may result in either death (acute) or other clinical complications (chronic). Chronic intake may lead to immunosuppression (Jiang et al., 2008), cancers, poor growth and abnormal foetal development (Gong et al., 2004; Probst et al., 2011).The United States Food and Drug Administration (FDA) has referred to aflatoxins as inevitable food contaminants, hence it has set the maximum allowable limit in human food products at 20 parts per billion. However, in the US, the levels vary between 10 parts per billion for humans and 20 parts per billion for livestock (Williams et al., 2004), while in Europe the limit is 4 ppb (Commission Regulation (EC) No 1881/2006, 2006). In Kenya, the limits are 10 parts per billion (Mutiga et al., 2014).

Over the last four decades, Kenya has experienced repeated cases of aflatoxicosis. In the year 2004, 317 people were hospitalised due to acute aflatoxin exposure 125 cases were fatal (CDC, 2004). It was hypothesised that there could be an underestimation of human aflatoxicosisburden in the country for four reasons: 1. Many cases may not be reported due to lack of infrastructure of capturing episodes in real time. 2. There is scanty clinical data to account for chronic sub-lethal exposure, 3. There is a systemic difficulty in enforcing residual limits policies among cereal traders and this ensures the circulation of cereals with high toxin levels. 4. There is a belief that aflatoxin contamination is a problem in only one region of the country yet it is more widespread covering other maize producing areas including Western and Rift Valley. Most of the human fatalities have been recorded in the Eastern region of Kenya, which is now considered a hot spot for aflatoxicosis (Probst et al., 2007; Muthomi et al., 2009). Fatalities common in the Eastern region is perhaps due to acute grain shortage, which force the populace to consume any available grain that is probably considered unfit for consumption. However, a survey by Mutiga et al. (2014) reported on high incidences of aflatoxin contamination in the Eastern region of Kenya following a bumper harvest in 2010. Preharvest drought and postharvest moisture are considered the most critical drivers of aflatoxin accumulation, which are factors that are largely influenced by cropping seasons. In Eastern Kenya, acute aflatoxicosis has been reported following periods  of  moisture  stress   during maize crop development, when rainfall occurs after the crop has attained physiological maturity and before it is harvested, and when harvests are stored incorrectly. For this reason, there has been renewed effort to study various aspects of the causal fungi and its interactions, which include intraspecific variations and competition, climatic and seasonal influence, toxigenicity (Probst et al., 2011), distribution in maize and soil (Muthomi et al., 2009).

Perhaps one of the most promising control efforts is in biological control whose success story has been reported in other countries like Nigeria (Fapohunda, 2009) and is undergoing initial stages of quarantined field trials (IITA, 2014). This approach uses non-toxigenic fungi to “competitively exclude” toxigenic ones from accessing maize kernels, thereby preventing toxin build-up (Cotty et al., 2007). As such, an inoculum of non-toxin producing fungi is introduced into the soil surface beneath the crop canopy 40-45 days after planting (two-to-three weeks before flowering) (Atehnkeng et al., 2014). However, considering the variations in both quantities and types of toxins produced by fungi of the genus Aspergillus, it is imperative to consider these variations alongside species composition, abundance and geographical distribution as would be affected by seasonal changes. This information would be important in understanding the possible toxin risks the population faces with each harvest and enable early preparedness. This study was conducted to determine the seasonal variation in the abundance and composition of putatively toxigenic Aspergillus species in maize and soil in Eastern Kenya. This has not been previously established in this region.

Fungi isolation and identification

Fungi isolation from maize was carried out following the procedure described by Muthomi et al. (2009) after surface sterilisation of kernels in 2% NaOCl. Fifteen kernels were randomly picked from the khaki bags and introduced into a conical flask containing NaOCl and swirled gently for one minute. Afterwards, they were removed and rinsed thrice in sterile distilled water. Five kernels were plated about 2 cm apart on Petri dishes (90 × 15 mm) containing Czapek Dox Agar (HiMedia Laboratories Pvt. Ltd) amended with 50 mg each of streptomycin sulphate and penicillin (Zhonghuo Pharmaceutical Shijazhuang Co. Ltd., China) per litre of medium. The set up was replicated thrice for each sample. As for soil samples, dilution plate technique was used. Briefly, 1 g of soil was suspended in 9 ml sterile distilled water and serially diluted to 1 x 10^-4. One ml of 10⁻³ and 10^-4 respectively were uniformly spread in duplicates in Czapek Dox Agar (amended as above). Afterwards, all culture plates were incubated in a growth chamber at 28°C for 7 days. Aspergillus species were isolated from colonies in Petri dishes and dilution plates then sub-cultured in Potato Dextrose Agar (HiMedia Laboratories Pvt. Ltd, India) and incubated as above. Upon maturation, fungi were classified based on cultural and morphological features such as colony diameter, colony colour on agar, front and reverse and colony texture (Klich, 2002a; Rodrigues et al., 2007). This was followed by the preparation of slide cultures and incubation in moist chambers at 28°C for 5 days before observation under a light microscope. For microscopic characterisation, microscopic features studied included conidiophores, conidial shape, phialides and metulae, presence and shape of vesicles. These are the common features used to identify Aspergillus fungi to species level (Diba et al., 2007; Klich, 2002a).

Screening for toxigenic Aspergillus species

Toxin producing ability of the fungi was tested following the ammonium hydroxide (NH₄OH) vapour test (Kumar et al., 2007). A single fungal colony was grown in the centre of a Petri dish containing yeast extract-sucrose medium for 5 days at 28°C. Then 2 drops of concentrated (27%) NH₄OH solution were added to the inverted lid of the Petri dish and allowed (30 min) to react. Toxin production (positive test) was evidenced by formation of a pink to plum-red colour on the underside of the fungal colony while negative tests had no observable colour changes (Zrari, 2013).

Data analysis

The data on the abundance of the fungi in each region were represented as a percentage of score total count. Analysis of variance (ANOVA) was performed to determine whether the distribution of the Aspergillus isolates in the four sites was significantly different. Student t-test was performed to determine whether the distribution of the species between the two seasons was statistically significant. SPSS (version 20.0) was used in the analysis of data.

The toxigenic Aspergillus isolates were of the species A. flavus (24), A. parasiticus (3), A. ochraceous (3), A. clavatus (8), A. ustus (1), A. niveus (1) and A. wentii (1). 

In the current study, seven putatively toxigenic species (A. flavus, A. parasiticus, A. ochraceous, A. clavatus,A. ustus, A. niveus and A. wentii) were reported. This corroborates earlier reports of toxin production by Aspergillus species other than A. flavus for example; A. parasiticus produces aflatoxins (Abbas et al., 2005); A. ochraceous produce ochratoxin A (Montessinos et al., 2015) while A. wentii produces the mycotoxins emodin (Pitt and Hocking, 2009). A polyisoprenoid toxin referred to as Austin was reported to be produced by A. ustus (Chexal et al., 1976) whereas A. niveus has been reported to produce fumonisins (Storari et al., 2012) and aspochalasin Z (Gebhardt et al., 2004). Aspergillus clavatus was isolated from soil only and was not isolated from maize, it was classified as toxigenic in this study. Since it was not isolated from maize, human risks appear to be reduced. Earlier studies have reported this species to produce a variety of secondary metabolites (SM) such as patulin, pseurotin A and cytochalasin E (Zutz et al., 2013). On the other hand, other species which did not produce toxin in this study have been reported to be toxigenic in earlier studies for example; A. candidus was reported to produce a mycotoxin known as AcT1 (Chattopadhyay et al., 1987), whereas A. awamori has been reported to produce fumonisins (Storari et al., 2012) and aspochalamins A-D (Gebhardt et al., 2004). Aspergillus terreus has been reported to produce territrems in bakery products and grains (El-Sayed Abdalla et al., 1998). The ammonium test was used to test the production of toxin in YES medium. It is a reliable method that has been published by Kumar et al. (2007). Another method is to include beta cyclodextrins in the culture medium, which would enhance the natural fluorescence of aflatoxins under ultra violet light (Fente, 2001; Yazdani, 2010). So far, three species of Aspergillus namely A. flavus, A. parasiticus and A. nomius have been reported to secrete aflatoxins (Varga et al., 2011). In this paper, additional putatively toxigenic species that may also be producing aflatoxins or its precursors are reported. For these reasons, there is need to confirm the toxigenicity of these fungi through analytical procedures such as HPLC, or through the use of molecular markers to confirm presence of toxin coding sequences in the various Aspergillus isolates’ genome. Follow-up studies such as molecular studies to confirm the species of the fungi reported herein is also recommended.

The findings of this study are vital in understanding the possible toxin risks the population faces with each harvest and enable early preparedness. There is very little information on the seasonal variations of toxigenic Aspergillus species in Kenya, particularly in the Eastern region where repeated cases of aflatoxicosis have been reported. Therefore, our findings contribute to new knowledge with respect to the Eastern region of Kenya.

The wide variety of toxigenic fungi reported in this study points out that the burden of mycotoxin exposure in Eastern Kenya is likely higher than previously thought. This is supported by the fact that the sampled maize grain was what was being consumed at the local household. The situation is further aggravated by lack of regulatory testing at that level. It is also possible that fatalities reported in Kenya could be due to cases of acute exposure to a cocktail of toxins.

In Kenya, the routine tests of toxins by the regulatory agencies (The Government Chemist and The Kenya Bureau of Standards) mainly aimed at aflatoxins (B1, B2, G1 and G2). While, this is proper due to the abundance of aflatoxins (Probst, 2007), we hold the view that consideration to include Ochratoxins and Fumonisins should be made since isolates of Aspergillus in this and previous studies point to higher risks of exposure (Bayman et al., 2002).

The authors have not declared any conflict of interests.

The authors acknowledge funding from the Bill and Melinda Gates Foundation (Grant ID# OPP1060246) from which reagents to carry out the analysis were purchased. Sampling was carried out with support from The National Commission for Science and Technology Foundation (NACOSTI/RCD/ST&I 5^th Call MSc 085). The authors express their heartfelt gratitude to the farmers in Eastern Kenya for their co-operation in this work. They are grateful to Collins Omondi (Egerton University) for technical support in microbiology work.