Distribution of indigenous strains of atoxigenic and toxigenic Aspergillus flavus and Aspergillus parasiticus in maize and peanuts agro-ecological zones of Kenya

The genus Aspergillus, a member of the phylum Ascomycota, includes over 185 known species. Members of Aspergillus section Flavi are characterized by their ability to produce flavine-derived secondary metabolites which include the aflatoxins. Aflatoxins are mycotoxins associated with hepatotoxicity, mutagenicity and carcinogenicity in humans and animals [1, 2]. Species capable of producing aflatoxins include Aspergillus flavus, Aspergillus parasiticus, and several less common taxa such as Aspergillus nomius, Aspergillus tamarii, A. pseudotamarii, A. minisclerotigenes and A. bombycis [3, 4]. Other Aspergillus outside of the section Flavi are also known to produce aflatoxins and species with this ability have been shown to be more diverse than previously thought [5].

Grains of important food and export crops in Africa, such as maize and peanuts, are susceptible to contamination with different toxin-producing moulds. Of particular importance are aflatoxins that pose serious health risks to humans and animals. In Kenya, the toll on human health from periodic outbreaks that record case fatality rates of 39 % [6] remain common. In addition, food grains contaminated with aflatoxin-producing fungi also lose their quality based on nutritional value, taste, smell, appearance, and are no longer suitable for consumption or sale. The major challenge is that the contaminated cereals such as maize may appear just like normal grains without any outward physical signs of fungal infection [7].

The earliest reported case of aflatoxicosis in Kenya was in 1977, where several dogs and poultry died in Nairobi, Coast and Rift Valley Provinces after feeding on poorly stored grains, while in 1978, samples of human and dog food (flour) were found to be contaminated with [8]. In 1984, twelve people died in Machakos district of Kenya after consuming aflatoxin contaminated maize, while in 1995, about 172 samples of maize analyzed were found to have aflatoxin. In 2004, there was an outbreak of acute aflatoxicosis in Kenya, one of the most severe episodes of human aflatoxin poisoning in history. A total of 317 cases were reported by July 2004, with a case fatality rate of 39 % [9, 10]. This epidemic resulted from ingestion of contaminated maize [11]. Other outbreaks occurred in 1981, 2001, 2004, 2005, 2006, 2007 and 2008 resulting in sickness, death and destruction of contaminated maize [8, 9, 11–13]. Maize from the affected areas contained as much as 4400 ng g⁻¹ aflatoxin B1, which is 440 times greater than the 10 ng g⁻¹ tolerance level set by the Kenya Bureau of Standards. Most of the aflatoxin poisoning outbreaks occurred in remote villages and, therefore, the number of people affected could have been higher than reported [11]. The aflatoxin contamination in maize has been associated with drought combined with high temperatures as well as insect injury [14].

Aspergillus flavus and the closely related subspecies A. parasiticus have a world-wide distribution and normally occur as saprophytes in soil and on many kinds of decaying organic matter. They readily infest several important crops such as corn, cottonseeds, peanuts and tree nuts. They are widely distributed with greater quantities of the fungus occurring in warm climates [4, 15].

Gene flow within A. flavus is primarily limited by a vegetative compatibility system [16, 17] that delineates the species into numerous genetic groups called Vegetative Compatibility Groups (VCGs). A. flavus VCGs clonal lineages [18] that exist in complex communities are composed of many VCGs. VCGs vary in many characteristics; including aflatoxin-producing ability. Populations of A. flavus in an individual agricultural field contain isolates of many VCGs [4, 19]. VCGs with aflatoxin-producing potential are known to vary less among isolates within a VCG than among isolates from different VCGs [18]. Communities resident in different fields, areas, and regions may vary widely in average aflatoxin-producing ability [20–22]. Isolates and VCGs that do not produce aflatoxin, called atoxigenic, are common within A. flavus communities [20–23]. Surveys of A. flavus isolates from various geographic regions have revealed differences in the proportions of isolates that produce low, medium, and high amounts of aflatoxins [5, 24, 25]. For instance, in Argentina [26] and Iran [27] less than 30 % of the A. flavus isolates were capable of producing aflatoxin whereas in Nigeria the number was much higher, exceeding 50 % [28]. In the southern USA most A. flavus isolates produce aflatoxin [24, 25]. In the USA, there are currently several atoxigenic strains of A. flavus used to reduce crop contamination.

Biological control strategies directed at utilizing non-aflatoxin (atoxigenic) strains to limit aflatoxin contamination on crops have been pursued for almost two decades [29]. These strategies seek to give atoxigenic strains a competitive edge and exclude their aflatoxin-producing relatives, therefore decreasing the potential for contamination in crops and the environment. Successful strategies have been accomplished by using native non-aflatoxin-producing strains such as A. flavus [30].

In this study, we sought to identify the natural occurence of different strains/species of Aspergillus and to identify the ecological prevalence of both aflatoxin-producing and non-producing strains. Identification of the proportion of non-aflatoxin-producing strains in natural environments is an important step towards estimating their applicability in the biological control of the more medically and economically important toxigenic strains in Kenya.

Aspergillus section Flavi were detected in all 57 soil samples collected from various agroecological zones of maize and peanut growing areas in Kenya. This widespread distribution and prevalence conforms to previous findings of similar studies in Israel, Thailand, North America and West Africa [19, 28, 35]. Several members of the section Flavi including A. niger, A. fumigatus, A. clavatus, A. ochraceus, A. terreus and A. versicolor have been shown to be major mycotoxin-producing contaminants in various agricultural produce in Kenya. Our study supports a high incidence of Aspergillus section Flavi with A. flavus being the most predominant (63.2 %), A. flavus could indeed be linked to B1 aflatoxicosis contamination that has been shown to be prevalent as was determined by Probst et al. [9]. Members of A. parasiticus were less prevalent but nevertheless produced high concentrations of both B and G aflatoxins (Tables 3, 4). They, therefore, contributed significantly to the aflatoxicosis potential of resident fungal communities, a situation similar to that observed in West Africa by Donner et al. [28]. While all species produced B aflatoxin in different quantities, only A. parasiticus produced G aflatoxins among all groups studied in Kenya.

The negative correlation of pH and proportions of L strains could be an indication of the poorer adaptation of these strains to higher pH optima compared to the S strains. However, the adaptations of the two strains is not mutually exclusive as each of the two strains occur in different proportions within all the pH ranges observed. In addition, the range of soil pH variation across the examined regions was narrow, ranging from weak acidic to weak alkaline. The higher incidence of S strains in more alkaline soils has previously been observed in North American soils among isolates of A. parasiticus, A. tamarii and A. flavus [24]. However, in Nigeria [28], a negative correlation was observed. The effect of pH has been shown to play a lesser role in A. flavus population dynamics, with climate being cited as a more important determinant [35]. Therefore, other shared factors within the study areas such as temperature maxima of 40 °C, average humidity, semi aridity and transitional savannah ecosystems provide a range of biophysical conditions conducive for elevated levels of aflatoxin-producing fungi.

The aflatoxin-producing potential of Aspergillus communities is higher when S strain is present, as L strain isolates produce on average only 33 % of the toxin yield from S strain isolates [24]. In West Africa, S strain isolates produce greater quantities of aflatoxins than L strain isolates. This corroborates the finding of this study, in which the S strains produced the highest concentration of aflatoxins in Kenya. All the isolates of A. flavus (both L and S strains) did not produce the G aflatoxins but produced the B toxins. The percentage of A. flavus L-strain isolates that produced aflatoxins varied with geography and climate (Fig. 3).

Aflatoxin-producing fungi vary widely in many characteristics, including virulence for crops and aflatoxin-producing capacity [33]. Aspergillus flavus and A. parasiticus are most commonly implicated as causal agents of aflatoxin contamination. Aspergillus flavus has two morphotypes, the typical or L strain (sclerotia of >400 µm in diameter) and the S strain (sclerotia of <400 µm in diameter) [33, 36]. S-strain isolates produce more aflatoxins than L-strain isolates, on average. Many L-strain isolates produce no aflatoxins (“atoxigenic”) [24].

All members of A. flavus lack the ability to synthesize G aflatoxins due to a 0.8–1.5 kb deletion in the 28-gene aflatoxin biosynthesis cluster [18]. In contrast to cases in the United States, studies conducted in West Africa found out that an unnamed taxon (sometimes called strain S_BG) is commonly implicated in contamination events [5]. Strain S_BG is morphologically similar to the S strain of A. flavus, but DNA-based phylogenies reveal strain S_BG to be a distinct species ancestral to both A. flavus and A. parasiticus [37, 38].

Aflatoxin levels unacceptable for human consumption may occur even in areas with relatively low frequencies of aflatoxin producers. In the current study, 59.3 % of aflatoxin-producing L-strain isolates produced more than 10 × 10⁵ ng g⁻¹ (14.3 ppb) aflatoxin B1. This combined with high incidences allows the L-strain to be the largest contributor to the average aflatoxin-producing ability of fungal communities in the five districts (Fig. 1; Table 4) and a potentially important causal agent of contamination in Kenya. Similar results were observed in Nigeria [28]. This is in contrast, with earlier studies conducted in Kenya where the S-strain was observed to be the primary cause of maize aflatoxin contamination [9]. The current study shows that S-strain is present in low frequency or absent in certain sites.

Incidences of atoxigenic strains of section Flavi varied widely among districts and agroecological zones (Table 1). The non-aflatoxin-producing strains are common in crop environments [21, 23, 24]. The atoxigenic strains of A. flavus and/or A. parasiticus have been employed as biopesticides directed at minimizing crop contamination with aflatoxins [4, 39]. Successful and effective biocontrol strategies necessitates that high ratios of atoxigenic to toxigenic strains be exhibited [40]. In the current study, high incidences of atoxigenic strains of section Flavi members were found in Kibwezi and Makueni districts both in lower midland zones (LM3, LM4 and LM5). These native populations provide potential biological control agents to manage the toxigenic strains.

Aflatoxin contamination of crops can be minimized by early harvest, prevention of insect damage and proper storage [41]. However, despite the careful management, unacceptable aflatoxin levels may occur from unpreventable insect damage to the developing crop or from exposure of the mature crop to moisture either prior to harvest or during storage in modules, handling, transportation or even use [33].

For many diseases, traditional chemical control methods are neither always economical nor are they effective, and fumigation as well as other chemical control methods may have unwanted health, safety and environmental risks in Kenya and many other developing countries. The antifungal abilities of some beneficial microbes have been known since the 1930s, and there have been extensive efforts to use them for plant disease control since then. However, they are only now being used commercially [33].

Aflatoxins cannot be readily removed from contaminated foods by detoxification. Therefore, there is interest in developing a biological control method that can increase crop safety by decreasing toxin content and this depends on the competitive displacement of toxigenic isolates using atoxigenic isolates of the same species. It has also been reported that aflatoxin production is inhibited by lactic acid bacteria, Bacillus subtilis and many moulds. This inhibition may result from many factors including competition for space and nutrients. In general, competition for nutrients required for aflatoxin production but not for growth and production of anti-aflatoxigenic metabolites by co-existing micro-organisms [42].

Currently, atoxigenic A. flavus L-strain isolates are used to competitively exclude aflatoxin producers during crop infection and thereby limit contamination in U.S. agriculture [4]. Such atoxigenic strains are highly effective against the S strain [4]. Adaptation and deployment of similar technologies in Africa could provide a promising strategy for prevention of future aflatoxicoses in East Africa while enhancing export possibilities for maize and peanuts [43].

Aspergillus section Flavi was resident in all sampled maize fields and quantities of section Flavi were higher on average in Kenya and compares to the ones in West Africa [35]. The incidences of the section Flavi in Kenyan soils demonstrates the fungal growth on crop-associated with organic matter. Corncobs, peanut pods and other crop debris harbor section Flavi for at least 3 years after harvest [44].

This study has reported high level of atoxigenic species of Aspergillus section Flavi. In vitro analysis showed that there is a growth competition between atoxigenic and toxigenic spp of Aspergillus section Flavi. The existing methods of biological control of aflatoxicosis by use of the atoxigenic strains can therefore be tested and adapted to Kenyan agriculture. The study has also established that the production of aflatoxins by these species depends on the prevailing physiological conditions such as temperature and moisture. Poor management practices such as poor storage, late harvesting, among others are predisposing conditions for the proliferation of the fungi, so there is need for education on the best practices so as to minimize on contamination. Furthermore, morphological analysis of Aspergillus section Flavi is limited with mechanisms of getting the true identity of the fungal species. There is need to adopt rapid and sensitive molecular techniques to support the morphological identifications. Finally, there is need to screen all the farm produce for possible aflatoxin contamination to reduce economic burdens related to the menace. We, therefore, hope this study will be taken into consideration by regulatory authorities in Kenya and beyond.

In conclusion, we report an ubiquitous presence of the members of the Aspergillus section Flavi within maize and peanut growing fields in Kenya. The diversity is characterized by a significant presence of atoxigenic A. flavus and A. parasiticus. Such atoxigenic variants have been previously applied in the biological suppression of natural populations of toxigenic strains and thereby minimizing aflatoxin contamination in peanuts [40], corn [45] and cotton [4]. The existing methods of biological control of aflatoxicosis by use of atoxigenic strains can therefore be tested and adapted to Kenyan agriculture. Control of natural populations of aflatoxin-producing fungi will lead to overall reduction of aflatoxicosis in farm produce as reported to be common in the regions studied [7, 9].