ABSTRACT
Groundnut (Arachis hypogaea L.) is an important food crop in Africa which is a source of nutrients and income in rural areas of Zimbabwe. It is considered to be a crop highly susceptible to aflatoxin contamination. Accordingly, the objectives of this study were to understand the presence of mycoflora, their association and the level of contamination by aflatoxins of groundnut from various markets in Zimbabwe. Thirty groundnut samples were purchased randomly from Bulawayo (Shashe and Main market), Gweru (Kudzanayi and Kombayi markets) and Harare (Mbare and Highfield markets). Identiï¬cation of various fungi was determined using the cultural method on Czapek Dox Agar. Fungi belonging to genera Aspergillus, Mucor, Penicillium and Rhizopus were isolated and characterised from six groundnut markets. Rhizopus species was the most dominant and negatively associated with other fungi species which is attributed to differences in environmental requirements or competition. Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger and Aspergillus parasiticus were identified in groundnut samples with A. flavus being the dominant and found in all markets. The range of AFB1 in groundnut samples analysed using a semi-quantitative immunochromatographic technique was within the safe limits for human consumption according to existing Zimbabwe (5 ppb) regulation. The presence of aflatoxigenic fungi (A. flavus and A. parasiticus) in groundnuts, however, means there is potential for aflatoxin production and fungal proliferation when conditions are favourable.
Key words: Aflatoxigenic, Arachis hypogaea, Aspergillus species, mycology.
The impact of aflatoxin contamination on agricultural commodities is immense and production losses and trade has been severely affected. In developing countries due to lack of storage infrastructure, poor harvesting and handling techniques and lack of effective monitoring mechanisms aflatoxins occur frequently in various agricultural commodities (Negash, 2018). One of these crops is groundnut that is widely grown in semi-arid areas and prone to aflatoxin contamination. There is low production of groundnut in African countries because of unreliable rains, lack of inputs, use of retained seed, poor agronomic practices, pests and diseases (Ajeigbe et al., 2015; SNV, 2012). Among other challenges is the frequent recurrence of droughts and variable rainfall patterns. Drought increases the probability of aflatoxin contamination on groundnut at any stage of production cycle and thereafter affecting international exports from Africa. Warm climate and high humidity experienced in most tropical countries predispose crops to aflatoxin contamination (Pazderka and Emmott, 2010).
Groundnut is an important crop in the livelihoods of many Zimbabweans and comes second after maize. Majority of farmers grow groundnut during rainy season which is a risk factor during harvest and post-harvest handling that predisposes the crop to aflatoxin contamination. The ideal environmental conditions for aflatoxin production in stored seed are high temperatures up to 45°C, high humidity 65 to 90%, moisture in excess of 9%, damage by insect pests, rodents and lack of genetic inheritance in the host plant (Okello et al., 2010; Ncube and Maphosa, 2020).
Dube and Mtetwa (2015) showed that the majority of stakeholders involved in the groundnut value chain in Mutare, Zimbabwe had never heard about aflatoxins. Several researchers showed that most commodities were contaminated by aflatoxins in Zimbabwe especially groundnuts and maize (Nleya et al., 2018). Dried traditional foods in Zimbabwe were found contaminated by the aflatoxigenic fungus from Bulawayo markets (Dangwa et al., 2014). Similarly, Mupunga et al. (2014) observed that groundnuts from Bulawayo markets in Zimbabwe were contaminated with high levels of aflatoxin. Dube and Maphosa (2014) reported aflatoxin prevalence in groundnut samples collected from seven districts of the Matebeleland provinces in Zimbabwe. Aflatoxin results for groundnuts from Bulawayo showed that 17% of the samples were contaminated with total aflatoxins ranging from 6.6 to 622.1 ppb (Mupunga, 2013). Accordingly, there is need for more research and understanding the prevalence of the aflatoxigenic fungi and current levels of aflatoxin contamination of groundnut on the market. There has been no attempt to identify which fungal species are more prevalent in groundnut as well as their association. Accordingly, the knowledge of aflatoxigenic fungal diversity, their association and prevalence of aflatoxin contamination will lead to informed breeding for resistance and aflatoxigenic management.
Collection of groundnut samples
Thirty samples of groundnuts were purchased from six open markets in three cities of Zimbabwe, Bulawayo (Shashe and Bulawayo main markets), Harare (Mbare and Highfield markets) and Gweru (Kombayi and Kudzanayi markets). These markets were purposively selected because they receive groundnuts from most places of the country. Five groundnut vendors were randomly selected from each market and raw, shelled 1 kg groundnut samples were collected per vendor. Samples collected represented the whole consignment and was done by collecting groundnuts from different sections of the sack. Collected samples were packaged in polyethylene bags, sealed, labelled and transported to Lupane State University Laboratory and kept in a cool refrigerator at 4°C.
Preparation of media
Czapek Dox Agar (CDA) (Thermoscientific, UK) was used to isolate and identify the fungi and was prepared according to manufacturer’s instruction.
Fungal isolation
Isolation of fungi from groundnuts was done under laboratory conditions using Czapek dox agar. Groundnut samples were surface sterilised for one minute using 80% ethanol and air dried for 30 min. Sterilized and unsterilized samples were plated on petri-dishes (10 kernels per petri dish) containing CDA. Ten dried seeds were placed in each petri-dish and replicated three times. Plates were incubated at 25°C for 72 h and replicated three times in completely randomised design. After incubation, morphological and growth characteristics were observed under a microscope. Colony characteristics (colour, shape) that grew and number of seeds infected with the same type of fungus were recorded. The individual isolates were transferred to new CDA plates in order to obtain pure cultures. Inoculation was done using flame sterilised inoculating needle dipped into a spore formed of the suspected fungi. The Petri-dish containing the CDA medium was spot inoculated and incubated at 25°C for 72 h in an incubator. Slides were made for examination of morphological characters under a microscope and colony characteristics were recorded (Cappuccino and Sherman, 1999).
Characterisation and identification of fungi
Identification was done using microscopic and macroscopic examination based on colony and morphological characteristics of pure cultures of the isolates. The micro morphological characteristics has been used for fungal identification including the shape of conidia heads, serration, the number of branching points between vesicle and philiades (uniseriate or biseriate), stripes (colour, shape, texture, and dimensions), vesicles shape and diameter, presence of metulae and conidia (Samson et al., 2014; Nyongesa et al., 2015). The Aspergillus consists of swollen conidiophore tips forming vesicle with philiades and metulae with chains of conidia. However, conidiophore tip of Penicillium lacks vesicles and has a number of metulae followed by philiades (Campos, 2019). The macro morphological features were used for identification of species based on the colony colour and texture (yellow green for Aspergillus flavus and dark or nearly ivy green for Aspergillus parasiticus) using the identification keys illustrated in a manual (Cappuccino and Sherman, 1999). However, the following characteristics of colonies were also considered; colony growth rates, texture, colour of mycelia, colony reverses and degree of sporulation (Samson et al., 2014; Nyongesa et al., 2015). Based on macro and micro morphological characters, the following species were identified, A. flavus, Aspergillus fumigators, Aspergillus niger, A. parasiticus, Mucor species, Penicillium species, and Rhizopus species. Different Aspergillus species with varying morphologies were identified. Macro and micro morphological characteristics were used for fungal identification from genus to species level together with taxonomic keys (Cappuccino and Sherman, 1999; Samson et al., 2014; Nyongesa et al., 2015).
Semi-quantitative detection of AFB1 using immunochromatographic technique
AFB1 Rapid Test based on competitive lateral flow immunochromatographic assay (Krska and Molinelli, 2009) was performed as a semi-quantitative step to identify whether aflatoxins AFB1 were present. This method has a lower limit of 5 ppb for AFB1. Thirty samples of groundnuts were brought to the laboratory and each sample was mixed thoroughly to achieve complete homogenization. About 10 g of groundnuts were picked from each sample and grounded to the particle size of the instant coffee using the pastel and mortar. A 2 g groundnut sample, 2 ml of pure water, 8 ml of ethyl acetate were added into a 15 ml centrifugal tube and mixed for 10 minutes. After emulsification, the sample was centrifuged for 5 minutes at 4000 rpm. About 4 ml of supernatant (ethyl acetate layer) was transferred into a 250 ml beaker and evaporated to near dryness and diluted in water. The results of AFB1 test were interpreted within 5–10 minutes after placing the droplets into the assay hole (Krska and Molinelli, 2009).
Data collection
Genus and species of fungi found growing on the surface of groundnuts samples collected from six groundnut markets were recorded together with their frequency of occurrence (Algabr et al., 2018). The percentage frequency of occurrence was calculated using the following formula:
% Frequency = Number of seeds on which fungi are growing / Total number of seeds × 100
Experimental design and statistical analysis
The experimental design used in this research is a 6×2 factorial replicated three times arranged in Completely Randomised Design (CRD). The two factors were divided into two categories which are markets and sterilisation. Principal component analysis was used to detect fungal species association and level of importance. All data were analysed using Genstat 13 (Payne et al., 2010).
Incidence of fungal contamination in groundnuts
Prevalence of fungal species significantly differed (p<0.05) across the six markets. Rhizopus species were the most prevalent fungus followed by A. flavus (Figure 1).
A. flavus was found in both sterilised and unsterilised groundnut from the six markets (Table 1). Rhizopus spp. were isolated from both non-sterilised and surface sterilised samples of five groundnut markets with a higher percentage of non-sterilised than in sterilised groundnut samples. Penicillium species were found in Harare Mbare market and isolated in 0.6% of surface sterilised groundnuts. Bulawayo Shashe market was the only market were Mucor spp. (4%) was found in non-sterilised groundnuts.
Principal component analysis of fungal distribution in groundnut samples of six markets of Zimbabwe
The principal component biplot analysis showed high correlations between A. flavus and A. fumigatus and A. parasiticus and Penicillium spp. as evidenced by the same direction dimension vectors and small angles between them (Figure 2). A. flavus and A. parasiticus were the most prominent fungal species as shown by the long vector. Rhizopus was negatively associated with all the other aflatoxigenic fungi with negative vector loadings (Figure 2).
Four principal components were important (eigen value >1) and explained 68% of the total variation. The first principal component explained 23% of the variation and was strongly positively associated with A. flavus and negatively associated with Rhizopus (Tables 2 and 3).
In the second, third and fourth principal components explaining 38%, 53% and 68% of variance respectively A. parasiticus had the highest positive loading (Table 2). These results suggest the high prevalence of the two fungal species, that is, A. flavus and A. parasiticus.
The results obtained in thirty groundnut samples collected from all six major markets of Zimbabwe, showed that AFB1 was not detected after analysis. This showed that AFB1 level was below the cut-off point of 5 ppb (Table 4). The results of each sample were recorded as positive or negative.
Occurrence of mycoflora in six selected groundnut markets in Zimbabwe
Different types of fungi were found in groundnuts sourced from six selected markets of three major cities of Zimbabwe which are Bulawayo Shashe, Bulawayo Main, Gweru Kombayi, Gweru Kudzanayi, Harare Mbare and Harare Highfield. The presence of Aspergillus spp. (A. flavus, A. parasiticus, A. niger, A. fumigatus), Mucor spp., Penicillium spp. and Rhizopus spp. in groundnuts have also been reported (Mupunga, 2013; Njoroge et al., 2016). Abuga (2014) isolated Mucor, Penicillium, Rhizopus and Aspergillus spp. with different frequencies on groundnut seeds. Other studies done in Kenya, Nigeria and Yemen also revealed the occurrence of Aspergillus, Mucor, Rhizopus and Penicillium spp. in groundnuts and other products (Menza and Muturi, 2018; Salau et al., 2017; Tobin-west et al., 2018). This suggests that conditions that favour one fungal species may support another species as well (Bayman et al., 2002). Results of the present study revealed higher fungi occurrence on groundnut from Bulawayo Shashe market and the lowest was found in Gweru Kombayi market. The variations could be caused by poor storage, poor sanitation and handling by the vendors though groundnuts are prone to aflatoxin contamination (Tobin-west et al., 2018). Groundnuts from all the six markets were stored in uncovered sacks exhibiting them to consumers thereby being exposed to airborne fungal spores.
Surface sterilisation was done to determine the presence of internal fungi in groundnuts and gave variable results. In all instances A. flavus was not affected by surface sterilisation suggesting that it was not superficial but had established itself in the groundnuts (Bayman et al., 2002). Presence of fungi was observed on both sterilised and non-sterilised groundnuts from all markets with non-sterilised having high frequency compared to sterilise suggests a high risk of aflatoxin.
Prevalence and association of Aspergillus spp. in selected groundnut markets
All selected groundnut markets from Bulawayo, Gweru and Harare were contaminated with at least two or more of Aspergillus spp., A. flavus, A. parasiticus, A. niger and A. fumigatus. In this study, A. flavus was identified in all groundnut markets showing that most groundnut species grown are susceptible to contamination from Aspergillus spp. (Salau et al., 2017). There were differences in occurrence of Aspergillus among Bulawayo, Harare and Gweru markets because of different weather conditions. There was high contamination from hot and dry climatic conditions prevalent in Bulawayo and Gweru compared to low incidence of fungi to cool and wet areas of Harare. However, the risk of aflatoxin is present countrywide given that Aspergillus spp. mainly A. flavus and A. parasiticus are considered as aflatoxin producers (Boli et al., 2014). Previous studies revealed that A. flavus is mostly prevalent in foods including groundnuts and contains aflD toxigenic genes producing aflatoxin frequently in dry weather conditions (Menza and Muturi, 2018). A. parasiticus rarely occurred (0.3 - 6%) in these selected markets and furthermore was reportedly not a challenge on stored groundnuts (Bediako et al., 2019). A. niger had high occurrence in non-sterilised groundnuts from Bulawayo Main, Bulawayo Shashe and Gweru Kudzanayi while A. fumigators was detected in groundnuts from Bulawayo main market (8%). Bediako et al. (2019) reported that A. niger does not produce aflatoxins, it produces other toxins such as ochratoxin A and malformins. This study showed that some groundnuts are contaminated with Aspergillus and the predominant species was A. flavus. The presence of Aspergillus spp. in groundnuts exposes human to aflatoxin making it unsafe for consumption. Al-Amodi (2016) suggested that presence of these fungi causes groundnut seed to decay, reduces germination and causes damage of stored groundnuts. The widespread distribution of A. flavus, A. niger, A. fumigatus and A. parasiticus shows their importance on stored groundnuts in Zimbabwe. Even distribution of A. flavus across all selected markets implies that management strategies should be implemented on all markets under investigation. From this study A. flavus was more prevalent in the samples collected from six markets while Mucor and Penicillium spp. were less prevalent. Rhizopus was always negatively associated with other fungi species which can be due to differences in environmental requirements or competition. This was however contrary to Bayman et al. (2002) who observed a positive association of A. flavus and A. niger in tree nuts.
The range of AFB1 in groundnut samples analysed in this study were within the safe limits for human consumption according to existing Zimbabwe (5 ppb) regulation (Table 2). Though groundnuts were reported as a good substrate for aflatoxins producing fungi, the results showed that none of the thirty groundnut samples from six major markets of Zimbabwe contained any detectable levels of AFB1 (≥5 ppb). In contrast with Mupunga et al. (2014), Dangwa et al. (2014), and Siwela et al. (2011) reported high contamination levels of aflatoxins in Zimbabwean groundnuts. Previous studies on aflatoxin in groundnuts from Bulawayo showed that 2/18 contaminated samples had detectable levels of AFB1 above 4 ppb the maximum allowable limit set by the EU and Codex Alimentarius Commission (7 ppb) (Mupunga, 2013; Kamika et al., 2014). The levels of AFB1 were below the cut-off point of 5ppb this might be because the conditions were not conducive for aflatoxin production. Furthermore, previous studies have shown that the interaction of A. flavus with other fungal species decreases its aflatoxin production or degrades it (Mann and Rehm, 1976; Mislivec et al., 1988). The presence of aflatoxigenic fungi (A. flavus and A. parasiticus) in groundnuts, however, means there is potential for aflatoxin production and fungal proliferation when conditions are favourable. Though levels were low, chronic exposure to aflatoxins remains a health concern (Williams et al., 2004; Okoth and Kola, 2012).
The authors have not declared any conflict of interests
The authors thank ICRISAT-Matopos, Zimbabwe for availing the AFB1 assay kits and their laboratory technician for assisting during aflatoxin detection.
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