1. Introduction

Groundnut (Arachis hypogaea L.) is an important food and feed crop, which also serve as component of crop rotation in many countries ^{[1, 2]}. Groundnuts are also significant source of cash in developing countries that contribute significantly to food security and alleviate poverty ^[3]. Developing countries account for 97% of the world’s groundnut area and 94% of the total production ^[4]. However, groundnut yield in this part of the world and particularly in Africa is lower than the world average due to prevailing abiotic, biotic and socioeconomic factors ^{[1, 2, 5]}.

In warm climates grains are easily infected with toxigenic microorganisms like Aspergillus species. Aspergillus species are facultative parasites. They invade host plant tissues directly or attack tissues that have been predisposed by environmental stresses such as dry weather or damages caused by insects, nematodes, natural cracking, and harvest equipment ^[6]. They are distributed worldwide, mainly in countries with tropical climates that have extreme ranges of rainfall, temperature and humidity ^[6]. Many strains of this fungus are capable of producing aflatoxins that render the seed unacceptable due to high toxicity for human or animal consumption ^[7].

Aflatoxins are highly toxic metabolites associated with Reye’s syndrome, Kwashiorkor and acute hepatitis ^{[8, 9]}. The Eastern lowland areas of Ethiopia have considerable potential for increased oil crop production including groundnut. Particularly areas such as Babile, Fedis and Gursum are the major producers of groundnuts for local and commercial consumption ^{[10, 11]}. Nevertheless, the area may also be very conducive for toxigenic fungi like Aspergillus species owing to its warm and dry climate. Moreover, farmers’ practices of production and handling of groundnut at pre- and pos-harvest stages may provide favorable conditions for outbreaks of fungi and their mycotoxins. As Chala et al. reported, groundnut from Eastern Ethiopia is heavily contaminated by aflatoxins at levels much more than international standards, and this might be associated with infection of the crop with Apsergillus species, mainly A. falvus and A. parasiticus that are known producers of aflatoxins ^[11]. However, up to date information on the incidence of aflatoxigenicfungi, which could be basis for the reduction of mycotoxins are limited under Ethiopian conditions. On the other hand, such studies are of paramount importance to give valid recommendations for safe consumption and marketing of groundnut.

Therefore, the present study was conducted with the objective to determine the effect of storage time on occurence of Aspergillus species in groundnut in eastern Ethiopia.

2. Materials and Methods

The study dealt with field work and laboratory characterization. The field work was conducted in major groundnut growing areas (Babile, Gursum and Fedis Districts) of East Hararghe Zone, Oromia Regional State, eastern Ethiopia (Figure 1) in 2014 crop season. The areas were selected purposively as they represent the bulk of groundnut production in Ethiopia ^[12]. These areas have high potentials for rain-fed groundnut production nationally. Description of the study area is described in Ephrem Guchi et al ^[13].

A total of fourty five groundnut seed samples with different time of storage were collected from the three districts of Eastern Ethiopia in 2014. The collected samples with their storage time were depicted in Table 1 below.

Fourty five groundnut seeds per sample were surface sterilized with 10% Chlorox solution for 1 min, followed by immersion in sterile distilled water for 1 min. Surface sterilized seeds were then placed on freshly prepared potato dextrose agar (PDA) plates (five seeds per plate) and incubated for three days at 25°C. Pure cultures of different out growing fungi were obtained by transferring fungal colonies to new PDA plates using sterile toothpicks, and incubating the plates for 5-7 days at 25°C. Pure cultures of each isolate werethen stored at 4°C in vials containing 2.5 ml of sterile distilled water for further use.

Isolates were identified to a species level based on morphological (phenotypic) features as described by Cotty, Egel, Kurtzman, and Okuda ^{[14, 15, 16, 17]}. For this purpose: Isolates representing each pure culture were grown on Czapek Dox Agar (CZDA) and PDA at 25°C for 5-7 days. Fungal colonies that grew rapidly and produced colors of white, yellow, yellow-brown, brown to black or shades of green, mostly consisting of a dense felt of erect conidiophores were broadly classified as Aspergillus species while those that produce blue spores were considered as Pencillium species ^[18]. Isolates with dark green colonies and rough conidia were considered A. parasiticus ^[19]. The major distinction currently separating A. niger from the other species of Aspergillus is the production of carbon black or very dark brown spores from biseriate phialides ^[20]. Those that showed brown colony with orange and cream reverse sides were considered A. sojae and A. oryzae, respectively ^[15]. Those, which produce conidia with smooth surface on CZDA and colonies typical of A. flavus were recorded as A. flavus.

Table 1. Description of collected samples in the three districts of Eastern Ethiopia in 2014

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After the initial isolation, data were recorded on the number of infected and non-infected kernels. The frequency of Aspergillus species in groundnut samples was determined as proportion of kernels contaminated by each fungal species to the total number of kernels plated.

The data were analyzed using Statistical Package for Social Studies (SPSS) Version 16 for Windows. Frequency (out of the total samples) of occurrence of each species and relative density of each pecies was determined. Poisson regression was used in the analysis of frequency of Aspergillus species.

3. Results

The research results on seed moisture showed that the moisture content of samples ranged between 3 and 15%. According to Codex Alimentarius Commission, the maximum allowable moisture content in groundnut is 10% and it is known that above this maximum range can support mould growth during storage and can lead to aflatoxin contamination ^[21].

The present finding is consistent with the investigation by Eshetu who reported that the moisture content of groundnut seed samples ranged between 7 and 15%; the lowest moisture content was obtained from Babile area where the groundnut was stored in sack for one year and the highest was from Garamuleta area where the wet groundnut was shelled from newly harvest product ^[22]. Also, the current finding is comparable to the findings of Sseruwu who sampled groundnut seeds, from five districts in Uganda, that were stored for three to eight months, and reported moisture content of 10.5 to 14.6% ^[23]. Comparison between the results of moisture contents with other reported data by Amare et al. shows higher difference in the percentage of seed moisture content, which ranged between 3.0 to 6.8%. The difference could accrue from the methods employed to determine the seed moisture content, i.e. oven drying method ^[24].

To better isolate the various fungal species from the infected groundnut samples, simple growth analyses made on agar plates were carried out. Several fungal species became obvious from the growth media after seven days of incubation. From the seven days onwards, infected samples displayed fungal species, which were obvious enough for isolation (Figure 1).

Figure 1. Aspergillus species isolated from groundnut seed samples

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Table 2. Fungi isolated from total samples (surface sterilized and unsterilized)

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From these tests, it was easy to evaluate the percentage of various fungal occurrences in the collected groundnuts. Table 2 showed isolated species/genera, percentage frequencies from total samples, total number of isolates and percentage relative dominancy of fungi from all samples using agar plate methods.

Of the several fungal species isolated from the groundnuts, Aspergillus nomius were the most predominant with 88.63% occurrence followed by A. niger with 85.65% occurrence then A. flavus with 81.80% and Aspergillus tamari with 63.57% occurrence. The other Aspergillus species account was 16.40 % of the total non-surface sterilized samples (Table 2).

Aspergillus flavus and Aspergillus niger species were the most prevalent mycotoxigenic fungi in all time of storage. The highest occurrence of A. flavus and A. niger were in Gursum one year stored groundnut seed samples. From the detected Aspergillus species, A. niger and A. flavus were the most prevalent species of the genus in groundnut seed samples examined and they were isolated from 46 and 43 on non-surface sterilized samples respectively. Their relative dominancy in number of isolates from the total fungi was 21.67% and 20.85% respectively.

4. Discussion

Similar study in Sudan by Abdela showed that A. niger and A. flavus were isolated from all samples of investigated groundnut; A. niger occurred in 29–60% and A.flavus in 4–52% of kernels. Rhizopus spp., Penicillium spp. and Sclerotium bataticola were fairly abundant in samplesas reported in similar study ^[25]. The difference in the frequency of occurrence for A. flavus and A. niger possibly due to the difference of methods, here we used simple plate count using colony and morphology of the organism but molecular techniques (PCR) there. To ascertain whether the fungal infection is on the surface or within the groundnut seeds, the collected samples were surface–sterilized prior to incubation, and a comparative analysis was carried out with the non–sterilized sample cultures. Aspergillus nomius had the highest occurrence rate in both surface–sterilized and non–sterilized peanuts but this occurrence was higher in the non–sterilized samples. However, A. flavus and A. niger had markedly higher incidence relative to the total isolates of occurrence in the surface–sterilized samples than the non–sterilized ones. The occurrence rate of other Aspergillus species were 16.40% of occurrence in both surface–sterilized and non–sterilized sample. Aspergillus tamari was moderately existed in both surface sterilized and unsterilized samples. Unlike surface unsterilized samples, the relative dominancy of A. flavus and A. niger were increased in surface sterilized samples and the results were 28.74% and 23.35% respectively. The result showed that aflatoxigenic fungi are not such a simple surface phenomenon rather it showed the infection of seed matrices.

5. Conclusion

All samples of new harvest were above codex standard for moisture content is due to moistening may be the major contribution for allowing appropriate water activity for aflatoxigenic fungi growth. Water activities above 0.70 at 25 degrees Celsius (77 ° Fahrenheit) are ‘unsafe’ as far as growth of Aspergillus flavus and Aspergillus parasiticus and possible aflatoxin production is concerned (codex, CAC/RCP 55-2004). The high incidence of A. flavus isolated from all infected kernels strongly suggest that A. flavus might be the main producer of aflatoxins. Other fungal isolates found associated with groundnut have never been reportedly associated with aflatoxin production. The study has showed that aflatoxins are common contaminant in groundnut of growing in the study area of eastern Ethiopia. Comparing the obtained value of aflatoxigenic fungi with different international standards showed, as time of storage increased the groundnut quality in relation to international standards are also affected. From the result, it is possible to conclude that storage time are the major factors for aflatoxigenic fungi contamination. It is imperative to avoid a long–term storage system in eastern Ethiopia.

Acknowledgements

We are grateful to the Ministry of Education, Ethiopia for financial assistance.

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