Incidence of Aspergillus contamination of groundnut ( Arachis hypogaea L . ) in Eastern Ethiopia

The production of groundnut is constrained by several factors, among which is Aspergillus spp. In addition to causing quantitative losses, Aspergillus spp. produce highly toxic and carcinogenic chemical substances known as aflatoxins. This study was conducted with the objectives to (i) identify Aspergillus species associated with groundnuts, (ii) determine the frequency of seed contamination, and (iii) survey agro-ecological conditions related to groundnut contamination by Aspergillus spp. About 270 groundnut samples were collected from farmers’ storage, fields and local markets of three districts that is, Babile, Darolabu and Gursum of Eastern Ethiopia for mycological analysis in the year 2010. Results of the mycological analysis suggested heavy infestation of groundnut samples by various molds including Aspergillus niger, Aspergillus flavus, Aspergillus ochraceus, Aspergillus parasiticus and Pencillium species. At the district level, the incidence of infected groundnut kernels ranged from 50 to 80%. Within the district kernel infection varied between 36.3 and 100%. The common Aspergillus symptoms (yellowing or chlorotic leaves, wilting, drying and brown or black mass covered by yellow or greenish spores) were also observed in groundnut fields. The current results were consistent with our earlier report of heavy aflatoxin contamination of groundnut from the same places, suggesting the urgent need to apply control measures against toxigenic fungi and associated mycotoxins.


INTRODUCTION
Groundnut (Arachis hypogaea L.) is an important food and feed crop, which also serve as component of crop rotation in many countries (Pande et al., 2003;Upadhyaya et al., 2006).Groundnuts are also significant source of cash in developing countries that contribute significantly to food security and alleviate poverty (Smart et al., 1990).Developing countries account for 97% of the world's groundnut area and 94% of the total production (FAOSTAT, 2010).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 (Pande et al., 2003;Upadhyaya et al., 2006;Caliskan et al., 2008).
In warm climates grains are easily infected with toxige-nic microorganisms like Aspergillus species.Aspergillus spp.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 (Pettit and Taber, 1968).They are distributed worldwide, mainly in countries with tropical climates that have extreme ranges of rainfall, temperature and humidity (Pettit and Taber, 1968).Many strains of this fungus are capable of producing aflatoxins that render the seed unacceptable due to high toxicity for human or animal consumption (Reddy et al., 1996).Aflatoxins are highly toxic metabolites associated with Reye's syndrome, Kwashiorkor and acute hepatitis (Wild and Hall, 1999;Wild and Turner, 2002).The Eastern lowland areas of Ethiopia have considerable potential for increased oil crop production including groundnut.Particularly areas such as Babile, Darolabu and Gursum are the major producers of groundnuts for local and commercial consumption (Getnet and Nugussie, 1991;Chala et al., 2012).Nevertheless, the area may also be very conducive for toxigenic fungi like Aspergillus spp.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. (2012) reported, groundnut from East 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 spp., mainly A. falvus and A. parasiticus that are known producers of aflatoxins.However, up to date information on the prevalence of fungi, and studies on environmental factors and farmers' practices that promote fungal contamination, 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.The objectives of this study were to: i) identify Aspergillus species associated with groundnut in East Ethiopia, ii) determine the frequency of seed contamination, and iii) survey agro-ecological conditions related to contamination of groundnut by Aspergillus spp.

Surveys of groundnut and sample collection
Groundnut samples were collected from three districts (Babile, Darolabu and Gursum) of Eastern Ethiopia in the year 2010.The samples were collected in three groups that is, from farmers' stores, fields and from local markets of selected locations of the three districts.Geographic description of survey locations are given in Table 1.In each district, five locations were chosen based on the groundnut productions potential.Six samples were collected from each location and hence 30 samples were collected per district making the total number of samples collected from all over the three districts 270 (3 districts × 5 locations per district × 6 samples per location × 3 sample groups per district).Data were also gathered on planting and harvesting dates of the crop, the variety, soil type, previous crop, and types of cultural practices.The common methods of storage structures, drying materials and grain moistures were observed.

Fungal isolation
Fifty 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 were then stored at 4°C in vials containing 2.5 ml of sterile distilled water for further use.

Species identification
Isolates were identified to a species level based on morphological (phenotypic) features as described by Cotty (1994), Egel et al. (1994), Kurtzman et al. (1997), andOkuda et al. (2000).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 spp.while those that produce blue spores were considered as Pencillium spp.(Okuda et al., 2000).Isolates with dark green colonies and rough conidia were considered A. parasiticus (Klich, 2002).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 (Raper and Fennell, 1965).Those that showed brown colony with orange and cream reverse sides were considered A. sojae and A. oryzae, respectively (Cotty, 1994).Those, which produce conidia with smooth surface on CZDA and colonies typical of A. flavus were recorded as A. flavus.

Data on frequencies of kernel infection by Aspergillus and
Penicillium species for samples collected from different locations of the districts were subjected to analysis of variance (ANOVA) using the SAS computer package, version 9.11 (SAS, 2003).LSD test at the 0.05 probability level was used for mean comparison.

Identification of Aspergillus species associated with groundnut
Four different Aspergillus spp.were found to be associated with groundnut samples collected from Eastern Ethiopia (Figure 1).The first species isolated from the collected samples was A. niger.The major distinction currently separating A. niger from the other species of Aspergillus is the production of carbon black or dark brown spores of biseriate phialides (Raper and Fennell, 1965).The current study also confirmed the production of black or brown-black or black conidia by this species (Figure 1).
A. flavus was the second species identified in this study.Colonies of this fungus were characterized by yellow to dark, yellowish-green pigments, consisting of a dense felt of conidiophores or mature vesicles bearing phialides over their entire surface (Gourama and Bullerman, 1995).In general, A. flavus was known as a velvety, yellow to green or the old colony was brown mould with a goldish to red-brown on the reverse.The conidiophores were variable in length; walls of A. flavus conidia were smooth to finely roughened or moderately roughened, pitted and spiny.These observations were consistent with the findings of Gourama and Bullerman (1995), who reported A. flavus colonies as being initially   yellow, turning to yellow-green or olive green with age and appearing dark green with smooth shape and some having radial wrinkles.
The third species identified in the current work, A.
ochraceoroseus, produced yellow-gold conidia (Bartoli and Maggi, 1978).The A. ochraceus was characterized particularly by its pale yellow conidial heads, orange-red conidiophores with coarsely roughened walls, light colored sclerotia, and salmon-pink mycelial turf on the reverse side of CZDA.Colonies of this species also produced near white to light yellow pigment and were dull yellow to dark yellow or sometimes brown on the reverse.
A. parasiticus was the fourth species isolated from groundnut samples tested in the current study.Colonies representing this species produced dark green and rough conidia on CZDA at 25 and 37°C after 5-7 days of incubation.Similar study by Peterson et al. (2001) distinguished A. parasiticus from A. bombycis by its typically dark green color on CZDA.
Groundnut plants infected by Aspergillus spp.showed typical symptoms of infection as shown in (Figure 2).These include yellowing of leaves or chlorosis and premature death and dropping of leaves, wilting, drying single plant and patching or drying of localized areas within the fields.Underground grains were rotten and distorted, and the plants were pulled out of the ground easily.On some dried plants, brown or black mass covered by yellow or greenish spores were observed in infected fields.A study by Subrahmanyam and Ravindranath (1988) stated the presence of shriveled and dried grains covered by yellow or green spores, when groundnut plants are infected by A. flavus (Aflaroot or yellow mold).The same study reported highly stunted seedlings; reduced leaf size with pale to light green; crown rot or collar rot, with germinating seeds covered with masses of black conidia; and rapid drying of plants due to infection of groundnut by Aspergillus spp.

Frequency of kernel contamination
Proportion of kernel contamination by Aspergillus spp.varied from 50% at Babile market to about 80% at farmers' fields in Gursum district and storage houses in the district of Darolabu (Figure 3).Groundnut samples collected from storage houses in the districts of Gursum and Babile, and farmers' fields at Babile had the second highest kernel contamination (70%).
When samples were analyzed at the location level (within district), kernel contamination varied significantly (p<0.05) from 36% at Bishan Babile market (Babile district) to 100% in farmers' fields of Harobate (Gursum district) (Table 2).Samples from stores in Bishan Babile and Shekabdi kebeles (Babile district), stores in Sororo location of Darolabu district, farmers' field in Kasa Oromiya of Gursum district were infected at a proportion of 93%.
In addition, groundnut samples collected from farmers' fields in Ausharif and Shekabdi location of Babile district, stores of Sakina and Odalaku (Darolabu district), and stores of Audal and Harobate in Gursum district were found to be contaminated at a rate of 90%.On the other hand, samples from markets at Shekusman location (Babile district), Sororo of Darolabu district and Oda Oromiya (Gursum district) had only 43% kernel infection.Generally, the current results suggest heavy contamination of groundnut kernels with Aspergillus spp.Groundnut samples collected from markets had the lowest level of Aspergillus infection compared to samples from farmers' fields and stores across the survey districts and locations.This may suggest possible sorting of kernels by farmers to avoid those with visible symptoms of infection (discoloration) before bringing the samples to the local markets.The results were in agreement with the study by Pitt and Hocking (2009) in which Aspergillus spp.were more prevalent in the field and stored foods than in the markets.
The frequency of Aspergillus isolated from the groundnut samples analyzed in the current study is presented in Table 3.Of the several species isolated from the ground-nut samples, A. niger and A. flavus were the most prevalent mycotoxigenic fungi across the storage, field and market samples in East Ethiopia.These two species were isolated at a rate of 21-48% (A.flavus) and 35-66% (A.niger).On the other hand, A. ochraceus accounted for 0-14%, while A. parasiticus was associated with 2-21% of kernel infection.Pencillium spp.were also isolated from 6-13% of samples analyzed (data not shown).In another experiment, Eshetu (2010) reported the most frequent occurrence of Aspergillus spp.(A.flavus, A. niger and other Aspergilli) in wet shelled one year stored peanut sample from Gursum district of Hararghe region in East Ethiopia.
In this study; A. flavus and A. niger were isolated at higher frequencies from samples collected from farmers' fields and stores than markets while A. parasiticus was consistently isolated at higher frequencies from market samples.In contrast to these, isolation frequency of A. ochraceus was not consistently high or low in any of the sample collection sites (fields, markets and stores) across the survey districts.The current results were consistent with Chala et al. (2012), who detected 5-11,900 µg/kg total aflatoxin from same samples suggesting heavy groundnut contamination by Aspergillus spp.and associated aflatoxins in the region.Aspergillus flavus and aflatoxins contamination was also prevalent in stored groundnuts in Ghana (Awuah and Kpodo, 1996).Abdela (2009) also reported contamination of groundnut samples from Sudan by A. niger and A. flavus, which were isolated at frequencies of 29-60% for A. niger and 4-52% for A. flavus.Fusarium oxysporum, A. niger, R. bataticola and S. rolfisii were the predominant species of fungi associated with diseased plants indicating the involvement of these fungi in pre-and post-emergence death of groundnut plants in Babile district (Tefera and Tana, 2002).

Description of local groundnut varieties around selected areas
Description of groundnut varieties in around Babile and Gursum districts are different from that of Darolabu districts.Their naming was based on the morphology standing from the ground and leaf shape, size, color and seed size.Around Babile and Gursum districts, the common local groundnut varieties were "Oldhale", "Sartu", and "Jawsi" (Data from Agricultural and Rural Develop-ment Bureaus of the distritcs).Another similar study re-ported that predominant groundnut local variety (Oldhale) was commonly produced by farmers around Babile district (Tefera and Tana, 2002).The shape and leaf size of these three varieties were different from each other.Two groundnut varieties that is, "Basuqa"and "Qacine" are found to grow commonly around Darolabu district.They are identified based on the plant morphology and seed size.The "Basuqa" variety has larger seeds and leaves than "Qacine".It gives higer yield than "Qacine".But it is more susceptible to diseases and drought as revealed by the Agricultural and Rural Development Bureau of the district.

Cultural practices
Across the study areas, the groundnut shell is removed and left in farms, on the roads or water furrows then irrigation water takes it to the farms and gardens.Such a scenario generally favors over seasoning of plant pathogens including Aspergillus for subsequent contamination of the next crops.Besides, groundnut in the survey districts is cultivated either as a sole crop or intercropped with different crops like sorghum (Sorghum bicolor), maize (Zea mays), haricot bean (Phaseolus vulgaris), and under the shade of chat (Khata edulis), coffee (Coffea arabica) and mango (Mangifera indica) trees.Although intercropping is generally known to decrease disease pressure in agricultural fields, the companion crops in groundnut fields may not be very effective against Aspergillus spp.
Mechanical damages caused during pulling and digging out groundnuts may also have predisposed kernels to infection by Aspergillus and associated aflatoxins.Seeds in split pods are frequently invaded by A. flavus and subsequently become contaminated with aflatoxin as suggested by (Graham, 1982).Shriveled kernels contain higher amounts of aflatoxin as a result of higher A. flavus infection (Hill et al., 1984).Other factors, which may aid the contamination of groundnut with Aspergillus and then aflatoxins might be exchanging of equipment or plowing materials and groundnut seeds between households.

Time of planting and harvesting of groundnuts
Rain fall is usually unpredictable at the time of planting and harvesting around the study areas.The planting time of groundnut around the study areas usually lasts from the beginning of April to the end of May, and the harvesting time is from the end of September to the first half of November according to the field conditions and availability of labor.However, pods may overstay in the field after optimum maturations due to lack of labor, which further increases Aspergillus infection.Drought stress during late stages of pod development favors inva-sion of groundnut by A. flavus and subsequent aflatoxin contamination.End of season drought was very critical as it affects grain filling and pod formation in groundnut (Hill et al., 1984).

Drying method of groundnut around the study areas
Groundnut crops harvested in the survey districts are usually sun dried on materials such as matting.Curing of groundnut was done for few days in the farms before drying and removing of the seed from the hulms, to remove some moisture content from the kernels.In cases, where the moisture content of threshed unshelled and shelled pods was too high, the pods were sometimes bagged and every day the bags were brought out from the stores and left in the open.Although such practices may help reduce Aspergillus invasion, it would have been made more effective had it been coupled with fumigation with burning cow dung fumes and sun drying for one day as suggested by Gehewande et al. (1986).

Traditional groundnut storage system
Storage structures commonly found in the survey districts are made from mud and animal dung.In mud house there was no improved aerations in some stores and in some rain could percolate from the top and from the sides.Groundnuts in such houses are usually stored in sacks from the previous years, and this may also increase the contamination of groundnuts by Aspergillus and aflatoxins.Dickens et al. (1973) associated moisture condensation on roofs, improper application of insecticide sprays or leaking hoses and application equipment, conveyance of water from flooded elevator dump pits into warehouse, and storage of peanuts on concrete floors that are damp or have no vapor barriers with increased A. flavus growth.According to Bankole and Adebanjo (2003), traditional storage structures used for on farm storage include containers made of plant materials (woods, bamboo, and thratch).

Conclusion
Results of the current survey revealed heavy contamination of groundnuts by Aspegillus spp.Infection of kernels were higher in farmers' store and farmers' fields than markets with incidence of kernel infection at district level ranging from 36% at Babile market to 100% at Gursum field.A. niger and A. flavus were the most dominant species infecting groundnuts in East Ethiopia.
In combination with our earlier report on aflatoxin contamination of groundnut from East Ethiopia, the current results should serve as a wakeup call to create aware-ness on toxicogenic fungi and associated mycotoixns in the country.As aflatoxins are associated with health risks, reducing their level in food stuff to a level accepted by international standard is paramount importance to ensure the safety of these food stuff to consumers thereby facilitate trade both within and between countries.
Five local groundnut varieties are found to grow in the survey districts.These are "Sartu", "Oldhale", "Jawsi", "Basuka" and "Qacine".Although these varieties vary in terms of morphological features and disease resistance, their resistance to Aspergillus and aflatoxins under differing environmental conditions is not established beyond ambiguity.Thus future work should also focus in testing varieties for Aspergillus resistance under different environmental conditions and management practices.
This study has identified some important factors that may contribute for aflatoxin contamination of groundnuts both pre-and post-harvest.These include: weather conditions, seed and equipment sharing, planting time; harvesting time and methods; curing, drying and storage practices.The roles of additional factors that contribute to aflatoxin contamination down in the value chain further investigation.In addition, the role of none chemical seeds treatments especially essential oils and that of biological control agents in reducing groundnut contamination should be studied to come up with a more effective and sustainable management strategy.Farmers' association and extension agents should also be encouraged in creating awareness about aflatoxins and management techniques.

Table 1 .
Geographic description of locations included in the survey.

Table 2 .
Proportion of groundnut seeds infected by Aspergillus spp. in different districts of East Ethiopia.

Table 3 .
Frequency of Aspergillus species isolated from groundnut seeds collected from three districts in East Ethiopia.