IJCRR - Vol 11 Issue 21, November, 2019
Date of Publication: 09-Nov-2019
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Fungal Infections and Aflatoxin Contamination in Maize Grains Collected from West Showa and East Wallega Zones, Ethiopia
Author: Solomon Yilma, Kassahun Sadessa, Denberu Kebede
Category: Life Sciences
Abstract:Mycotoxin affects the world's food crops and creates a large economical loss in the developed and developing countries. Aflatoxins are a group of mycotoxins that mainly produced by Aspergillus species viz., A. flavus, and A. parasiticus. An aflatoxins contamination of maize grains has exhibiting a serious threat to human and animal health over the past two decades. Toprotect the safety of food commodities, regular monitoring and diagnosis of the presence and amount of non-permissible levels of aflatoxins in food is necessary to take appropriate management measures. Maize grain samples were collected from Ilu Galan and Bako districts of West Shoa and Gobu Sayo district of East Wollega zones of Oromiya; from different grain storage types. About 500 gr of maize grains were sampled from each sampling spot. PDA media was used for isolation of associated maize grains sample associated fungi. Sun-culturing and purification of the associated fungi were done and preserved using agar slant technique. The associated fungal mycoflora were characterized based on morphological and growth sporulation properties. Enzyme Linked Immuno Sorbent Assay (ELISA) diagnostic kit were used for identification and quantification of aflatoxins. Aspergillus, Fusarium Penicillium and Trichoderma species were identified and characterized. Aflatoxin B1 was identified and quantified from zero to 381.6\?g/kg. About 34.4% of the samples were positive to aflatoxin B1 compared to Food and Drug Administration (20\?g/kg) and European Union (4\?g/kg), respectively. The management of mycotoxigenic fungi, improvement of storage methods, development of resistant maize varieties and awareness creations could be possible solutions
Keywords: Maize grain, Storage methods, Fungi, Identification, Detection, Quantification and AflatoxinB1
Mycotoxins are toxic secondary metabolites produced by certain fungi viz., Aspergillus, Fusarium, Alternaria and Penicillium spp. in agricultural products that are susceptible to mold infestations (Morenoaet al., 2009). Now a day mycotoxin effectis attracting the worldwide attention because of the significant economic losses associated with their impact on human health, animal productivity and trade (Wagacha and Muthomi, 2008). These toxins are highly toxic and carcinogenic to animals and humans which lead to hepatotoxicity, teratogenicity, immunotoxicity and even death (Wen et al., 2004).Children below five years remain most vulnerable, with exposure damaging their immunity and causing stunted growth (www.aatf-africa.org). Mycotoxins contaminate and reduce crops quality through discolorations and reduction of nutritional values (Waliyaret al., 2008). Regulations on mycotoxins have been set and strictly enforced by most agricultural commodities importing countries, thus affecting international trade(FAO, 1988). In some developing countries where agricultural commodities account for about 50 percent of the total national exports, the economic importance of mycotoxins is considerable. FAO (2014) has estimated that about 25% of agricultural crops worldwide contaminated by mycotoxins. Similarly, the Center for Disease Control (CDC) has estimated that more than 4.5 billion people in the developing world are exposed to aflatoxins (CDC, 2004). The total allowable level of aflatoxin (µg/kg) in human food in different countries were reported i.e., Australia, china, European union, India, Kenya, Taiwan and USA is 15, 20, 4-15, 30, 20, 50, and20, respectively (FAO, 2004). The estimated crop lost due to aflatoxin is $225 million per year, out of the $932 million crop lost in each year in the USA (Betran and Isakeit, 2003).
Many studies across the world showed that the maize grains highly contaminated with aflatoxins that mostly caused by Aspergillus flavus and Aspergillus parasiticus(Patten, 1981; Munkvold, 2003). Aflatoxins can produced by fungal action during production, harvest, transportation, storage and food processing (CAST, 2003; Murphy et al., 2006). According to Befekadu and Berhanu (2000) the maize crop is the third most important crop in Ethiopia after wheat and teff which accounts for the largest share in total crop production. The maximum quantities of maize produced are stored under poor storage conditions. The traditional storage of maize in Ethiopia made up of mud, bamboo strips, and pits (Chauhan et al., 2016). Storage of maize grains under poor storage methods enhances the growth of fungi and promotes the production of mycotoxins (Chauhanet al.,2008).Despite the fact that maize is a crucial food to Ethiopia and it’s vulnerable to aflatoxin risk due to different geographical and climatic conditions and poor handling (Alemu et al,2008). There are limited reports on the relationship between fungal infections and aflatoxins contamination in maize crop in Ethiopia.
Materials and Methods
Description of the study areas and sample collections
The maize grain samples were collected from West Showa and East Wallega Zones of Oromia regional state which is located at about 250km to the West of Addis Ababa along the main road to Asossa. Ilu Gelan and Bako Tibe districts from West Shoa Zone; Gobu Sayo district from East Wallega Zone were selected for this studies based on their maize production potential and available storage systems. These areas annual rainfall and temperature range from 800 – 1000 mm and 15°C – 29°C, respectively. A total of 90 maize grain samples were collected; about 30 samples were collected from each district i.e., Ilu Gelan, Bako Tibe and Gobu Sayo. Maize grains were sampled from six traditional grain storage types. The samples were collected from different storage positions across different storage types. About 500gm maize grains were sampled from each sampling spot from the top, middle and bottom of each type of storage. The samples were temporary stored in the paper bag and transported to Ambo plant protection research center laboratory within 72 hour for fungal Microflora and Aflatoxins analysis.
Mycotoxigenic fungi isolation, characterization and identification
Agar plate method was used to determine the number and kind of fungi present. About 48 undamaged kernels of each sample was taken by a spatula directly into sterilized flasks and surface sterilized with 2% hypochlorite solution for 3 minutes and then rinsed in sterile distilled water. Potato dextrose agar (PDA) medium containing 100μg chloramphenicol per ml was used. About 8seeds per plate were cultured to isolate and detect the associated fungi. The plates were incubated at 25oC and the presence of A. flavus and other common fungi were observed after one week. The suspected mycotoxigenic fungi colonies were further purified individually by sub culturing on PDA plate and then on PDA slant. Isolated fungi were then identified according to Raper and Fennel (1965), Nelson et al. (1983), Rechard (1996), and Klich (2002) based on colony characteristics and morphology under light microscope.
Detection, identification and quantification of Aflatoxin B1
Specific ELISA kit was used for the detection, identification and quantifications aflatoxin B1. The samples preparation, extraction and purification were done according to the instruction given by the company (RIDASCREEN®Aflatoxin B1, Germany). ELISA reader was employed for the quantification of aflatoxin B1. Finally, detected and quantified aflatoxin B1 was used for analysis across each samples, grain and storage types.
Statistical analysis on aflatoxin B1 concentration and standardized curve of determination was performed using mini tab version 20 and all the graph and percentage was done by excel.
Assessment and identification of fungal microflora
The assessment of associated and identified fungi spps. were provided below in the Fig. 1. A total of nine species of fungi were isolated and identified as Aspergillus flavus, Aspergillus parasiticus, Fussarium verticilliodes, Penecillium notatum, Penecillium verrucosum, Fussarium proliferatum,Fussarium gramminearum, Aspergilus niger, and Trichoderma Spp. The most common fungi isolated from the maize grains were Aspergillus flavus (25.7%) and Aspergillus parasiticus (18.9%) from the total of ninety maize grain samples.
Occurrences of mycotoxigenic fungi in different maize storage types
Mycotoxigenic fungal occurrence were assessed in all maize samples collected from the six of storage types; namely open above ground(OAG), sack in open air(SOA), sack in house(SH), open sorghum stalk (OSS), improved gottera(IG), and in house ground (IHG) storages (Table1). Accordingly, the highest occurrence of mycotoxigenic fungi were seen in OAG storage typewhich has been accounted about 44(29.7%) followed by SOA 27(18.2%) and few mycotoxigenic fungal 5(3.4%) was isolated from IG. Aspergillus species; A. flavus, A. parsiticus and Fussarium species; F. gramminearum, F. verticilliodes were the most prevalent mycotoxigenic fungi in OAG and SOA storage types.
Aflatoxins detection and identification in maize grain samples
Aflatoxins B1 has been identified from the maize samples detected in laboratory using Enzyme Linked Immuno Assay (ELISA). The results of ELISA has reveal that the mean31 (34.4%) of 90 maize grain sample were above and shown toxicity higher than those recommended by Food and Drug Administration (2004) and European Union (2018)standards which states that maximum permissible level of afaltoxin B1 in maize should be 20µg/kg and 4µg/kg, respectively. Higher aflatoxin B1 concentration (73.3%) was observed in maize grains sampled from Gobu Sayo district followed byIlu Gelan(20%)(Table 2). The determination of aflatoxinB1 concentration was done by developing a curve from the supplied aflatoxin B1standard which has ranged from 0-4.5ppb (Fig. 2).
Aflatoxin B1 concentration across storage and grain types
The level of aflatoxin B1 concentration is presented in Table 3. It was observed that the level of aflatoxin B1 in sample 2,14, 57,71,74,75,78,82 and 83 was detected and measured between 3.9-381.6 ppb and its corresponding storage type were open above ground, open sorghum stalk and sack in house. It is also observed that the aflatoxin B1 level was varied with the maize grain storage types. Open above ground, open sorghum stalk and sack in house storage types were exposed to rain and high temperature which has been created conducive environment for mycotoxigenic fungi growth and development.
Notice: OAG; open above ground, OSS; open sorghum stock, SH; sack in house, SOA; sack in open air, IG; improved gottera, IGH, in house ground storage
The highest concentration of aflatoxinB1was detected and quantified in LIMU variety (8.9 ppb) followed by BH660 (4.0ppb) and BH540 (1.6ppb). Out of the total 90 sample about 34.4 % (31 samples) were possessed more than 0.05ppb concentration of aflatoxins B1 while 65.6 % (59 samples) have had the aflatoxin B1 level less than 0.05 ppb. Whereas, results of ELISA has demonstrated that 0.2 ppb as a mean aflatoxin B1 concentration for all 90 maize grain samples tested(Table 4).
The effect of maize grain storage types on the growth of mycotoxigenic fungi which in turn favor the production of aflatoxin B1were analyzed from the data of mycological results. There were six types of maize grain storage were observed during sample collection (Fig. 3). The occurrences of aflatoxin B1in different storage types were recognized different in concentrations. Accordingly, the highest aflatoxin B1 concentration were recorded in open ground (18.03 ppb) and the lowest aflatoxin B1 concentration were observed in maize grain stored in improved gottera (0.16ppb) as stated in Table 5.
Figure 3: open above ground(A), improved gottera(B), sack in house(C), sack in open air(D), Open sorghum stalk (E), in house ground(F)
Several fungal species have been isolated from the maize grain sampled from three districts. Mycotoxigenic fungi were isolated and identified from the maize grain samples as well as the associated aflatoxin B1 was detected and quantified. Aspergillus spp. were the most predominant mycotoxigenic fungi with 50.7% frequency of occurrence followed by Fussarium spp. with 26.4%, and Penicillium spp with 22.3%.Trichoderma species was also isolated in trace amount(1.07%). Similar studies were done in Gedeo zone by Chauhan et al. (2016)and in Kewot Provence by Geremew Tassew et al. (2016). The higher frequency of fungal infection specifically Aspergillus spp. was due to poor storage types and longtime storage greater than two yearsas similarly reported by Habtamu et al.(2001).The maize grain aflatoxigenic fungi contamination started from the fields before harvest and continued across storage, consumptions and marketing which is similarly reported by Bhat et al. (1997) and Gao et al. (2007)in different maize growing countries like Ethiopia Kenya, Somalia, Uganda and Sudan. The prevalence of maize grain aflatoxin B1 contamination has reached 34.4% and higher aflatoxin B1 concentration 22 (73.3%) were observed in Gobu Sayo province followed by Ilu Gelan6 (20%).About 3.3 %and 7.7% maize samples had aflatoxinB1 higher than those recommended by Food and Drug Administration (FDA; 20µg/kg) and European Union (EU; 4µg/kg) regulatory levels respectively. The observed aflatoxin B1 concentration was very low compared to the reports of Chauhan et al. (2016) that has stated mean aflatoxins concentration for a two year stored maize grain samples53 ppb with 100% contamination in aflatoxin. The highest aflatoxin B1 concentration were recorded in open ground (18.03 ppb) this could be due to the exposure of the grain to favorable temperature and rain which in turn facilitate the growth of aflatoxigenic fungi, and the lowest aflatoxin B1 concentration were observed in grain stared in improved gottera(0.16ppb).Generally, there should be the management of mycotoxigenic fungi starting from the field, harvesting, transport, and storage through the development of mycotoxins resistant maize varieties, improvement of grains storage types and awareness creations.
The fungi isolated in the present study were from the different genera that are common in maize grain. A. flavus was the predominant one while other toxin-producing species such as Aspergillus parasiticus, Fussarium verticiloide and Penecillium notatum occurred at relatively at the higher levels. The aflatoxin B1 concentration in the majority of the sample are below the recommended level however, in few of the sample its level is much higher than the EU standard. The level of aflatoxinB1 concentration is higher in maize grain stored in open ground field.
Authors acknowledge the immense help received from the scholars whose articles are cited and included in references of this manuscript. The authors are also grateful to authors / editors / publishers of all those articles, journals and books from where the literature for this article has been reviewed and discussed.
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