1 Department of Biological sciences, Egerton University. 2 Department of Chemistry, Egerton University

Size: px

Start display at page:

Download "1 Department of Biological sciences, Egerton University. 2 Department of Chemistry, Egerton University"

Ann Pierce
6 years ago
Views:

1 Identification, Characterization and Distribution of Aspergillus and Fusarium species isolated from maize kernels from three agro-ecological zones in Western part of Kenya. Nyukuri, N. J. 1, Wagara, I. N. 1 and Matasyoh, J. C. 2 1 Department of Biological sciences, Egerton University 2 Department of Chemistry, Egerton University 1

2 ABSTRACT Moulds destroy more than 30% of crop yields and produce potentially poisonous mycotoxins. The most prevalent on foods are Aspergillus, Fusarium, Penicillium, Rhizopus and Mucor. The aim of the study was, to isolate and characterize moulds associated with maize from LVB. Thirty samples of good and mouldy maize were collected from Trans-nzoia, Kakamega and Kuria districts to determine the mould s distribution. These areas are in mid altitude agroecological zones with warm and humid conditions which favors development of moulds and mycotoxins. Two genera isolated and identified were Aspergillus and Fusarium. Among the Aspergillus, 12 mycotoxigenic species were identified whereas among the Fusarium 14 mycotoxigenic species were identified. When the Aspergillus population was separated according to geographical areas, the predominant species in Trans-nzoia was A. flavus at 23.1%, In Kakamega district, the most prevalent was A. flavus at 15% and in Kuria district, the most frequent species isolated was A. flavus at 20%. Overall infections of genus Aspergillus on maize in Trans-nzoia district was substantially higher than those collected from Kuria and Kakamega district. Among the Fusarium, the most frequent in Trans-nzoia was F. solani at 23.1%. In Kakamega, F. nivale and F. chlamydosporum were the highest at 10%. The most prevalent Fusarium in Kuria district was F. solani at 8.1%. Among the Fusarium, F. solani was the most prominent at 23.1%. In their distribution, Fusarium species was the most prominent in Kakamega district compared to Aspergillus species. Key words: Mycotoxins, moulds, maize, Aspergillus and Fusarium 2

3 Introduction Moulds are opportunistic biological agents of ubiquitous nature (Ryan and Ray, 2004). Because of their powerful arsenal of hydrolytic enzymes, these microorganisms can cause a high degree of deterioration when present in foods and are responsible for considerable economic losses (Souza et al., 2005). They are known to destroy 10 to 30% of the total yield of crops and more than 30% of perishable crops in developing countries by reducing their quality and/or quantity (Matasyoh et al., 2009). Moulds cause extensive damage on foods, feeds and other agricultural commodities in the field, during transportation, storage and processing, leading to postharvest losses. Toxigenic fungi can attack maize prior to harvest and further decay the crop during storage. As a result, mycotoxins may form both during crop development and in storage. Aspergillus, Fusarium, Penicillium and Cladosporium are the predominant fungal genera associated with grain in storage. Of all mycotoxins, aflatoxins probably cause the most concern. This is due to their carcinogenic and immune suppressing effects in both humans and domestic animals (Turner et al., 2003) and economic losses due to significant reductions in export value. Mycotoxins are chemicals produced by fungi that are harmful to humans and domestic animals. These chemicals may contaminate staple foods and feeds worldwide, posing a number of significant food safety concerns (Schmale and Munkvold, 2009). They contaminate 25% of agricultural crops worldwide and are a source of morbidity and mortality throughout Africa, Asia and Latin America (Smith et al., 1994). They cause diseases referred to as mycotoxicoses in humans and animals (Agrios, 1997). Most mycotoxicoses are caused by the common and widespread moulds namely Aspergillus, Penicillium and Fusarium. They cause acute liver damage, liver cirrhosis, induction of tumours and attack on the central nervous system, skin disorders and hormonal effects (Erkekoglu et al., 2010). The most important mycotoxins are aflatoxins, deoxynivalenol, fumonisins, ochratoxins and zearalenones. Among these the most famous are the aflatoxins which are produced by Aspergillus flavus, A. parasiticus and several other species of Aspergillus in a wide variety of agricultural commodities including grains, legumes and nuts (Patten, 1981). Aflatoxins are produced by A. flavus and A. parasiticus, and are among the economically most important mycotoxins (Brodhagen and Keller, 2006). Acute aflatoxicosis epidemics 3

4 occur in several parts of Africa and Asia leading to death of several hundred people (Varga et al., 2009). Aflatoxins are known to be potent hepatocarcinogens in animals and humans. Ochratoxin A, which is has been experimentally shown to be teratogenic, a potent renal carcinogenic and immunosuppressive is largely produced by A. ochraceus and less frequently by A. niger (Nielsen et al., 2009). As an enzyme inhibitor, ochratoxin A affects lipid peroxidation and has been implicated in Balkan Nephropathy (BENO in humans (Atrosh et al., 2000). Toxin-producing fungi may invade at pre-harvesting period, harvest-time, during postharvest handling and in storage. Toxigenic fungi can be divided into three groups: (a) field fungi namely, genus Fusarium, e. g F. moniliforme, F. roseus, F. trincinctum and F. nivale; (b) storage fungi which include the genera Aspergillus and Penicilium, e.g A. flavus, and A. parasiticus; and (c) advanced deterioration fungi which normally do not infect intact grains but easily attack damaged ones and require high moisture content. Examples of the third group are A. clavatus, A. fumigatus, Chaetomium, Scopulariopsis, Rhizopus, Mucor, and Absidia (Makun et al., 2009). Materials and Methods Source of maize samples A total of 30 samples categorized as good (maize without moulds) and mouldy maize were collected randomly from various rural households and markets in Trans-nzoia, Kuria and Kakamega district were used in this study. In Trans-nzoia, samples were collected from Kitale central and Kiminini divisions. In Kakamega, samples were collected from Kakamega municipality division whereas in Kuria district, samples were collected from Kehancha and Masaba divisions. Most of these areas are in mid altitude agroecological zones with warm and humid conditions which favour development of moulds and production of mycotoxins (Kaaya et al., 2006). These areas have unpredictable rainfall patterns making it difficult for small scale farmers to efficiently dry their produce. Ten samples (each weighing a half a kilogram) were collected from each district in properly labeled khaki paper bags to minimize saprophytic fungal contamination and transported in a cool box to the laboratory for analysis. The samples were stored at 4 o C. 4

5 Isolation of the moulds Moulds were isolated from these samples using direct plating technique. For each sample, 20 seeds were picked randomly and surface sterilized by soaking for 1 minute in 2.5% of sodium hypochlorite, and rinsed in three changes of sterile distilled water. The grains were blotted with sterile filter paper and plated on potato dextrose agar containing 7.5% sodium chloride and 1gm streptomycin sulphate (for 1litre of media). Addition of streptomycin sulphate is very effective in the inhibition of fast-growing "spreader" moulds such as Trichoderma. Yet, it does not inhibit the growth of other mould species, including the mycotoxin producers. Thus, identification is not compromised (Diba et al., 2007). The plates were incubated at 25 o C and monitored daily for fungal growth for seven days. The resulting cultures were identified based on cultural and morphological characteristics using taxonomic keys (Matasyoh et al., 2009; Klich, 2002). Target moulds were sub-cultured to obtain pure single-spore cultures. Identification and characterization of the moulds The resulting cultures were identified based on cultural and morphological characteristics using taxonomic keys (Pitt, 1979). Morphological features of moulds were studied and the major and remarkable macroscopic features that were looked at are colony diameter, colony color on agar and reverse, exudates and colony texture. Microscopic characteristics that helped in the identification process were conidia heads, stipes, color and length, vesicles shape and seriation, metula covering, conidia size, shape and roughness (Diba et al., 2007). The number of seeds showing each type of mould growth were determined and compared for each sample. Data analysis One way analysis of variance (ANOVA) was used to test whether the moulds affecting maize from the three districts are significantly different. Least significant difference (LSD) was used to discriminate which maize and from which district is highly contaminated with moulds. Students t-test was used to test if the two genera of moulds are statistically significant on the maize from the three districts. The statistical level of significance was fixed at p< 0.05 (95%). 5

6 Results and Discussion Five different fungal genera, Aspergillus, Fusarium, Penicillium, Rhizopus and Mucor were identified from the maize samples collected during the survey. The moulds isolated and identified among the genus Aspergillus were 14 mycotoxigenic species which include A. parasiticus, A. flavus, A. wentii, A. ustus, A. nidulans, A. ochraceus, A. sparsus, A niger, A. versicolor, A. flavipes, A. terreus, A. fumigatus, A. humicola and A. tamarii. The resulting cultures were identified based on cultural and morphological characteristics using taxonomic keys (Pitt, 1979). Among the genus Fusarium, 17 species were identified and these include F. solani, F. merismoides, F. subglutinans, F. sporotrichioides, F. moniliforme, F. scirpi, F. chlamydosporum, F. trincinctum, F. semitectum, F. oxysporum, F. nivale, F. avenaceum, F. proliferatum, F. graminearum, F. culmorum, F. crookwellence and F. lateritium. Out of the 17 species, 11 of them were mycotoxigenic. Isolates that had dark green colonies and produced rough conidia were considered A. parasiticus. Isolates with brown to yellow-brown colonies on agar were classified as belonging to A. tamarii. In all the 3 districts, the most frequent Aspergillus species on maize was A. flavus. When the Aspergillus population from the maize seeds were separated according to geographical areas of Western part of Kenya, the predominant Aspergillus species in Trans-nzoia was A. flavus (23.1%) followed by A. parasiticus (15.4%), A. wentii (7.9%), A. ochraceus (7.9%), A. versicolor(7.9%) and A. flavipes, A. niger, A. terreus all at 4.5%. In Kakamega district, the most prevalent Aspergillus species were A. flavus (15%), followed by A. niger (10%), A. flavipes (8%) and A. terreus, A. ochraceus and A. versicolor at 5%. In Kuria district, the most frequent Aspergillus species isolated were A. flavus (20%), followed by A. niger (15.8%), A. parasiticus, A. wentii, A. ochraceus, A. versicolor and A. nidulans isolated were 4.5 %. Aspergillus flavipes and A. terreus were not isolated. Overall, infection of genus Aspergillus on the maize seeds in Trans-nzoia district were substantially higher than those collected from Kuria and Kakamega district. The less prevalent Aspergillus species throughout all the sites were A. fumigatus, A. sparsus, A. tamarii, A. ustus, A. humicola, and A. terreus. In Trans-nzoia district, the most frequent Fusarium species was F. solani (23.1%), followed by F. proliferatum 6

7 (8%). Fusarium nivale, F. merismoides, F. oxysporum, and F. graminearum were not isolated as shown in Table 1 below. In Kakamega district, F. nivale and F. chlamydosporum were the highest at 10% followed by F. oxysporum and F. solani at 5%. The most prevalent Fusarium in Kuria district was F. solani at 8.1% followed by F. merismoides, F. oxysporum, F. nivale and F. proliferatum at 4.5%. Among the Fusarium, F. solani was the most prominent at 23.1%. In their distribution, Fusarium species was most prominent in Kakamega district compared to Aspergillus species. Relative level of incidence ranged from 0% to 23.1% from the samples. However, these infection limits were not significantly different from each other. The distribution of the moulds isolated and identified from the three districts were not significantly different (at p>5%) as shown in Table 2. When the data from the moulds isolated in the three districts were analysed separately using Tukey HSD, there was no significant differences in the number of the moulds in the three districts at p<0.05. The mean difference between Trans-nzoia and Kakamega was 0.365, between Trans-nzoia and Kuria was 0.335and between Kuria and Kakamega was (Table 3). This explains why the distribution of the moulds in the three districts is the same. Fungal species belonging to the two genera were isolated and identified in maize kernels from the three agro-ecological zones in the Western part of Kenya and Aspergillus species were the most predominant. High levels of Aspergillus species have previously been reported in Nigeria albeit in pre-harvest maize (Bankole and Adebanjo, 2003). It is likely that preharvest infections greatly influence the mycoflora in storage (Hell et al., 2003). This co-occurrence of toxigenic fungi on products in storage is commonly exacerbated as storage conditions become conducive for mycotoxin production. Although the distribution of members of Aspergillus species varied across agro-ecological zones, Aspergillus flavus was the most dominant species in all the three agroecological zones. Similar and higher frequencies of A. flavus in stored maize have been reported previously in Benin (Hell et al., 2003). The high frequencies of A. flavus compared to other members of Aspergillus species can be explained by the occurrence of correspondingly high levels of Aspergillus species resident in the soil, plant debris and insects (Nesci and Etcheverry, 2002), which acts as the reservoir of inoculums for infection of kernels in the field. 7

8 Species of the genus Aspergillus are found almost everywhere on every conceivable type of substratum. Many types are known to cause diseases in man and animals and some are known to produce toxins and be involved in problems of mycotoxicoses. Most of the species isolated are mycotoxigenic ones because the three districts are found in warm and humid region which predisposes the maize to the moulds after being harvested. The warm and humid conditions are due to the Lake Victoria. ACKNOWLEDGEMENTS The authors are grateful to the Lake Victoria Basin Research Initiative (VicRes) for the financial support. 8

9 REFERENCES Agrios, G. N. (1997). Plant Pathology, 4 th edition. Academic Press, New York. Pp 635. Atroshi, F., Biese, I. and Saloniemi H. (2000). Significance of Apoptosis and its relationship to Antioxidants after Ochratoxin A administration in Mice. Journal of Pharmaceutical Sciences 3(3): Bankole, S. S. and Adebanjo, A. (2003). Mycotoxins in food West Africa: Current situation and possibilities of controlling it. African Journal of Biotechnology 2: Brodhogan, M. and Keller, N. P. (2006). Signalling pathways connecting mycotoxin production and sporulation. Journal of Molecular Plant Pathology 7(4): Diba, K. K., Mirhendi, S. H. and Mahmoude, M. R. (2007). Identification of Aspergillus species using morphological characteristics. Pakistan Journal of Medical Science 23: Erkekoglu, P., Sahin, G. and Baydar, T. (2008). A special focus on mycotoxin contamination in Baby foods: their presence and regulations. Journal of Pharmaceutical Sciences 33: Hell, A. B. (2003). Controlling Aflatoxins and Fumonisins in maize by Crop management. Journal of Toxicology 2 &3: Kaaya, A.N., Kyamuhangire, W. and Kyamanwa, S. (2006). Factors affecting aflatoxin contamination of harvested maize in the three agroecological zones of Uganda. Journal of Applied sciences 6: Klich, M. (2002). Identification of common Aspergillus species. ASM Press, Washington, DC, pp Makun, H. A., Gbodi, T.A., Akanya, O. H., Salako, E. O and Ogbedu, G. H. (2009). Fungi and some Mycotoxins found in Mouldy Sorghum in Niger state, Nigeria. World Journal of Agricultural Sciences 5:

10 Matasyoh, J. C., Maiyo, Z.C., Ngure R.M. and Chepkorir, R. (2009). Chemical composition and antimicrobial activity of essential oil of Coriandrum sativum. Food Chemistry 101: Nesci, A. and Etcheverry, M. (2002). Aspergillus section Flavi populations from field maize in Argentina. Letters in Applied Microbiology 34: Nielsen, K. F., Mogensen, J. M., Johansen, M., Larsen, T. O. and Frisvad, J. C. (2009). Review of secondary metabolites and mycotoxins from the Aspergillus niger group. Anal Bioanal Chem 395: Patten, R. C. (1981). Aflatoxins and disease. American Journal of Tropical Medicine and Hygiene 30: Pitt, J. I. (1979). The genus Penicillium and its teleomorphic states Eupenicilium and Talaromyces. Academic Press (London, New York). Pp 634. Ryan, K. J and Ray, C. G. (2004). Sherris Medical Microbiology (4 th edition). Mc Graw Hill. Pp Schmale, D. G. and Munkvold, G.P. (2009). Mycotoxins in Crops: A Threat to Human and Domestic Animals Health. The Plant Health Instructor D01: /PH Smith, J. E., Solomons, G. L., Lewis, C. W. and Anderson, J. G. (1994). Mycotoxins in human health. Brussels: An article of European Commission. Souza, E. L., Lima, E. O, Freire, K. R. and Sousa, C. P. (2005). Inhibitory action of some essential oils and phytochemicals on the growth of various moulds isolated from foods. Brazilian archives of Biology and Technology 48: Turner, P.C., Moore, S. E., Hall, A. J., Prentice, A.M. and Wild, C. P. (2003). Modification of immune function through exposure to dietary aflatoxin in Gambian children. Environ Health Perspective 111(2): Varga, J., Frisvad, J. C. and Samson, R. A. (2009). A reappraisal of fungi producing afaltoxins. World Mycotoxin Journal 2(3):

Tables showing the incidence of the moulds and their significance wihtin the three districts Table I: Incidence of the moulds in the three districts 25.00% 20.00% 15.00% 10.00% 5.00% 0.00% A.flavus F.

11 Tables showing the incidence of the moulds and their significance wihtin the three districts Table I: Incidence of the moulds in the three districts 25.00% 20.00% 15.00% 10.00% 5.00% 0.00% A.flavus F.solani F.chlamydosporum A.flavus A.parasiticus A.wentii A.niger A.ochraceus A.versocolor A.flavipes A.terreus F.solani F.merismoides F. nivale F. proliferatum A.nidulans F.oxysporum A.ustus A.sparsus F.chlamydosporum 11

12 Table II: The Test of Analysis of Varience for Significance of moulds distribution within and between the three districts ANOVA Number of moulds Source of variation Sum of Squares df Mean Square F Sig. Between Groups Within Groups Total df=degree of freedom, F=freedom, Sig=significance Table III: Comparison of the moulds in the three districts Multiple Comparisons Numberofmoulds Tukey HSD Mean 95% Confidence Interval (I)Sampling place (J)Sampling place Difference (I-J) Std. Error Sig. Lower Bound Upper Bound Kakamega Trans-nzoia Kuria Trans-nzoia Kakamega Kuria Kuria Kakamega Trans-nzoia

Overview of Mycotoxins in India with special reference to Aflatoxins. F Waliyar

Overview of Mycotoxins in India with special reference to Aflatoxins F Waliyar Important Mycotoxins Aflatoxins Ochratoxins Fusarial toxins Fumonisins Zearalenone T2-toxin Deoxynivalenol Nivalenol Mycotoxin