Agricultural Science Digest

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Agricultural Science Digest, volume 43 issue 1 (february 2023) : 75-80

Isolation of Fungi from Maize Samples Collected from Selected Counties in Kenya

Mwancha S.N. Okioma1,*, Japhet Mburugu Muthamia1, Isabel Nyokabi Wagara1, Eliud Mugu Gathuru1, Paul Njenga Waithaka2, Benson Muriuki Githaiga1
1Department of Biological Sciences, Egerton University, P.O. Box, 536 Egerton.
2School of Pure and Applied Sciences, Kirinyaga University, P.O. Box, 143-10300, Kerugoya.
Cite article:- Okioma S.N. Mwancha, Muthamia Mburugu Japhet, Wagara Nyokabi Isabel, Gathuru Mugu Eliud, Waithaka Njenga Paul, Githaiga Muriuki Benson (2023). Isolation of Fungi from Maize Samples Collected from Selected Counties in Kenya . Agricultural Science Digest. 43(1): 75-80. doi: 10.18805/ag.D-338.
Background: Maize is the stable food in Kenya. However, its production has been jeopardized by the high prevalence of fungi in most developing countries and the whole world at large. Some fungi produce mycotoxins which threaten human lives.

Methods: This study aimed at isolating fungi from selected Counties in Kenya. Maize samples were ground using a kitchen blender and cultured on Potato Dextrose agar at 28°C for 7d. The fungal isolates were identified using morphological and cultural characteristics. Data was analyzed using Statistical Package for Social Sciences (SPSS) version 25.0 software.

Result: The mean fungal isolates varied from 10.8±0.2 CFU/g in Wallenia spp. to 47.4±0.2CFU/g in Aspergillus Spp. The most common fungal isolates were Aspergillus spp., Fusarium spp. and Penicilium spp. The Mean fungal isolates from the selected Counties varied from Kitui (9.6±0.2 CFU/g), Machakos (9.7±0.3CFU/g), Bungoma (25±0.1CFU/g), Migori (25.3±0.3CFU/g), Kericho (25.3±0.2CFU/g), Kisumu (27.1±0.1CFU/g), Meru (27.1±0.2CFU/g), Kisii (28.5±0.1CFU/g) and Trans-Nzoia (30.1±0.3CFU/g). The number of spores in maize samples from baskets varied from 13.6±0.2-19.6±0.2, polypropylene (24.0±0.2-24.0±0.2), Jute (13.6±0.2-21.6±0.2) and polythene bag (48.0±0.2-72.0±0.2) CFU/g.

Conclusion: This study established that maize in the selected Counties is highly contaminated by pathogenic fungi. The most prevalent fungal spp. were Aspergillus, Penicillium and Fusarium. The best maize storage material for maize were baskets. There is need of identifying the most prevalent fungi up to the molecular level. 
Maize is an important staple food in most parts of the world (Zhao et al., 2017). Its growth is expansive due to its adoptability to different farming systems (Samson et al., 2014). Besides, the crop is adjustable to different regions of the world (Paterson et al., 2017). Maize contributes to high food security especially in developing countries. With the rising human population all over the world, maize production needs to be supported by all means (Jančič et al., 2016).

In Kenya, farming is the key driver of the country’s economy (Okioma et al., 2020). To small scale farmers, maize provides 60% of dietary calories and over 50% of proteins (Tobin-West et al., 2018). Small scale farmers grow about 2 million hectares annually with an average yield of 1.2-1.6 tons per annum (Odhiambo et al., 2013). The biggest drawback to maize production is infestation by fungi (Chaubey et al., 2015). Some fungi produce mycotoxins which impact negatively to human and animal health (Surendra et al., 2011). Mycotoxins are carcinogenic, mutagenic and teratogenic (Salau et al., 2017). In addition, they cause impaired growth in children and sometimes immunosuppression (Sangares et al., 2016). Mycotoxins cause leuco-encephalomalacia in horses, pulmonary oedema in pigs, liver and kidney cancer in mice and rats.

Fungal growth is increased by high temperature, humidity, monsoons, low rains and flooded conditions (Affeldt et al., 2014). Poor harvesting and postharvest practices predisposes maize to fungal attack (Zajc and Gunde-Cimerman, 2018). The highest producer of mycotoxins are in the genera Aspergillus, Fusarium and Penicillium. The fungi are ubiquitous and cosmopolitan (Medina, 2017). Additionally, the fungi are filamentous proliferating in a vast range of environmental conditions (Krulj, 2019). Damage by fungi in maize is manifested by colour change, decrease in nutritional value and reduction in overall quality and quantity of maize (Negash, 2018). This study aimed at isolating fungi in stored maize in selected counties in Kenya.
The study areas

The current is study was carried out in Egerton University, Department of Biological Sciences laboratories in the year 2020. Maize samples were collected from Trans Nzoia, Kisii, Kisumu, Bungoma, Migori, Kericho, Machakos, Kitui and Meru Counties. Recently, the counties had reported mycotoxin infections (Okioma et al., 2020). Trans-Nzoia county is located in Western part of Kenya. The county lies at 2,100 m above sea level. It experiences a temperatures range of 9.05°C-26.85°C (Mutegi et al., 2018). Kisii County lies 0°41' 0 N 34°46' 0 E. The temperatures varies from 16°C-27°C. The County has a population of 1,152,282 (Okioma et al., 2020). Kisumu County covers an area of 2085.9 km2. The County is located at longitudes 33° 20'E and 35°20¢E and latitudes 0°20'South and 0°50'South (Josephat et al., 2015). Bungoma County has an area of 2,069 km2 and is located at 00°34'00²N, 34°34'00²E (Odhiambo et al., 2013). Migori County is located in Western Kenya and its temperature varies from 24°C to 31°C (Birgen et al., 2020). Kericho County lies at 35° 02' and 35° 40' E and 0 23' S with an altitude of about 2002 m above the sea level (Odhiambo et al., 2013). Machakos County is found at latitudes 0°45' S to 1°31' S and longitudes 36° 45' E to 37° 45' E. The county lies at an altitude of 1000 to 2100 m above sea level (Josephat et al., 2015). Kitui County is found in Eastern Kenya at co-ordinates 1°22' 0" South and 38° 1' 0" East and a temperature range of 26°C to 34°C (Birgen et al., 2020). On the other hand, Meru County is located in Eastern Kenya at 0.047035° N and 37.649803° E. The County has a temperature range of 8°C-32°C (Mutegi et al., 2018).

Collection of maize samples

Maize samples were collected from baskets, polypropyne, jute and polythene bags from the selected Counties. The sample size was calculate at 95% confidence level at a precision of 5% as proposed by Nleya et al., (2018).

n= Sample size.
Z= Confidence level at 95% (1.96).
p= Estimated proportion of the sample population.
q= (1-p).
e= Desired level of precision at 5% with a standard value of 0.05.

Substituting the values in the formula above, the sample size was determines as follows:
Samples were placed in sterile khaki bags and stored at 4oC in the Department of Biological Science Laboratories of Egerton University.
Isolation of fungi from maize samples

The maize samples were surface sterilized using 70% ethanol for 2 minutes. The samples were rinsed with distilled water to remove ethanol on the surface (Chukwudi et al., 2021). The Maize samples were blot dried (Pemingo et al., 2016). A dry mill kitchen blender (BL335, Kenwood, UK) was used in grinding one kilogram of each maize sample. One gram of each ground maize sample was separately placed in 9mL of sterile distilled water, shaken using an orbital shaker and serially diluted up to 10-2. Aliquots of 0.1 mL were each plated on potato dextrose agar (PDA) and incubation at 28°C for 7 d (Menza and Muturi, 2018). The fungal isolates were sub-cultured on PDA. The fungal isolates were enumerated using a formula provided by Mezzomo et al., (2018);

Identification of the mycotoxin producing mycoflora

Macroscopic and microscopic characteristics were used in identifying the fungal isolates (Kumar and Kalita, 2017). Length and type of spores were used to morphologically characterize the isolates while cultural characteristics considered colour and margins of the colonies (Lee et al., 2013). In addition, the fungal isolates were characterized using fungal identification keys (Abdel-Sater et al., 2017).

Spores count of the fungal isolates

Fifty grams of each maize sample from baskets, polypropyne, jute and polythene bags was weighed and placed in a glass beaker containing 200 mL of double distilled water and stirred for two minutes (Okayo et al., 2020). Using a teat pipette, the spore suspension was transferred into a heamocytometer. The number of spores from each sample was determined with the help of a microscope (Abe et al., 2015).

Data analysis

Data on the number (CFU/g) of fungal isolates from the selected Counties, mean fungal isolates and spore count per gram of maize sample were subjected to analysis of variance (ANOVA) using PROC ANOVA procedure of Genstat Discovery 2 statistical software version 25.0. The means were compared using Fisher’s protected LSD test at 5%.
Fungal isolates from the selected counties

The fungal isolates from Trans-Nzoia varied from 12±0.3 CFU/g to 63±0.3 CFU/g, Kisii (11±0.2-62±0.3), Kisumu (11±0.2-59±0.2), Bungoma (12±0.1-55±0.1), Migori (13±0.2-54±0.3), Kericho (11±0.2-59±0.2), Machakos (4±0.1-15±0.2), Kitui (5±0.3-14±0.1) and Meru (6±0.2-57±0.3) CFU/g  (Table 1). The mean fungal isolates varied from 10.8±0.2 CFU/g in Wallenia spp. to 47.4±0.2CFU/g in Aspergillus spp. The most common fungal isolates were Aspergillus spp., Fusarium spp. and Penicilium spp. (Fig 1). The number of fungal isolates from the selected counties varied significantly (F=1.987 P=0.0484). These results differed with a previous study carried out by Tola and Kebede, (2016). The difference could be attributed to variation in fungal control methods used by farmers in the study sites. Marroquin-Cardona et al., (2014) asserted that some farmers use long term fungal control methods which influence the fungal species that infect maize. In addition the most common fungi are known to produce a variety of mycotoxins (Al-Defiery and Merjan, 2015). This may explain the high cases of mycotoxins poisoning outbreaks reported in the study areas (Njoroge et al., 2016).

Table 1: Number (CFU/g) of fungal isolates from the selected counties.

Fig 1: Most common fungal isolates from maize samples from the selected Counties; A and B; Aspergillus spp., C; Penicillium spp. and D; Fusarium spp.

Morphological and cultural characteristics of the fungal isolates

Identification of the fungal isolate was carried out using colony morphology (cell size, shape, pigmentation and arrangements) and cultural characteristics (Table 2). Use of morphological and cultural characteristics in identification of fungi is the oldest and most widely used tool for fungal identification (Tai et al., 2020). The morphological and cultural characteristics concurred with a previous study carried out by Mousa et al., (2016). This may be attributed to isolation of the same fungal species (Ncube et al., 2021). Okayo et al., (2020) successfully isolated and characterized maize fungi using morphological and cultural means.

Table 2: Morphological and cultural characteristics of the fungal isolates.

Mean fungal isolates from the selected counties

The mean fungal isolates from the selected Counties varied from Kitui (9.6±0.2 CFU/g), Machakos (9.7±0.3 CFU/g), Bungoma (25±0.1 CFU/g), Migori (25.3±0.3 CFU/g), Kericho (25.3±0.2 CFU/g), Kisumu (27.1±0.1 CFU/g), Meru (27.1±0.2 CFU/g), Kisii (28.5±0.1CFU/g) and Trans-Nzoia (30.1±0.3 CFU/g) (Fig 2). The mean fungal isolates between the selected counties varied significantly (F=1.877 P=0.0474). Although these results indicated that fungi can grow in a wide range of ecological conditions, Kitui presented the lowest fungal isolates while Trans-Nzoia County presented the highest. This disagreed with other studies curried out elsewhere (Maryam, 2017; Okayo et al., 2020; Tai et al., 2020) which can attributed to differences in humidity conditions of various study sites (Medina, 2017). According to Ncube et al., (2021), fungal growth is favoured by warm, moist and humid conditions.

Fig 2: Mean fungal isolates from the selected Counties.

Spores count of the fungal isolates

The number of spores in maize samples from baskets varied from 13.6±0.2-19.6±0.2, polypropylene (24.0±0.2-24.0±0.2), Jute (13.6±0.2-21.6±0.2) and polythene bag (48.0±0.2-72.0±0.2) CFU/g (Table 3). The number of spores obtained from maize stored in baskets, polypropylene, jute and polythene bag varied significantly (F=3.815 P=0.03945). The results were in agreement with a previous study by Nyongesa et al., (2015). Dube and Mutewa (2015) observed that baskets allows further drying of maize in storage conditions. According to Peremingo et al., (2016) maize is in most cases harvested when moisture content is still high (Njoroge et al., 2016). Tai et al., (2020) recommended storage of maize in well aerated containers to further reduce the moisture content in the grains. Further, Negash et al., (2018) established that post-harvest practices of most farmers expose maize to infection by fungi prior to storage.

Table 3: Number of spores per gram of maize sample.

This study established that maize in the selected Counties is highly contaminated with fungi. The most prevalent fungal spp. were Aspergillus, Penicillium and Fusarium. The best maize storage material for maize were baskets. There is need of identifying the most prevalent fungi up to the molecular level. The mycotoxins produced by the most prevalent fungal spp. need to be established.

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