ISOLATION AND IDENTIFICATION OF FUNGI ASSOCIATED WITH IRVINGIA SPECIES KERNELS FROM SOME MARKETS WITHIN ZARIA METROPOLIS AND THEIR AFLATOXIN CONTENT

ABSTRACT

This study was carried out, in order to isolate, identify the various fungi associated with Irvingia kernels and to ascertain whether or not there was significant aflatoxin content in Irvingiakernels sold in five markets in Zaria. The two species of Irvingiastudied were Irvingiagabonensisand Irvingiawombolu. Commercially available Potato Dextrose Agar (PDA) served as the general purpose culture mediaandSabouraud Dextrose Agar (SDA) served as the selective media, which was used in the preparation of pure cultures. The cotyledons and thetesta were examined separately. Aspergillusflavus, Aspergillusfumigatus, Aspergillusniger, Penicilliumspecies and Rhizopusstolonifer were isolated from both Irvingiagabonensisand Irvingiawombolutesta and cotyledons. Four fungal species, namely, Absidiacorymbifera, Aspergillusversicolor, Mucor species and Phomaherbarum were only isolated from Irvingiawombolu cotyledons and testa.More A.flavus, A .nigerand Penicillium sp. were isolated from the testa of both I. gabonensis and I. wombolu,than from the cotyledons. Rhizopusstolonifer had the highest percentage occurrence in the cotyledons of both I.gabonensis (69.44%) andI.wombolu (47.43%). Aspergillusnigerhad the highest percentage occurrence (37.59 %) of all the fungi isolated, and was closely followed by R.stolonifer(33.81 %).Similarly, A. nigerhad the highest percentage occurrence in the testa of both I.gabonensis (45.71%) andI.wombolu (42.94 %). Also, I.gabonensis samples from SabonGari market had the highest mean number of fungal species (12), while Dan Magaji/Wusasa market had the least (2). Similarly, I.wombolu samples from A.B.U Community, Dan Magaji/Wusasa and Tudun Wada markets all had the highest mean number of fungal colonies (24), while Samaru market had the least (22). Enzyme-Linked Immunosorbent Assay (ELISA) of the kernels showed that,I.gabonensis and I.wombolu kernels from SabonGari market had the highest aflatoxin concentrations (108.36 and 95.60g/kg) respectively. Similarly, I.wombolu kernels from Dan Magaji/Wusasa market had the least (70.28mg/kg) aflatoxin concentration, while I.gabonensis kernels from Samaru market had the least (75.02mg/kg). They were all beyond the World Health Organization (WHO) standard for food samples, which is 20mg/kg, hence not safe for human consumption. The study concludes that,the two Irvingia species kernels sold in some markets within Zaria metropolis were contaminated by several post-harvest fungi. One of these fungi isolated,Aspergillusflavus, had the capacity to produce aflatoxins, therefore the consumption of these kernels puts the consumers at risk.

CHAPTER ONE

1.0   INTRODUCTION

1.1 General Introduction/Background Information on Irvingia species

The natural forests of West and Central Africa are rich in resources, and have tremendous biodiversity (FAO, 1983), particularly in trees that provide food, fuel, fibre, medicines and various other products, including construction and building materials (Ladipoet al., 1996).

The tropical rainforests of West and Central Africa is the home to many native economically important trees such as the Irvingia species, namely Irvingia gabonensis, and Irvingiawombolu(Harris, 1996; Lowe et al., 2000).

The Irvingia plant is dicotyledonous, and belongs to the Irvingiaceae family, and order Malpighiales. It is known by a variety of local names, both within and outside Nigeria; among which are bush mango or African mango for English speakers. In Yoruba language, the tree is known as “Oro”, while the kernel is known as “Apon” (Adebayo-Tayoet al., 2006). Ndigbo call it Adu, Ugiri or Ogbono depending on the species (Okafor, 1978). In Nupe, it is called “Pekpeara”, “Ogwi” in Bini (i.e. Edo), “Uyo” in Efik (Okafor, 1984), „Oro-Akpele‟ in Igala. The Eton people of Lekie division in Cameroon call it „Azang‟, „Andok‟ or „Beti‟. The Mpong Mpong people of Haut Nyong division in Cameroon call it “Ogno‟k”, while the Bulu people of Mvila division in Cameroon call it „Andoh‟. In Gabon, it is known as the “Dika” nut (Ayuk et al., 1999). In french, it is known as „Manguier sauvage‟. The paste produced from the kernels is termed “dika bread” in Gabon, while in Cameroon, it is known as “etima” (Ndoye et al., 1997), it is also called „Borborou‟ by the Abbey (Cote D‟ivoire) and „Ewewe‟ by the Bolon tribe (Gabon) (Vivien and Faure, 1988; 1996; Ake-Assi,1991; Ndoye and Tchamou, 1994; Tabuna, 1997).

All these names cut across South East, South South, South Western and North Central Nigeria, as well as some other Western and Central African countries where the Irvingia plants are grown, or where the kernels are eaten in one form or the other. The generally accepted common name in Nigeria is “Agbono” or “Ogbono” (Festus and Nwala, 2012).

The Irvingia tree is large, attaining a height of about 50m when it grows naturally in the wild, but under improved agricultural practices, especially when budded seedlings are planted, it could attain a height of about 22.5m, with a straight bole, cylindrical, slightly buttressed (Festus and Nwala, 2012). The buttresses reach a height of 3m (9.8ft) (Orwaet al., 2009). The cylindrical trunk exceptionally exceeds one metre in diameter, with a well developed and maintained support (Festus and Nwala, 2012). It has a hard, heavy and durable wood, with a fine grain, easy to polish, as well as being resistant to termite.

The leaves are about 1.5 – 1.7cm long, spirally arranged and grouped towards the tips. It is alternately arranged, simple, elliptic and shiny on both surfaces (Festus and Nwala, 2012).Under natural conditions, the Irvingia tree matures (reproductive maturity) between the ages of 8 – 10 years in some cases, while in some others maturity is delayed till between 15 – 20 years (Festus and Nwala, 2012). However, with modern improved cultural management practices, which include budding and topping, especially with the use of budded seedlings, the vegetative phase is considerably reduced, and flowering and fruiting can start from 4 – 5 years, while economic yield can be attained after 7 – 8 years from planting (Ladipo et al., 1996).

Maturity of the Irvingia tree refers to the stage when the plant begins to produce flowers, in readiness for fruitification. The first stage in the flowering process is floral induction or evocation (Sedgley and Griffin, 1989). It is not known what triggers this process in Irvingia species, but a substantial variation was observed in the number of floral flushes within the population of 182 trees planted in 1990, with most trees not flowering at this age, or flowering only once a year, with a few flowering 2 – 4 times per year (Ladipo et al., 1996).The flowers produced during the flowering period are arranged in axillary racemes, fragrant, small, greenish and hermaphrodite. They bear disks that become bright and yellow during ripening stage (Alston, 1992).Irvingia gabonensis flowers in February – March, and fruits in the rainy season (July – September), while Irvingia wombolu flowers in October, and fruits in the dry season (January – March), (Franzel et al., 1996).Fruiting on the other hand, sets in when the flowers produced at the stage of flowering are pollinated by insects such as Coleopterans, Dipterans, Hymenopterans and Lepidopterans (Orwa et al., 2009), as well as mammals and birds.The maturedfruit is greenish when unripe, but turns yellow when ripe, with a fleshy mesocarp. The fruits are broadly ellipsoid, about 4 – 7 cm long, and 3 – 5 cm in diameter. The fruits of the two Irvingia species resemble those of the common mango (Mangifera indica), hence the name African or bush mango (Okafor, 1975; Simons and Leakey, 2004; Matoset al., 2009).The fruit of Irvingia gabonensis is usually bigger than that of Irvingia wombolu.When ripe, the fruit of Irvingia gabonensis has a sweet edible mesocarp, hence it is usually consumed by man and animals fresh, whereas Irvingiawombolu has a bitter or sour taste, and very slimy mesocarp, hence rarely eaten by man and animals (Etukudo, 2000; Fajimi et al., 2007).

1.2         Uses of Irvingia

Every part of the Irvingia gabonensis and Irvingia wombolutree is useful, ranging from the stem, fruits and kernels, to the leaves. The stem supplies durable timber for construction purposes. The branches supply firewood, as well as chewing stick. The fruit mesocarp of Irvingia gabonensis in particular, is consumed by both man and livestock (Ayuk et al., 1999).

It serves as dessert fruit or snack throughout Western and Central Africa. The fruit mesocarp is equally used for the preparation of juices, jams, jellies, wine, as well as in soap making (Okafor, 1985; Shiembo et al., 1996; Leakey et al., 2003). The fruit juice obtained from the pulp is rich in vitamin C (Ejiofor, 1994; Leakey and Newton, 1994).The leaves and stem bark are employed as purgative, for gastro-intestinal and liver conditions, for hernias and urethral discharge, or for sores and wounds (Ayuk et al., 1999).In some traditional/cultural practices, the split seed shell is used in prediction or fortune telling (Abbiw, 1990). The mature fruits serve as ripening agent for bananas and plantains. Environmentally, the Irvingia tree serves as wind break (Ladipo, 2000).

1.3         Fungal Attack of Irvingia kernels

Irvingia gabonensis(Plate I) and Irvingia wombolu(Plate II) kernels are economically valuable due to the fact that they have both health and medicinal benefits (Duguma et al., 1990; Ndoye et al., 1997; Van 2010). However, the sales and consumption of Irvingia kernels has a major set back, since the kernels are susceptible to post-harvest spoilage fungi, with their attendant health risks (Etebu and Bawo, 2012). Fungal contaminated Irvingia kernels are potentially harmful to those who consume them (Adebayo-Tayo et al., 2006), as it has been observed that these contaminated kernels could possess aflatoxin.

Mycotoxins are toxic secondary metabolites naturally produced by molds (fungi), (Pittet, 2005). Mycotoxin is a term derived from the Greek word “Mykes” meaning fungus, and the Latin word “toxicum” meaning by poison (Adjou et al., 2012).Aflatoxin is just one out of the many mycotoxins produced by harmful fungi. Aflatoxin is a group 1 carcinogen, proven to cause liver cancer, and also suppresses the immune system and ultimately death when consumed in high doses (Lewis et al., 2005; Strosnider et al., 2006)......

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