Incidence of mycoflora complexes on retail kolanuts (C. nitida and C. acuminata) in North Central Nigeria

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1 Journal of Applied Biosciences 56: ISSN Incidence of mycoflora complexes on retail kolanuts (C. nitida and C. acuminata) in North Central Nigeria Adeniyi D. O*, Kolawole O. O, Oduwaye O. F, Adejobi K. B, Adenuga O. O, Adepoju A. F, Olaniyi O. O and Anagbogu C. F. Cocoa Research Institute of Nigeria, Km 14, Ibadan-Ijebu Ode Road PMB 5244, Ibadan, Nigeria *Corresponding author: modeleadeniyi@gmail.com Original submitted in on 20 th June Published online at on August 30th ABSTRACT Objectives: The prevalence of mycoflora associated with retail kolanuts (C. nitida and C. acuminata) obtained randomly across the North Central States of Nigeria was studied. Methodology and results: Surface sterilized tissues of the kolanuts samples were cultured on acidified Potato Dextrose Agar (PDA) using standard laboratory methods. Twelve fungi isolates, mostly toxigenic and belonging to nine genera (Aspergillus, Fusarium, Mucor, Lasiodiplodia, Paecilomyces, Chlamydomyces, Rhizopus, Penicillium and Curvularia) were isolated from the kolanuts tissues. L. theobromae and F. pallidoroseum were the most predominant isolates and the incidence of L. theobromae on C. nitida was significantly higher (p<0.05) than other fungi isolates. However, the occurrence of F. pallidoroseum was more frequent than L. theobromae in C. acuminata, but the difference was not significant. Conclusion and application of results: Mycoflora complexes especially of toxigenic potentials were found associated with retail C. nitida and C. acuminata in the north central states of Nigeria, raising questions on the safe consumption of the crop. Key words: Mycoflora, retail, C. nitida, C. acuminata, north central, Kolanuts. INTRODUCTION Cola species are widely cultivated in tropical countries especially in Africa. Cola nitida was originally distributed along the west coast of Africa from Sierra Leone to the Republic of Benin with the highest frequency and variability occurring in the forest areas of Cote d Ivoire and Ghana (Opeke, 1992). C. nitida is planted through Senegal, Guinea, Liberia, Cote d Ivoire and Ghana towards the western part of Nigeria. However, Southern Nigeria is considered the centre of occurrence of Cola acuminata, with its original area of distribution stretching from Nigeria to Gabon. It also occurs spontaneously in the mountainous areas of Angola, Zaire and Cameroon, and has been cultivated in Islands of Principe and Sao Tome (Opeke, 1992). The fruit of the kola tree contains seeds known as kola nuts. Kolanuts are the cotyledons of some species of Cola, a genus of trees belonging to the family Sterculiaceae (Purseglove, 1974). They are a major stimulating masticatory in West Africa. They are used for pharmaceuticals and for flavoring soft drinks and in the preparation of coca-cola and wine (Opeke, 1982). These nuts are consumed for their taste and are traditionally used in Western and Central Africa during weddings, funerals and rituals. Kola nut (C. nitida) is a central nervous system stimulant which has been shown to mediate some 4075

2 physiological effects that are similar to the action of caffeine (Carrillo and Bennitez, 2000). Kola nut is chewed in Africa for its alkaloid properties which dispel sleep, thirst and hunger. The nuts are also used in the pharmaceutical and food industries to produce cardiac stimulants, laxatives, sedatives and sodas. The cultivation, processing and storage of the nuts are undertaken in the warm, humid rain forest zone where there is high mould infection. However, so highly esteemed are the nuts that fairly moldy samples are commonly ingested (Adebajo and Popoola, 2003). Mould growth is a vital problem encountered by farmers and retailers in the process of preservation and storage of kolanut. Storage of kolanut has always been a laborious and delicate task. Kola traders were reportedly using the traditional methods of storage (Agbeniyi and Ayodele, 2010). However, kolanut retailers have a measure of regular removal of infested nuts during storage and get them separated to control and minimize spread. Peasant processing and packaging MATERIALS AND METHODS Collection of kolanut samples: Samples of seemingly healthy kolanuts (C.nitida and C. acuminata) were purchased from retailers in different locations in the North Central States of Nigeria. The selected locations were; Ilorin and Offa (Kwara), Kabba and Ayingba (Kogi), Gboko and Otukpo (Benue), Bida and Minna (Niger), Lafia and Akwanga (Nassarawa) and Jos and Barkin- Ladi (Plateau). The kolanuts samples were collected in a sterile polythene bags, labeled appropriately and assayed in-vitro within a week of collection. Isolation and Identification of mycoflora: The Potato Dextrose Agar (PDA) was routinely prepared in the laboratory and acidified with 10% lactic acid. With the use of sterilized scalpel, the kolanut samples were cut into small pieces of about 3mm in diameter, surface sterilized in 2% Sodium hypochlorite for 2 minutes and rinsed in predisposes the kola nuts to mould contamination which is worsen especially during hawking. Odebode (1990) reported the susceptibility of kola nuts to fungal infection. However, Agbeniyi et al. (2000) observed that the contamination of healthy nuts with spores from rotted nuts is often a greater economic problem in fresh market producing areas. Botryodiplodia theobromae has been found to be the most common single species of pathogen associated with kola. Lasiodiplodia theobromae has also been described as the causal organism of storage rot which is said to be the most severe post-harvest disease of kola nut. This fungus can also initiate latent infections on nuts in the field when harvest is delayed, consequently resulting in rot during storage. Hence, mycotoxicoses is a serious concern, which is aggravated by reluctance of consumers to discard fairly moldy nut samples for its economic value. The objective of this study is therefore to establish the array of mycoflora complexes associated with retail kolanuts in the North central states of Nigeria. four changes of sterile distilled water. The sterilized tissues were blotted dry in between sterile Whatman No 1 filter paper (125mm) and inoculated into petridishes containing about 20ml of solidified PDA. Each of the inoculated samples was replicated five times. The inoculated plates were incubated at 28+2 o C, colony-growth was observed and emerging fungal colonies were subcultured onto PDA plates to obtain pure cultures. The isolates were identified after microscopic examinations and fungi occurrence was recorded. Frequency of occurrence of the fungi isolates: The frequency of occurrence of the fungal isolates from the nuts of C. nitida and C. acuminata was determined by calculating the percentage frequency of occurrence of isolates by the formula: Freq. of Occurrence = No. of Plates containing Species 100 No. of Plates in Sample 4076

3 RESULTS Twelve (12) fungi isolates were cultured from the C. nitida and C. acuminata samples. They include Lasiodiplodia theobromae, Aspergillus niger, A. flavus, A. fumigatus, Mucor spinosus, Paecilomyces variotii, Chlamydomyces sp., Rhizopus spp. F. pallidoroseum, F. moniliforme, Penicillium sp. and Curvularia sp. (Table 1). Table 1: Mycoflora distribution of sample locations Fungi Ilorin Offa Kabba Ayingba Gboko Otukpo Bida Minna Lafia Akwanga Jos Barkin- Ladi A. niger A. flavus A. fumigatus F.pallidoroseum F. moniliforme M. spinosus L. theobromae P. variotii Chlamydomyces sp. Rhizopus spp Penicillium sp Curvularia sp Present (+), Absent (-) L. theobromae and F. moniliforme were isolated in nine locations within the selected areas. A. niger, Rhizopus spp. and F. pallidoroseum were found in seven of the locations. Penicillium spp. and A. fumigatus occurred in six locations, while Curvularia spp was intercepted in five locations. A. flavus was also isolated in four locations. However, each of P. variotii and Chlamydomyces spp. were found in two locations. Generally, the highest incidence of fungi complexes associated with the kola nuts samples was found in Kwara State. The analysis of the data obtained from the study showed that the fungi isolates cultured from C. nitida differ significantly (p<0.05) from each other in their occurrence (table 2). Table 2: Occurrence of mycoflora of C. nitida and C. acuminata Fungi Frequency of occurrence (%) C. nitida C. acuminata A. niger 11.4c 10.2c A. flavus 18.1b 5.2cd A. fumigatus 0.0d 9.1c F.pallidoroseum 22.8b 39.7a F. moniliforme 12.1c 8.2c M. spinosus 10.2c 9.5c L. theobromae 32.2a 34.5a P. variotii 12.0c 2.5d Chlamydomyces sp. 0.08d 0.0d Rhizopus spp. 9.5c 11.4c Penicillium sp. 11.8c 21.5b Curvularia sp. 0.09d 0.0d Mean not followed by the same letter in the same column are significantly different (P = 0.05) according to Duncan s Multiple Range Test. 4077

4 L. theobromae was significantly higher than other fungi isolates and there was no significant difference between the occurrence of A. flavus and F. pallidoroseum. The occurrence of F. moniliforme, A. niger, M. spinosus, P. variotii, Penicillium sp and Rhizopus spp. were not significantly different from each other. Curvularia spp and Chlamydomyces spp were also not significantly different in their occurrence. However, the incidence of A. fumigatus was not observed in C. nitida. Unlike the observation made from C. nitida, the incidence of L. theobromae and F. pallidoroseum differ significantly (p<0.05) from DISCUSSION The fungi isolates on kolanuts reported in this study was in concordance with the findings of Dongo et al., (2007) and Agbeniyi (2004). They reported the occurrence of Botryodiplodia theobromae, Fusarium pallidoroseum, F. moniliforme, F. cavispermum, Aspergillus niger, A. fumigatus, A. flavus, Paecilomyces variotii and species of Penicillium. The incidence these fungi are not restricted to kolanuts only, as Adebajo and Diyaolu (2003) reported their occurrence in retail cashew nuts. Some species of the isolated fungi from the kolanuts especially species of Aspergillus and Penicillium are known to have strains that produce toxic metabolites, thus they pose a potential hazard to consumers health. This observation corroborates the findings of Adebajo and Popoola (2003) which reported the isolation of Aspergillus niger, A, flavus, Penicillium funiculosum, Fusarium moniliforme, A, tamarii, Botryodiplodia theobromae, F. oxysporum and A. orchraceus as most commonly isolated fungi from kolanuts. The findings of Dongo et al. (2007) showed the isolation of fungal species belonging to the genera Penicillium and Aspergillus and the subsequent detection of Ochratoxin A (OTA) from REFERENCES Adebayo A A (1966). Kola diseases. Ann. Rept. Cocoa Research Institute of Nigeria, pp other fungi isolates obtained from C. acuminata, although the occurrence of F. pallidoroseum was more frequent than L. theobromae, but the difference was insignificant (p<0.05) (able 2). Occurrence of Penicillium spp. also differs significantly from other fungi isolates. There was no significant difference among the percentage occurrence of A. niger, A. fumigatus, F. moniliforme, M. spinosus, A. flavus and Rhizopus spp. However, A. flavus and P. variotii were not significantly different in their occurrence. The incidence of Chlamydomyces spp. and Curvularia spp. was not observed in C. acuminata. the kolanut samples. Earlier findings also reported the major fungal causing mould and rots of the nuts as Botrytis spp; B. theobromae; Paecilomyces variotii; Mucor spp and Fusarium spp, which are all known to be favored by high relative humidity (Adebayo, 1966; Olunloyo, 1979; Agbeniyi and Fawole, 1999; Agbeniyi et al., 2000). The conditions generally known to influence production of mycotoxins in foods and allied agricultural products include presence of a toxigenic mould, a suitable substrate for the growth of the mould, and an environment conducive for toxin production by the mould. Thus, toxin production might not be far fetched in these retail kolanuts samples as a number of toxigenic fungi were isolated. Hence, findings in this study can be harnessed as a rung for further investigations on retail kolanuts in Nigeria. Botanicals indigenous to the study locations can be assessed for their potentials to prevent or reduce the incidence of these fungi on kolanuts below toxin and disease threshold quantity, which can thereafter be utilized traditionally for packaging and preservation kolanuts. Adebajo LO and Diyaolu S A (2003). Mycology and spoilage of retail cashew nuts. African 4078

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