Botanicals and the Growth of Aspergillus flavus from Stored Groundnuts

Transcription

1 World Journal of Agricultural Sciences 13 (1): 39-44, 2017 ISSN IDOSI Publications, 2017 DOI: /idosi.wjas Botanicals and e Grow of Aspergillus flavus from Stored Groundnuts V.C. Okereke, M.I. Godwin-Egein and C.T. Oji Department of Crop and Soil Science, Faculty of Agriculture, University of Port Harcourt, PMB 5323, Port Harcourt, Nigeria Abstract: Sampling of stored groundnut was carried out in five different markets in Port Harcourt, Nigeria to determine e occurrence of mycotoxin producing fungi and e subsequent effects of some botanicals on most prevalent fungus in vitro. The fungi were isolated and identified by standard meods of culturing and subculturing. Aqueous leaf extracts were obtained from locally sourced Garcinia kola, Gongronema latifolium, Moringer oleifera, Azadirachta indica, Ocimium gratissimum and Vernonia amygdalina. The extracts were applied at ree different concentrations namely; 25, 50 and 75g per litre in sterile distilled water. The experiment was laid out in completely randomized design (CRD) replicated four times. Results showed at Aspergillus flavus, Aspergillus niger and Fusarium oxysporum were predominantly isolated from e grains, wi A. flavus having e highest occurrence. Mycelial grow of Aspergillus flavus was significantly (P<0.001) reduced by V. amygdalina, G. kola, O. gratissimium and G. latifolium in ascending order of magnitude, at 7 days after treatment. The concentration of 50g/litre showed e best inhibitory effects. V. amygdalina, G. kola, O. gratissimium and G. 50g/litre could form an integral part of active component for botanical based fungicide production to protect stored groundnuts against Aspergillus flavus and subsequent aflatoxin contamination. Key words: Groundnut Leaf extract Aspergillus flavus Mycelial grow Concentration INTRODUCTION considerable risk to human as well as livestock heal and has significant economic implications to e agricultural Wi e alarming increase of e world population industry worldwide [5, 6]. In Africa, aflatoxin by one billion people nearly every decade [1], maintaining contamination of agricultural crops, such as groundnut a stable food supply is a crucial goal for mankind. and cereals, causes annual losses of more an $750 Groundnut (Arachis hypogea L.), grown around e world million [7]. These constraints have resulted to e is a crop capable of contributing to a global diet. It is an unavailability or contamination of stored groundnuts. important food and forage crop because of its high Several management operations have been employed protein and oil contents [2, 3]. The grain contains 40-50% in e past in a bid to curb e devastating effect of fat, 20-50% protein and 10-20% carbohydrates [4]. In mycotoxin producing fungi ereby ensuring e Nigeria, groundnuts are widely grown in many parts of e availability of groundnut after harvest. The increasing country, but one of its major constraints is e infection demand for e consumption of groundnut and of e grains in bo e field and e store by fungi. regulations on e use of agrochemicals and e Grow of commonly occurring filamentous fungi in emergence of fungi resistant has necessitated e search foods may result in e production of toxins known as for new control strategies. Alough, ere are a number mycotoxins, which can cause a variety of ill effects in of synetic fungicides available in e market for humans and livestock. Some common mycotoxicoses checking ese losses, ey have been shown to have caused by common and widespread fungi such as several side effects such as high toxicity, long Aspergillus, Penicillium and Fusarium species result in degradation periods and residue problems [8]. Plant severe illness and dea. Aflatoxins produced by materials are very rich sources of bioactive chemicals; Aspergillus spp on agricultural commodities pose ey are volatile, biodegradable, eco-friendly and easily Corresponding Auor: V.C. Okereke, Department of Crop and Soil Science, Faculty of Agriculture, University of Port Harcourt, PMB 5323, Port Harcourt, Nigeria. chykeoky@yahoo.com. 39

2 available in local environments [8]. Several studies have powder using a Philips blender (HR 2815 Holland). They been conducted on e use of ese plant materials in e were weighed and packaged in an air-tight container control of diseases in e store. labeled properly and stored in a refrigerator until used. Some tropical aromatic plants have shown to exert high antimicrobial activities and since ey are natural Isolation and Identification of Fungi: The grain samples products, mostly consumed by man, ere is little or no gotten from e markets were washed and first sterilized in fear of poisoning even at very high concentrations [9]. 2% sodium hypochlorite for ree minutes and rinsed in According to [8, 10, 11] extracts from ese plants contain several changes of sterile distilled water. The sterilized a wide range of bioactive secondary metabolites which grains from each market were en placed on two include alkaloids, flavonoids, tannins, saponins, phenols, moistened filter papers in 90 mm five grains phlobatannins, quinones, lecitins, polyphenols, per plate and a total of ten Petri dishes per market. The glycosides, terpenoids, polypeptides and steroids and Petri dishes were en kept in e inoculation chamber at ese bioactive substances have been given as e reason room temperature and observed for fungal grow. Subfor e inhibitive roles against fungi in enobotany, drug cultures were made from emerging colonies repeatedly application and plant heal management [12-14] Leaves until pure cultures were obtained. The fungi were of O. gratissimum have been found to exhibit high identified using Illustrated Genera of Imperfect Fungi by antifungal activities against Fusarium moniliforme, [19]. The experiment was repeated five times. The fungal Aspergillus flavus and Aspergillus fumigatus [15] and it isolates were furer checked for eir viability and purity is said to be a potential food preservative [16]. using standard biochemical test. Aframomum danielli has been reported to show antimicrobial activities and also reduced aflatoxin Preparation of Aqueous Leaf Extracts: Leaf powder of concentration in maize [17, 18]. It has also been used in Garcinia kola, Moringer oleifera, Ocimum gratissimum, e storage of maize and soybean [9]. Gongronema latifolium, Vernonia amygdalina and Furermore, literature is scanty on e distribution of Azadirachta indica (Neem), were each soaked in sterile mycotoxin producing fungi in stored products in some distilled water at ree different concentrations as follows; markets in Port Harcourt, Nigeria. This study was erefore, aimed at (i) investigating e occurrence and 2.5g/100ml distribution of mycotoxin-producing fungi on groundnuts 5.0g/100ml and sold in e markets of Port Harcourt (ii) evaluating e 7.5g/100ml of sterile distilled water. efficacy of plant leaf extracts on in vitro grow of Aspergillus flavus associated wi stored groundnuts. They were left for 24 hours and en filtered using cheese clo into well labeled containers and used for MATERIALS AND METHODS treatment application immediately. Study Site: Market survey was carried out in five different Treatment Application: 10 ml of Potato Dextrose Sugar markets along e East-West Road in Port Harcourt, (PDA) and 1.5 ml of each of e leaf extracts were poured Nigeria namely; Rumuokoro, Slaughter/Eliozu, Tank, into e Petri-dishes and allowed to gel. Using a 5 mm cork Rumuokwurusi and oil mill markets. The in vitro borer, e pure cultures of e test fungus was introduced experiment was carried out in e Department of Crop and at e center of each plate. The plates were kept at a room Soil Science major laboratory, University of Port Harcourt, temperature and daily measurement of e mycelial grow Port Harcourt, Nigeria. of e fungus was taken. The control plates comprised of PDA, sterile water and e test fungus wiout e leaf Experimental Materials: Fresh leaves of Garcinia kola, extracts. Moringer oleifera, Azadirachta indica, Ocimium gratissimium, Vernonia amygdalina, Gongronema Data Collection: Data was collected on e following:- latifolium, were locally sourced, while e groundnut Percentage occurrence of e fungi across e grains were procured from e markets. The plant materials sampled markets. were washed oroughly, air-dried on e laboratory Daily measurement of e mycelial grow of e test bench and in e oven at 55 C for 24h and ground em to fungus for 7 days. 40

3 Experimental Design and Data Analysis: The experiment extracts on e in-vitro grow of A. flavus. The result was laid out in a 6 (leaf extracts) 3 (concentrations) in showed a significant effect of e plant extracts on completely randomized design (CRD) replicated four e mycelial grow of A. flavus from day 2 to day times. The control had four plates. Analysis of variance 7 (P < 0.001), but day 1 showed no significant effect of e (ANOVA) was obtained using GenStat (GenStat 16 plant extract (P= 0.5). Plates treated wi V. amygdalina Edition; VSN International Ltd, UK) and means separated had e lowest mycelial grow rate of 13 mm on e 7 using e standard error of difference (SED) at 5% level of day, followed by G. kola, O. gratissimum and G. probability. latifolium wi mycelial grow of 15 mm on e 7 day of treatment. Higher mycelial grow was observed in M. RESULTS Oleifera and A. indica. The fungus covered e control plates 4 days after treatment. Percentage Fungi Occurrence: Table 1 shows e percentage distribution of fungi across e markets Effect of Concentration on Mycelial Grow: Fig. 2 shows sampled. Three different species of fungi were e effect of concentration of e plant extracts on e predominantly isolated from grains namely; Aspergillus mycelial grow of A. flavus. No significant effect of e niger, Aspergillus flavus and Fusarium oxysporum. concentration on mycelial grow was observed at day 1 The results showed at A. flavus has e highest (P=0.65), day 2 (P=0.94) and day 3 (P=0.43). However, percentage occurrence in e sampled grains wi e ere was significant effect (P<0.05) of concentration from highest percentage occurring at Rumuokwurushi (62%), 4 day after treatment, wi 5g/100ml and 7.5g/100ml followed by Tank market (50%) and en Rumuokoro concentration showing e least mycelial grow of 23 mm market (45%). and 24 mm, respectively, 7 days after treatment. Plates treated at concentration of 2.5g/100ml and e control Effect of Leaf Extracts on Mycelial Grow of Aspergillus plates had higher mycelial grow rate of 30 mm and flavus in vitro: Fig. 1 shows e effect of different plant 47mm, respectively. Table 1: Percentage occurrence of fungi paogens isolated from e different markets. Market/Paogen Aspergillus niger Aspergillus flavus Fusarium oxysporum Rumuokoro Slaughter/Eliozu Tank Rumukwurushi Oil mill Fig. 1: Effect of leaf extracts on mycelial grow of Aspergillus flavus 41

4 Fig. 2: Effect of concentration on e mycelial grow of Aspergillus flavus DISCUSSION of botanicals has been focused on as a viable pest control strategy. Significant work has been done on e use of After treating e fungus wi aqueous leaf extracts botanicals as control agents against fungal paogens to determine eir efficacy on e grow of A. flavus in of stored products. However, less work has been vitro, it was revealed at V. amygdalina, G. kola, G. reported on e occurrence and prevalence of latifolium and O. gratissimium, significantly inhibited mycotoxin producing fungi in stored products in mycelial grow of Aspergillus flavus. The inhibitory Port Harcourt Nigeria. In e present study, A. flavus effect of O. gratissimium corroborates e reports of was e most prevalent in e sampled grains, wi [31,32] at leaf extract of O. gratissimum exhibited high Rumukrushi market showing e greatest occurrence. antifungal activities against Fusarium moniliforme, The high occurrence of A. flavus in Rumukrushi Aspergillus flavus and Aspergillus fumigatus. [33] have market which is a daily market might probably be also reported reduction in seed mycoflora of wheat when attributed to e presence of a big weekly market treated wi extracts of Vernonia amygdalina. The leaf close to it. People from far and near come to trade extracts of A. indica and M. oleifera may have had low resulting in e high build of disease causing inhibitory effect on e grow of A. flavus contrary to e agents in e market. The big market wi e least reports of [34]. In eir work aqueous extract of Moringa occurrence may be attributed to e fact at it is a oleifera and emulsified seed oil of Azadirachta indica weekly market where people from different places seed oil were reported to have inhibitory effect on come to sell eir products and go wiout leaving Aspergillus flavus in tomatoes. Probably e neem seed eir stock behind so ere is less build-up of fungi. could contain more antifungal substances an e leaf [20-24] have stated at groundnut is one of e most against Aspergillus flavus. susceptible host crops to Aspergillus flavus which produces aflatoxin in e products. The works of CONCLUSIONS [25-27] also confirmed at A. flavus is a paogen of several crops including maize, groundnuts, soybean and The results obtained from is study showed at oer stored products in Africa. The incidence of Aspergillus flavus, Aspergillus niger and Fusarium sp. Aspergillus spp., Penicillium spp., Rhizopus spp. and were associated wi stored groundnut wi A. flavus Mucor spp. on oer grains such as sorghum and millet being e most prevalent. V. amygdalina, G. kola, G. have also been reported [28-30]. Aflatoxins produced by latifolium and O. gratissimium was efficacious in e Aspergillus spp on agricultural commodities poses inhibition of e grow of A. flavus at e application rate considerable risk to human as well as livestock heal and of 50g/litre of water. These botanicals show promise for have significant economic implications to e agricultural e management of A. flavus and could be incorporated in industry worldwide [5]. In recent years, e development integrated management programs. 42

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