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African Crop Science Conference Proceedings, Vol. 7. pp. 591-595 Printed in Uganda. All rights reserved ISSN 1023-070X/2005 $ 4.00 © 2005, African Crop Science Society

Inhibitory effects of neem kernels on major mould species of harvested maize in Uganda

A. N. KAAYA, W. KYAMUHANGIRE & S. KYAMANYWA1 Department of Food Science and Technology, Makerere University, P.O. Box 7062, Kampala, Uganda 1 Department of Crop Science, Makerere University, P.O. Box 7062, Kampala, Uganda Abstract Mould and aflatoxin contamination of maize is a major problem in Uganda. This study was conducted to establish the inhibitory effects of neem kernels (Azadirachta indica) on mould species commonly isolated from harvested maize in Uganda. Moulds were grown on potato dextrose agar (PDA) with or without neem kernels. To determine the effect of neem kernel size, different sizes of neem fragments were used and the ratio of inhibition zone to fragment weight was calculated. Neem kernels significantly inhibited growth of the majority of moulds apart from Rhizopus spp. Values of the inhibition zones showed that P. expansum was the most inhibited followed by P. italicum and A. fumigatus, while A. flavus and A. niger were the least inhibited. A similar trend was observed when the inhibition zone ratio values were computed. These results indicate that if neem kernels are used by farmers or traders, they have the potential of improving maize quality during storage. Key words: Aflatoxin, inhibition, neem kernels, quality Résumé La contamination du mais par la moisissure et l'aflatoxine est un problème majeur en Ouganda. Cette étude était conduite pour établir l'effet inhibitoire des kernels de neem (Azadirachta indica) sur les espèces de moisissures communément isolées des mais récoltés en Ouganda. Les moisissures étaient plantées sur le dextrose agar des patates avec ou sans kernels de neem. Pour déterminer l'effet de la dimension du kernel, différentes dimensions des fragments de neem étaient utilisés et le taux des zones d'inhibition par poids du fragment était calculé. Les kernels de neem inhibant significativement la croissance de la majorité des moisissures a part le Rhizopus spp. Les valeurs des zones d'inhibition montra que P. expansum était le plus inhibé suivie par P. italicum et A. flavus et A. niger était les moins inhibé. Une tendance similaire était observée quand les valeurs des zones d'inhibition étaient calculées. Ces résultats indiquent que si les kernels de neem sont utilisés par les fermiers ou commerçants, ils ont le potentiel d'améliorer la qualité de mais pendant le stockage. Mots clés: Aflatoxine, inhibition, kernels de neem, qualité


In Uganda, maize is ranked as the most important cereal crop contributing approximately 40% of the per capita calorie intake in both rural and urban areas. However, mould and aflatoxin contamination of this crop has been reported to be a major problem in the country (Kaaya et al., 2002). Various techniques have been recommended to control and prevent mould and aflatoxin contamination of maize. Although preharvest prevention techniques should be considered as strategies to control moulds and mycotoxins (Munkvold and Desjardin, 1997), postharvest techniques involving use of plant products are also recommended (Bankole and Adebanjo, 2003). Several postharvest technologies intended to improve the quality and extend the shelf life of harvested produce have been developed in Uganda. At Kawanda Agricultural Research Institute (KARI), pest management techniques that can be used at subsistence level have been tested and recommended (Agona and Nadhy, 1998). For example, botanicals like tobacco, Tephrosia, Tagetes and neem (Azadirachta indica) have been found to be effective against storage insect pests of dry cereals and legumes. Neem kernel and leaf powders at 2% dosage rate have

been reported to control Sitophilus spp. and Acanthoscelides obtectus which are common insect pests of maize and beans respectively (Agona and Muyinza, 2000). However, the antifungal and aflatoxin inhibition properties of these botanicals have not been well established in Uganda. Many plant extracts have been shown to possess fungitoxic properties (El-Maraghy, 1995; Awuah, 1999; Yin and Tsao, 1999) and some have been proven to inhibit aflatoxin production. For example ElMaraghy (1995) reported that cloves, thyme, Chinese cassia, cinnamon and a mixture of the four spices inhibited aflatoxin production. Inhibition of aflatoxin production by carrot root extract was investigated by Batt et al. (1980) who found that the extract contained a compound that inhibited differentiation and aflatoxin production by Aspergillus parasiticus. However, Awuah and Kpodo (1996) reported that studies to assess the efficacy of plant extracts against aflatoxin synthesis have been cumbersome due to labour intensive techniques required for the extraction and quantification of aflatoxins. The objective of this study was to establish whether neem kernels can inhibit mould growth so that in addition to controlling insect pests, they can also be recommended to farmers for control of moulds and mycotoxins.


A. N. KAAYA et al.

Materials and methods

Neem kernels. Neem (Azadirachta indica) kernels (20 kg) were purchased from Uganda National Tree Seed Centre, Kampala, and another lot (10 kg) was purchased from Kawanda Agricultural Research Institute (KARI). All kernels were tested for viability at KARI laboratory using the method described by Agona and Nadhy (1998) and their average germination percentage was 98% implying that they were still viable and potent against insect pests. Studies on the inhibitory effects of neem kernels on fungal growth. Before using neem kernels (Azadirachta indica) to control aflatoxins in maize grain, it is important that the inhibitory effects of fungi, including Aspergillus flavus, by these kernels is studied in the laboratory. It has been reported that if growth of toxigenic fungi can be prevented, subsequent contamination with mycotoxins can also be prevented (El-Maraghy, 1995). Studies on the inhibitory effects of neem kernels on fungal growth were carried out in the Department of Food Science and Technology laboratory, Makerere University following the method described by Bagchi et al. (1999) with some modifications. Inoculum preparation. Maize kernels (Azadirachta indica) suspected to be naturally contaminated with fungi were assayed by direct plating technique (Pitt and Hocking, 1997) for internal mould infection. The kernels were surface sterilised for 1 minute with 10% Jik, a commercial bleach (Rickitt Benckiser, East Africa Ltd), washed three times with sterile distilled water and placed directly on malt extract agar media. Ten kernels were placed directly on each agar plate. The plates were incubated upright at 30oC for 42 ­ 72 hours. Nine mould species (Aspergillus flavus, A. niger, A. fumigatus, A. penicilliodes, A. wentii, Fusarium verticillioides, Penicillium italicum, P. expansum and Rhizopus stolonifer) prevalent in maize grain in Uganda (Kaaya et al., 2000; Ssebukyu, 2000) were isolated and identified. Inoculum was produced by growing each of these organisms at 28oC on Potato Dextrose Agar (PDA) plates into pure cultures until well sporulated. Using a sterile aluminum coke borer (5 mm diameter) and inoculating loop, the spores were harvested and suspensions of fungi prepared in 0.1% Tween 80 to give a spore concentration of approximately 106 spores/ ml. The spore concentration was estimated using a Brightline Hymatocytometer (Hausser Scientific, 0.100 mm deep, USA). Using a sterile pipette, 0.1 ml of inoculum was added to 3 ml of unsolidified PDA cooled to 50oC. Seeded agar plates were prepared in duplicates by pouring 20 ml of PDA into each plate. After solidification of medium, each plate was overlaid with the PDA containing inoculum of each fungal species. The inoculum was added aseptically, swirled and the contents (3.1 ml) were immediately poured onto PDA surface. The seeded plates were swirled to spread the inocula evenly. Fungal inhibitory test. The neem kernels were carefully shelled to avoid breakage and graded into three categories of small/shrivelled, medium and large size. The medium

size grade kernels, which were the majority, were used in the study and each kernel was split longitudinally into two fragments. Each fragment was weighed using a digital balance (Sartorius Basic, BA 210 S, Germany) and the weight (mg) recorded. The fragments were surface sterilized for 1 minute using sodium hypochlorite solution (10% commercial bleach-Jik, Rickitt Benckiser, East Africa Ltd), and washed three times with sterile distilled water, dried on sterile filter paper and placed upside down (cut surface on agar) on media plates previously seeded with the test fungi. Three to four fragments were placed on each agar plate. The test plates were incubated at 28ºC for 72 hrs to observe the antifungal activities. The distances (mm) between the outer boundaries of each of the three kernel fragments and fungal growth (fungal growth inhibition zone) was measured and mean distance used to quantify antifungal activity. For comparison of the kernel fragments of different sizes the ratio of inhibition zone to fragment weight was calculated and used to quantify the inhibitory activity in mm/ mg of fragment material. Properly dried and degermed maize kernels known to have no fungal inhibitory effects were used as control. Data analysis. Data were subjected to ANOVA using Genstat 5, Release 3.2, PC/Windows NT, Lawes Agricultural Trust, Rothamsted Experimental Station (1995) and the means were separated using LSD (P = 0.05).


Neem kernels significantly inhibited growth of the majority of moulds apart from Rhizopus spp (Table 1). Values of the inhibition zones show that P. expansum was the most inhibited mould followed by P. italicum and A. fumigatus, while A. flavus and A. niger were the least inhibited. A similar trend was observed in the results of the inhibition zone ratio and the mean weights of the different fragments used in the experiment did not differ significantly. Maize kernel used as a control had no inhibition effect on the growth of the moulds (Fig. 1).


Neem (Azadirachta indica) is used as an anti-malarial, anti-infirmmatory, anti-pyretic and anti-tumour herbal treatment (Ragasa et al., 1997). The plant is well known for its insecticidal and pesticidal properties (Bhartnagar and McCormick, 1988) and, neem seed kernel extract (5% in 1 litre of water with 2% soap or teepol as surfactant, sticker) is the most recommended pesticide by agricultural scientists, all over the world (Anon. 2000). In addition, neem oil and its isolates ­ nimbidin, nimbidiol and nimbin have been reported to inhibit fungal growth on humans and animals (Anon, 1997). In the current study, neem kernels were found to show excellent inhibitory effects to the growth of six out of nine mould species that are commonly isolated from stored maize in Ugandan. Different parts of neem tree have been tested for their antifungal activity during storage of grain. These include leaf extracts (Bhatnagar and McCormick, 1988; Sinha et al., 1999), kernels (Bagchi et al., 1999; Sinha

Inhibitory effects of neem kernels on major mould species


et al., 1999) and kernel extracts (Awuah and Kpodo, 1996; Srinivasan et al., 2001) among others. These authors reported promising results on the use of neem to inhibit fungal growth which is in agreement with the findings of the current study. According to Bagchi et al. (1999) seed fragments of A. indica were very effective against the plant pathogen Fusarium moniliforme (currently F. verticillioides) and had better antifungal activity than 10 ìg amphotericin and bavistin which are well-known antibiotics for fungi. Similarly, F. verticillioides was among the six fungal species whose growth was inhibited by the neem kernels in this study. In the current study however, neem kernels were found to have a minor inhibitory effect on A. flavus agreeing with the findings of Awuah and Kpodo (1996). Since A. flavus is the major producer of aflatoxin, failure by neem kernels to inhibit its growth may imply that they may not

adequately inhibit aflatoxin formation in maize grain. Sinha et al. (1999) and Bankole (1997) reported that extracts including essential oils from neem kernels inhibited aflatoxin B1 and B2 synthesis in SMKY liquid and in inoculated maize grain, respectively. This suggests that kernel extracts may exhibit greater A. flavus and aflatoxin inhibition than intact kernels or kernel fragments. The concern here is the practical applicability of the neem extracts by farmers compared to the kernels. The neem kernels were used in the current study because they have been recommended for adoption by farmers in Uganda as storage natural pesticides (Agona and Muyinza, 2000). Although neem kernels did not exhibit strong inhibition against growth of A. flavus, they could have an added advantage of reducing insect damaging effects if applied to stored maize thereby indirectly reducing aflatoxin contamination.

Table 1. Effect of neem kernels on growth of nine mould species isolated on PDA. Mould species Aspergillus flavus A. niger A. fumigatus A. penicillioides A. wentii Fusarium verticillioides Penicillium italicum P. expansum Rhizopus sp LSD (P d" 0.05) CV (%)

a b

Mean fragment wt (mg) 81.17 68.33 74.13 84.67 73.33 76.43 76.57 73.07 68.83 17.85 12.7

Mean inhibition zone (mm) a 0.846 0.755 4.967 3.233 3.717 3.133 5.567 7.050 0.000 0.5522 15.4

Inhibition zone ratio (mm/mg)b 0.0104 0.0111 0.0670 0.0382 0.0507 0.0410 0.0727 0.0965 0.000 0.00543 11.4

Width (radius in mm)of inhibition zone produced by a fragment. Inhibition zone computed from the ratio of width (mm)/fragment weight (mg).

Figure 1. Inhibition effects of neem kernels (Azadirachta indica) on Aspergillus niger, A. flavus and Penicillium. expansum.


A. N. KAAYA et al.

Moulds are known to affect the quality of grain by reducing the nutritional value and germination potential, imparting off-flavours and off-colours as well as producing mycotoxins (Sauer, 1988). The neem kernel fragments inhibited growth of six important moulds, therefore this may contribute to improved grain quality if they are used. In addition, the kernels greatly inhibited growth of F. verticillioides and P. expansum which are moulds known to produce highly potent mycotoxins. F. verticillioides produces fumonisins and moniliformin while P. expansum produces patulin (Richard, 2000). It should however, be noted that despite the vast literature on the efficacy of plant materials in controlling mycotoxigenic moulds, there has not been any concerted effort towards large-scale use of these plants in the field by farmers (Bankole and Adebanjo, 2003). Caution must be exercised in using plant materials to control mycotoxins because some of these materials are excellent substrates for fungal growth (Efutoye, 1999; Udoh et al., 2000).


Neem kernels effectively inhibited growth of A. fumigatus, A. penicillioides, A.wentii, F. verticillioides, P. italicum and P. expansum. The inhibitory effect of these six major mould species infecting maize in Uganda by the neem kernels may have the potential of improving maize quality through prevention of mycotoxin production and other related quality attributes. Since neem kernels are excellent pesticides, their use in stored grain may have an added advantage of controlling insect infestation which has been related to aflatoxin contamination. Neem kernels therefore should be used by farmers during storage of maize.


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