Introduction

India is a country of vast population and about 70% of people are engaged in agriculture. Rapid population growth in the developing countries leads to an ever-increasing demand for food. Because of rising demand, production has steadily increased over the past years. In the production of food crops losses occur during the growth cycle in the field. Further losses occur during harvest, where up to 5% of grain weight can reduce the agricultural output¹. Further losses occur during storage, where found average damages of 30% in stored maize after six months storage in Togo². Storage pests are the main cause of these losses, and under the tropical climatic conditions the development of storage fungi, especially species of the genera Aspergillus spp. and it produced toxin, known as aflatoxin.

Aspergillus flavus is a soil-inhabiting, filamentous fungus that saprophytically utilizes a wide range of organic substrates. Though A. flavus is considered a saprophyte, it is also an opportunistic pathogen³ that can invade agronomically important oil seed crops such as corn, peanut and cottonseed that are under biotic or abiotic stress. Among the toxic secondary metabolites produced by this organism are the aflatoxins (AFs) with aflatoxin B1 being the most potent natural carcinogen known⁴. The foods at highest risk of aflatoxin contamination are corn, peanut and cotton seed⁵. Aflatoxin B1 has been detected in 80% of maize samples obtained from different locations in Southeast Nigeria⁶ and similarly 92 % of animal feed samples taken from commercial sources in Thailand were contaminated with aflatoxin B1⁷. In at least three parts of the world, East Africa, the Philippines and Thailand, good epidemiological evidence has been collected showing a correlation between the incidence of liver cancer and exposure to aflatoxins⁵.

Aflatoxins have also been identified as a potential biological weapon for food and water contamination⁸. Physical, chemical and biological methods have been investigated in order to prevent the growth of aflatoxin producing fungi and to eliminate or reduce the levels of aflatoxins or to degrade or detoxify aflatoxins in foods and feeds⁹. Control by naturally produced agents is becoming increasingly important because of consumers’ mistrust of food and feed treatments that involve using synthetic xenobiotic substances. Natural plant compounds have been used traditionally to preserve foods in countries like Japan, India and Russia¹⁰.  Extracts and powders of various spices, herbs and essential oils have been reported to have antimicrobial activity against aflatoxin producing fungi and some of them also inhibit aflatoxin formation¹¹.  Aim of the present study was to evaluate the inhibition of Aspergillus flavus growth and aflatoxin production with using Cassia alata and has a potential for use as a aflatoxin inhibitor.

Materials and Methods

Sample collection

The healthy and contaminated maize (Zea mays L.) seed samples were collected from storage facilities at Thanjavur District, Tamilnadu, India. Samples were placed directly in polyethylene bags and transferred immediately to the laboratory where they were stored in a cool place for further investigation. The contaminated maize seed sample was used as isolation of aflatoxin producing fungi.

Isolation and identification of aflatoxin producing fungi

The serial dilution procedures were followed. The potato dextrose agar was prepared and it was poured onto the sterile Petri plates. After solidification, the selected dilution factors 10^-2 to 10^-4 were spread on the medium respectively. Then the plates were incubated. The isolated fungal strain was identified based on their cultural and morphological characteristics.

Collection of plant and preparation of powder

The plant of Cassia alata (leaves) was collected from wild area of Thanjavur District, Tamilnadu, India. The leaves of Cassia alata was air dried and crushed to small piece using Mortar and Pestle and powdered in an electric grinder.

Experimental Design

T₁         –           Control (Healthy maize)

T₂           –           Healthy maize + Aspergillus flavus

T₃         –           Healthy maize + Aspergillus flavus + Plant powder

T_{4           –}               Healthy maize + Plant powder

The healthy samples were inoculated artificially with A. flavus as per the method ¹². The treated maize seeds were stored at room temperature in airtight polyethylene bags till analysis¹³. This stored maize was taken on 3^rd, 6^th, 9^th and 12^th (3 month interval) month after treatment for aflatoxin analysis and growth of Aspergillus flavus.

Fungal spore

Sample, 1 g from each flask was serially diluted 10-fold with sterile distilled water and 0.1 ml of each dilution was spread uniformly on potato dextrose agar plates and incubated in the dark for 3 days. On the 3rd day, colonies were counted and expressed as log colony forming units per gram (log CFU/g).

Estimation of Aflatoxins by High Performance Thin Layer Chromatography (HPTLC)

Aflatoxin determination was based on the Association of Official and Analytical Chemists (AOAC) BF method (AOAC, 2001) with some modifications. The experimental design treated maize samples (25g) were blended with 200ml Acetone: water (85 + 15, V/V), 2g NaCl and 5g Celite powder and mixed. Mixture was allowed for half an hour and then filtered. 25ml of hexane was added 2 times and the lower layer was separated and put into a free test tube. Lead acetate 20% in 0.3% acetic acid was added (Added 1 ml/10ml of extract) and allowed for setting for 15 min and then filtered.

Filtrate was collected in a separating funnel. 50 ml of chloroform was added and shaken for 2 min. Lower layer was removed in anhydrous sodium sulphate. Extract was evaporated in water bath and the final layer of solution was transferred to the tube containing chloroform of about 2 ml. Samples were injected into HPTLC and florescence intensity  was calculate under long UV lamp at 365nm wavelength.

Calculation

Concentration of Aflatoxin in µg/kg =

 S × Y × V

X×W

Where

S =  µl aflatoxin standard which matches the unknown

Y= Concentration of Aflatoxin standard µg/ml.

Results and Discussion

In this present study fungal species was isolated from the contaminated maize (Zea mays L.) seed samples. Number of fungal colonies was isolated from the soil samples. Among these isolated colonies of A. flavus was identified based on the cultural, morphological and biochemical characteristics. The results were presented in the Fig -1. The A. flavus as a major fungal contaminant in maize seeds and humans are exposed to its spores every day without any clinical outcome. But, in some cases when persons are exposed to intense spore dust, hypersensitivity reactions and lung disease (primary Aspergillus pneumonia, aspergilloma, allergic bronchopulmonary aspergillosis, and invasive Aspergillus) may be observed¹⁴. Many researchers have reported A. flavus as a dominant mycoflora from different substrates¹⁵.

The dried Cassia alata leaves powder was used for inhibition of aflatoxin production and growth of A. flavus. The effect of Cassia alata leaves powder on the growth of A. flavus and production is presented in Table – 1. An inhibitory effect on the A. flavus growth production was observed at all concentrations of the leaves powder sample used in the study and was found to be dose dependent. The pattern of inhibition shows that at lower concentration of 1% inhibition of A. flavus is relatively greater than inhibition of growth of the fungus. 10% concentration the Cassia alata leaves powder delayed the growth of A. flavus and complete inhibition was observed upto 120 days.

Table 1: Effect of certain Cassia alata leaves powder to prevent Aspergillus flavus growth.

Mean ± Standard Deviation

Efficacy of Cassia alata leaves powder to prevent aflatoxin production in maize was estimated to all treatments. The investigated results were presented in Table 2. The highest percentage reduction in aflatoxin production was observed in T₃ compare than other treatments. The percentage reduction in aflatoxin production (%) every three months increased in T₃. Similarly T₂ the spore count (log CFU/g) and (ppb) Aflatoxin were equally increased. The Fig – 1 illustrates T₃ and T₂ treatments were increased on opposite direction. The fungicidal activity of some plant extracts in controlling different plant pathogens have been reported by several workers^{16,17, 18}. Therefore, the present study is an important step toward development of plant based pesticides, which are eco-friendly for the management of storage fungi, today and in near future.

Table 2: Efficacy of Cassia alata leaves powder to prevent aflatoxin production in maize.

Mean ± Standard Deviation; ND – not detected

Acknowledgements

We kindly thank to Director, Muthaiyah Research Foundations for their valuable and enthusiastic encouragement at every state of this work.

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