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Aflatoxins are a group of secondary metabolites produced mainly by Aspergillus flavus and also by Aspergillus parasiticus, that are acutely toxic to warm blooded animals including human beings.Recently, the use of Biopesticides has been suggested for the safe control of aflatoxin production as well as growth of Aspergillus flavus. In view of the known biopesticidal and anti-aflatoxigenic properties of plant extracts, an attempt has been made in the present study to explore the Antifungal Activity of Plant root extracts against growth of Aspergillus flavus by some wild plants of family Asteraceae. The cold and hot water extracts of root of five wild plants of family Asteraceae viz., Ageratum conyzoides, Carthamus oxycantha, Eclipta alba, Echinops echinatus and Vernonia cinerea have been studied against growth of toxigenic strain of Aspergillus flavus by standard cup method on solid medium. Out of three concentrations viz. 10, 15 and 20 per cent of root extracts, the 20 per cent concentration was found most effective. The maximum inhibition (52%) in growth of test fungus was caused by cold water root extract of Ageratum conyzoides as well as hot water root extract of Vernonia cinerea.

To explore the Antifungal Activity of Plant root extracts against growth of Aspergillus flavus by some wild plants of family Asteraceae.

Biopesticides are natural materials derived from animals, plants, and bacteria, as well as certain minerals that are used for pest control [3, 4]. Several plants are known for their medicinal and antifungal properties since ancient times. Their use in controlling many fungal and bacterial diseases has been reported by various workers [5, 6]. The use of natural products like cinnamon and clove oil has also been suggested for the inhibition of aflatoxin production as well as growth of Aspergillus species [7].The essential oil found in Ageratum conyzoides can inhibit the growth and production of toxigenic strain of Aspergillus parasiticus (Patil et al., 2010). The ability to inhibit the aflatoxin production is a new biological activity of A. Conyzoides [8, 9].

Considering this fact, in the present investigation, biopesticidal and antiaflatoxigenic effect of root extract of 5 wild plants of family Asteraceae on growth of toxigenic strain of Aspergillus flavus was studied by standard cup method.

Five wild plants of family Asteraceae viz. Ageratum conyzoides, Carthamus oxycantha, Eclipta alba, Echinops echinatus and Vernonia cinerea have been selected in the present study for their biopesticidal and anti-aflatoxigenic nature. Roots of above mentioned plants have been collected from fields in sterilized polythene bags and stored at 4°C in refrigerator till processed. 2.1 Preparation of extracts In each case, 100 g of fresh root sample of selected plants were washed twice with tap water and sterilized distilled water. Subsequently, cold and hot water extracts were prepared according to the method of Ezhilan et al. [10]. In this method for cold water extraction, the surface sterilized 100 gm root of plant were crushed in 500 ml distilled water (2/10, w/v) with mixie. Later, macerates were squeezed through double layered cheese cloth and then centrifuged at 5000 r.p.m. for 5 minutes. The supernatant was filtered through Whatman No.1 filter paper and then sterilized by passing through the Seitz filter (G-5). The extract (20%) thus obtained was used for the in vitro experiments. The clear supernatants were diluted with sterile distilled water to arrive at the required concentration (10%, 15%). The plant root extracts obtained in this way were stored at 4°C for further use. For hot water extraction, 100 gm of sterilized root sample of each plant was chopped and plunged in required quantity of water (2/10, w/v) taken in a beaker and heated over a water bath at 80°C for 10 minutes. The materials were then processed with music and strained through cheese cloth. It gave the standard plant extract solution (20%). The extracts were subjected to low speed centrifugation (5000 rpm. for 5 min.) and the clear supernatants were diluted with sterile distilled water to arrive at the concentration of 10% and 15%. The root extracts obtained in this way were stored at 4°C for further use. 2.2 Preparation of spore suspension Highly aflatoxigenic strain of Aspergillus flavus (ATCC-15517) was subcultured on potato dextrose agar medium from the stock culture. The slants were incubated for seven days at 28±1°C. The harvesting of the spores and preparation of spore suspension @10 spores/ml was carried out according to the method of Hesseltine et al. [11]. 2.3 Standard Cup Assay Method It was studied by the Standard Cup Assay Method. In this method, 1 ml spore suspension of toxigenic strain of Aspergillus flavus was transferred by separate sterile pipettes under aseptic conditions to seed the assay plates. The sterilized and luke warm PDA medium was poured into petri-plates at the rate of 20 ml/ plate, The plates were gently shaken to disperse the spore suspension uniformly in the medium. After allowing the medium to solidify, cups were prepared by using 4 mm cork borer. The various plant extracts 10%, 15%, 20%) were added to cups in triplicate and then incubated at 28±1°C for four days. After incubation period, the diameter of zone of inhibition was measured in mm and percent inhibition in growth was calculated as suggested by Misra and Dixit [12].

Statistical analysis of results was performed through one way ANOVA (Analysis of Variance) at value p<0.05 followed by Tukey’s Post Hoc test with p≤0.05 was used to determine the significant differences between the results obtained in each experiment.

The root extracts of these plants were tested on growth of toxigenic strain of Aspergillus flavus (ATCC 15517) by standard cup method on solid medium. Three concentrations viz. 10, 15 and 20 percent of root extracts in cold and hot water were used.

The perusal of Tables 1 and 2 indicates that root extracts of different plants inhibited growth of Aspergillus flavus to varying extent. The maximum inhibition in growth of Aspergillus flavus was 52 per cent and it was noted in two cases, due to 20 per cent concentration of cold water root extract of Ageratum conyzoides as well as 20 per cent hot water root extract of Vernonia cinerea. The hot water root extract of Ageratum conyzoides at 20 per cent concentration could reduce the growth of Aspergillus flavus to the extent of 50 per cent. The next effective plant was Vernonia cinerea, which inhibited the growth of test fungus to the extent of 52 per cent in case of hot water root extract, while cold water extract of this plant inhibited the growth up to 44 per cent.

Table 1. Effect of cold water root extract of plants on growth of Aspergillus flavus (Standard cup method)

Control growth – 90±0.00 mm in diameter

Note: The values represent the mean ± SEM of triplet experiments. Statistical analysis through one way ANOVA at value p<0.05 followed by Tukey’s Post Hoc test with p≤0.05 was used. The means with different alphabets are significantly different with each other as indicated by Tukey’s Post Hoc test (row by row analysis) at alpha = 0.05.

Table 2. Effect of hot water root extract of plants on growth of Aspergillus flavus (Standard cup method) 

Control growth – 90±0.00 mm in diameter

Note: The values represent the mean ± SEM of triplet experiments. Statistical analysis through one way ANOVA at value p<0.05 followed by Tukey’s Post Hoc test with p≤0.05 was used. The means with different alphabets are significantly different with each other as indicated by Tukey’s Post Hoc test (row by row analysis) at alpha = 0.05.

 This study revealed that three plants viz., Ageratum conyzoides, Eclipta alba, and Vernonia cinerea showed more than 35 per cent inhibition in growth of Aspergillus flavus. The remaining plants viz., Carthamus oxycantha, Echinops echinatus, showed 19% and 17.7% inhibition respectively in case of hot water root extract at 20% conc.

It is interesting to note that root extracts of all experimental plants showed inhibitory effect on growth of Aspergillus flavus in solid media. Several workers like Varshney et al. [13], Gautam et al. [14]. Reddy et al. [15] and Suurbaar et. al., [16] Rangarajulu et al. [17] Dhaliwal et al. [18] noted antifungal properties of extracts of many flowering plants.

In the present study, Ageratum conyzoides and Vernonia cinerea were found to be most effective. Kamboj and Saluja [19] reported that extract of Ageratum species was most effective for inhibition in growth of fungus. Likewise, Fiori et al [20] reported that crude extract of Ageratum conyzoides was more effective in inhibiting the mycelial growth of Didymella bryoniae, where as the essential oil of A. conyzoides provided 100 per cent inhibition in the mycelial growth and germination of spores of D. bryoniae. The essential oil of A. Conyzoides have biological activity which indicates as a useful tool for a better understanding of the complex pathway of aflatoxin biosynthesis in the case of Aspergillus flavus (Nogueira et al., 2010) [21] These facts support the findings of present investigation in relation to Aspergillus flavus.

Kurucheve et al [22] also reported that cold water leaf extract of Another plant of family Asteraceae viz., Parthenium hysterophorus inhibited the growth of Rhizoctonia solani up 45 per cent. Reddy et al. [15] reported 46 per cent control of leaf spot of Mulberry caused by Cercospora moricola by leaf extract of Parthenium hysterophorus. It is suggested that parthenin'a constituent of Parthenium hysterophorus is having fungitoxic and biopesticidal activity. Sharma [5] indicated that growth of toxigenic strain of Aspergillus flavus in terms of dry weight of mycelium was decreased by all the plant extracts tested and maximum inhibition (44.2%) was caused by extract of Allium sativum and the minimum inhibition (23.5%) was shown by extract of Euphorbia hirta.

Crude Extract from the Rhizome and Root of Smilacina japonica has strong fungicidal activity against different fungi such as Candida species and Cryptococcus neoformans [24, 25].

Root extracts of all experimental plants showed inhibitory effect on growth of Aspergillus flavus in solid media. In the present study, Ageratum conyzoides and Vernonia cinerea were found to be most effective. During COVID-19, difficulties in management of food and grain storage godowns during lockdown period as well as adversely affected economic life of humans may also increase the consumption of aflatoxin contaminated foods [26].

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