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In the recent years, study of mycotoxins has gained a great importance as most of them are known to pose health ill effects in humans and domestic animals. More than a dozen important mycotoxins with their derivatives have been reported to contaminate different types of food materials. Among these, aflatoxins are extremely important, owing to the highest degree of carcinogenic potency [1]. Aflatoxins are highly toxic mycotoxins that are secondary metabolites derived from polyketides produced by fungal species such as Aspergillus flavus, A. parasiticus, and A. nomius but the occurrence of later is quite less in Indian sub continent. Due to deleterious effects of aflatoxin, attention is now, therefore, directed towards achieving effective control measures against aflatoxin production on food commodities. They cause deleterious effects on the various body organs and body systems including the development of cancers especially the liver cancer mainly due to aflatoxin B1 exposure [2].

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].

1. Sarma, U.P., Bhetaria, P.J., Devi, P. and Varma, A. Aflatoxins: Implications on Health. Ind J ClinBiochem 2017;32(2):124–133. DOI 10.1007/s12291-017-0649-2. 2. Singh U., Gupta S., and Gupta M. “A REVIEW STUDY ON BIOLOGICAL ILL EFFECTS AND HEALTH HAZARDS OF AFLATOXINS”. Asian Journal of Advances in Medical Science 2021;3 (1), 1-8, https://mbimph.com/index.php/AJOAIMS/article/view/1834. 3. Gupta, S. and Dikshit A. K., Biopesticides: An eco-friendly approach for pest control, Journal of Biopesticides 3(1 Special Issue) 186 - 188 (2010). 4. Kandpal, V. Biopesticides. International Journal of Environmental Research and Development. Volume 4, Number 2 (2014), pp. 191-196. 5. Sharma, Asha 2002. Studies on effect of some medicinal plant extracts on aflatoxin production and growth of Aspergillus flavus. PhD. Thesis, Dr. B.R.A. Univ., Agra. 6. Singh, U. 2009. Studies on antiaflatoxigenic potential of some wild plants of Asteraceae. Ph. D. Thesis, Dr. B. R. Ambedkar University Agra. 7. Alpsoy, L. Inhibitory effect of essential oil on aflatoxin activities. African Journal of Biotechnology.2010; 9. 2474-2481. 8. Patil RP, Nimbalkar MS, Jadhav UU, Dawkar VV, Govindwar SP. Antiaflatoxigenic and antioxidant activity of an essential oil from Ageratum conyzoides L. J Sci Food Agric. 2010;90(4):608-614. doi:10.1002/jsfa.3857 9. Chahal R, Nanda A, Akkol EK, et al. Ageratum conyzoides L. and Its Secondary Metabolites in the Management of Different Fungal Pathogens. Molecules. 2021;26(10):2933. Published 2021 May 14. doi:10.3390/molecules26102933 10. Ezhilan, G.J., Chandrasekar, V. and Kurucheve, V. Effects of six selected plant products and oil cakes on the sclerotial production and germination of Rhizoctonia solani. Indian Phytopathology,1994; 47: 183 - 185. 11. Hesseltine, C.W. Shatwell, O.L; Ellis, J.J. and Stubblefield, R.D. Aflatoxin formation by A. flavus. Bact. Rev., 1966;30: 395-805. 12. Misra, S.B. and Dixit, SN. Fungicidal properties of Clematis gouriana. Indian Phytopath.,1977; 30 : 577-579. 13. Varshney, V. Effect of plant extracts on Drechslera graminea, the causal agent of stripe disease of barley. Ind. Phytopath., 2001;54(1): 88-90. 14. Gautam, A.K. and Bhadauria, R. Occurrence of Toxigenic moulds and mycotoxins in Ayurvedic medicine Triphala churn. J. Mycol. Pt. Pathol., 2008;38(3): 664-666. 15. Reddy, C.S.; Reddy, K.R.N.; Prameela, M.; Mangala, U.N. and Muralidharan, K. Identification of antifungal component in clove that inhibits Aspergillus spp. Colonizing rice grains. J. Mycol. Pt. Pathol., 2007;37(1): 87-94. 16. Suurbaar, J., Mosobil, R. & Donkor, A., Antibacterial and antifungal activities and phytochemical profile of leaf extract from different extractants of Ricinus communis against selected pathogens. BMC Res Notes 2017;10, 660. https://doi.org/10.1186/s13104-017-3001-2. 17. Rangarajulu, S.K., Gomathi V. and Kannabiran B. Fungitoxic effects of root extracts of certain plant species on Colletotrichum capsici causing anthracnose in Capsicum annuum. Indian Phytopath.2003; 56 (1) : 114-116. 18. Dhaliwal HS, Thind TS, Chander M, Chhabra BR. Activity of some essential oils against Uncinula nectar causing powdery mildew of grapevine. Indian Phytopathology. 2002; 55(4):529-531. 19. Kamboj, A. and Saluja, A.K. Ageratumconyzoides L.: A review on its phytochemical and pharmacological profile. International Journal of Green Pharmacy. 2008; 59 – 68. 20. Fiori A.C.G., Schwan, Estrada, K.R.F.; Stangarlin, J.R.; Vida, J.B.; Scapim, C.A.; Cruz, M.E.S. and Pascholati, S.F. Antifungal activity of leaf extracts and essential oils of some medicinal plants against Didymella bryoneal. J. Phytopathology. 2000;148: 483-487. 21. Nogueira JH, Gonçalez E, Galleti SR, Facanali R, Marques MO, Felício JD. Ageratum conyzoides essential oil as aflatoxin suppressor of Aspergillus flavus. Int J Food Microbiol. 2010;137(1):55-60. doi:10.1016/j.ijfoodmicro.2009.10.017. 22. Kurucheve V, Gerard EJ. and Jayaraj J. Screening of higher plants for fungitoxicity against Rhizoctonia solani in vitro. Indian Phytopythology 1997;50 (2): 235-241. 23. Gupta S. and Singh U. Study of antifungal activity of leaf extract of some wild plants of Asteraceae on Aspergillus flavus. Journal of Biopesticides. 2020; 13(2): 145-149. 24. Wei Liu , Baozheng Sun, Manman Yang, Ziyue Zhang, Xiuxiu Zhang, Tingsong Pang and Shengzheng Wang. Antifungal Activity of Crude Extract from the Rhizome and Root of Smilacina japonica A. Gray. Hindawi Evidence-Based Complementary and Alternative Medicine. Volume 2019, Article ID 5320203, 9 pages https://doi.org/10.1155/2019/5320203. 25. Y. Cui, X. Yang, D. Zhang et al., “Steroidal constituents from roots and rhizomes of smilacina japonica,” Molecules, vol. 23, no. 4, p. 798, 2018. 26. Gupta S., COVID-19, a pandemic viral pneumonia is a global disaster in 2020. Asian Journal of Advances in Research. 2020;3 (1), 16-20, https://mbimph.com/index.php/AJOAIR/article/view/1506.