Vernonia cinerea (L.) Less. Var. cinerea, an Antifungal Weed Species of Asteraceae Family.

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1 Page 128 Journal of Basic Sciences, 2015, Special Issue on BioIPPF, Vernonia cinerea (L.) Less. Var. cinerea, an Antifungal Weed Species of Asteraceae Family. Kadlag S.D a, *, Zaware B.N b and Londhe A.B a JOURNAL OF BASIC SCIENCES a* Department of Botany, New Arts, Commerce and Science College, Ahmednagar , (M.S.), India. b Dept. of Botany, Anantrao Pawar Arts, Comm. And Science College, Pirangut, Pune. Abstract: Allelochemicals are the natural secondary metabolites produced by the plants. Plants contain varieties of chemicals that are produced as secondary metabolites. They are natural and easily biodegraded. Botanical biocides are eco-friendly i.e. biodegradable, renewable and abundantly available, there is a vast scope for research in Allelopathy and investigating allelochemicals and use them as biocides in sustainable agriculture for food production without polluting environment. Keywords: Vernonia cinerea (L.)Less. var. cinerea, weed, antifungal. 1 Introduction Allelochemicals are the natural secondary metabolites produced by the plants. Molisch (1937), a plant physiologist and the father of Allelopathy introduced the word Allelopathy for beneficial as well as harmful (detrimental) reciprocal biochemical interactions among plants including microorganisms. Allelopathy, a current area of research, may be useful in agriculture to control fungal and bacterial diseases of crops. Plants contain varieties of chemicals that are produced as secondary metabolites. They are natural and easily biodegraded. Since botanical source of biocides is eco-friendly i.e. biodegradable, renewable and abundantly available, there is a vast scope for research in Allelopathy and investigating allelochemicals and use them in agriculture to sustain food production without polluting environment. The present paper will focus on the aspects of Allelopathic potentials of a common weed of Asteraceae family as fungicide. 2 Materials and Methods Study area: Ahmednagar district is the largest district of Maharashtra state. It is located between and North latitude and and East longitude. It occupies an area of sq.km. Sahyadri ranges are present on northeast part of the district. This is region where forest are situated are situated.the Sahyadri has formed a naturally boundaries in between Ahmednagar, Thane and Nasik District. Balaghat is the prominent ranges of Sahydri that traverse the district. Plant species was identified by using Flora of Maharashtra, Almeida(2001). Vernonia cinerea (L.)Less var. cinerea Classification (as per Bentham and Hooker s system): Journal of Basic Sciences September 2015

2 Page 129 Print ISSN: E-ISSN Botanical name: Vernonia cinerea (L.)Less var. cinerea Kingdom: Plantae Order: Asterales Family: Asteraceae (Compositae) Genus: Vernonia Species: cinerea (L.)Less Variety: cinerea Erect, tall, annual herbs; distributed in all districts of Maharashtra; pubescent striate stem with few branches; alternate leaves of variable shapes, ovate-lanceolate, narrowed at base, entire or shallowly crenate or serrate, acute, shortly mucronate, pubescent; Fls and Frts in July to March, Heads, in lax divaricated terminal corymbose cymes, peduncles with small bract beneath the head; Receptacles convex, naked or with few bristles; Involucral bract many seriate, green, the outer ones shorter, ovate, pubescent, the inner ones linearlanceolate, awned, silky on the back. All florets have pink-purple, tubular, 5-lobed, 1-2mm long corollas. Anthers erect with obtuse bases. Style arms subulate. Achenes are mm in length, obovate-oblong, and hairy. Pappus bristles in two series, the outer ones shorter than the inner. Common weed in gardens, agricultural fields and grasslands; in waste lands; on old walls, and barren areas. Aspergillus niger van Tiegh: It belongs to class Ascomytae, order Aspergillales and family Aspergillaceae. It is popularly known as black mould. It is a saprophytic fungus which is widely distributed(cosmopolitan) and occurs as one of the most frequent contaminant of stored food, agricultural produce (figs, dates, nuts),jams and jellies, rice, bread and other food stuffs, cellulose materials. It also grows as animal and bird parasite. EXTRACT BIOASSAY: Bioassay experiments at room temp (25to28 0 c) were performed in the recognized Research laboratory of Botany Department, New Art s, Commerce and Science College, Ahmednagar (Maharashtra state) during the year Plants were collected from waste places around the college campus. Stock solutions of the fresh leaf samples were prepared. Extract was obtained by crushing leaves in a mortar and pestle using distilled water in a ratio of 1:10 w/v, 1: w/v and 1:30 w/v (leaves: distilled water) to obtain stock solutions of 10%, 20%, 30% concentrations (Narwal, S.S. & Tauro. P.(1994). Preparation of PDA medium : 200 g peeled pieces of potato tubers (Solanum tuberosum )were boiled in distilled water and filtered through muslin cloth in 100 ml conical flask.15 g dextrose was added in it and then 15 g agar was added slowly while stirring it. Final volume 1000ml was made. This PDA medium was then autoclaved and used for culturing fungi in sterile petridishes. Aspergillus niger procured from the Departmental laboratory was inoculated in agar medium under sterile conditions. Petridishes of 11cm diameter containing freshly prepared PDA medium were used and allowed the fungus to grow. Many such plates were prepared. With the help of cork borer four wells per petriplate were prepared. Leaf extracts of 10, 20 and 30 % concentration were added separately. In control plates sterile distilled water was added in the wells. Six replicates of each treatment were maintained. Extracts inhibited the fungus around the wells. Inhibition zones around wells were measured randomly by taking at least 24 readings per treatment. Results obtained (means) were analyzed by single factor ANOVA multiple range test using Microsoft Excel programme. Calculation of CD (critical difference): From summary table of single factor ANOVA, critical difference in between pairs of means is calculated by using following formula: estimated variance within groups/n CD= q* ( Where q i.e is the value from the studentized range (Table NO 7 from statistics book), n= df within groups). Tukeys HSD (Honestly significant Difference) test: The formula is: qs = (Y A - Y B )/SE (where Y A is the larger of the two means being compared and Y B is the smaller, of Kadlag et al. / Journal of Basic Sciences, 2015, Special Issue on BioIPPF,

3 Page 130 the two means being compared, SE= standard error of the data in question). Photographs were taken by using digital camera. 3 Results and Discussion Effect of aqueous and methanolic extracts of Vernonia cinerea (L.)Less var. cinerea on mycelial growth of Aspergillus niger van Tiegh: [Refer table No1,Graphs 1(A) and 1(B)] In the present work it was found out that the fresh leaf extracts of Vernonia cinerea (L.)Less var. cinerea of various concentrations inhibited mycelia growth of the fungus Aspergillus niger Van Tiegh. around the wells prepared in the agar plates. The inhibition went on increasing with increase in leaf extract concentrations. The inhibitory zones were measured. About 24 readings were taken randomly and entered in MS Excel worksheet. Statistical analysis (refer Plate IV) revealed following things: 1. P-value is less than 0.05% alpha value (taken during single factor ANOVA analysis). This indicated that all means (of extract conc. Treatment) are significantly different from each other. 2. CD values at 0.05% level are and CD at 0.05% is < diff.in means indicating all means are significantly different. 3. F value is more than F critical value (refer plate No IV) indicating all means are significantly different from each other. 4. Tukeys HSD test (q value) was performed to find out any similarity in the means obtained. q values calculated were more than q critical value (3.68 from table No 7, (where df: 96,treatments:4 ).It also indicated that all means were statistically significantly different from each other except mean values obtained at 20 and 30% methanolic extracts (denoted by alphabet c). Aqueous and methanolic extracts of Vernonia cinerea (L.)Less var. cinerea more or less equally inhibited mycelial growth of Aspergillus niger Van Tiegh. Vernonia cinerea (L.) Less var. Cinerea Leaf Extracts (A)Effect of aqueous leaf extract (B) Effect of methanolic leaf extract of Vernonia Journal of Basic Sciences September 2015

4 Page 131 Print ISSN: E-ISSN Table No 1: Effect of aqueous and methanolic leaf extract of Vernonia cinerea (L.)Less var. cinerea on growth of Aspergillus niger van Tiegh Sr No Extract 1 Aqueous 2 Methanolic Conc control (DW) 10% 20% 30% control (DW) 10% 20% 30% Inhibition zone (in mm) 0.00a ± b ± c ± d ± a ± b ± c ± c ±0.015 P-value 2.333E- 09 CD at 0.05% F value F critical value E Data presented are means of six replicates; values within the same column with different letters a(a,b,c,d)are significantly different at 0.05% P-level by Single factor ANOVA test followed by CD & Tukeys test., ±Standard error of means. In above table, Q values are more than q critical Value Hence means are signicantly different. Graph No 1: Effect of aqueous and methanolic leaf extract of Vernonia cinerea (L.)Less var. cinerea on growth of Aspergillus niger van Tiegh Discussion Polyacetylenes are largely found on the plants of Asteraceae family, for example, a-terthienyl in Tagetes erecta L., phenylheptatrine in Bidens pilosa auct.non. L., safynol and dehydrosafynol in Carthamus tinctdrius L., etc are antimicrobial (Rice, 1984). Arora and Kaushik (2003) recorded that the extracts of Conyza bonariensis (L.) Crong and Erigeron karvinskianus DC. (Family Asteraceae); Ghaneri (Lantana camara L.) and Nirgudi (Vitex negundo L.) (Family Verbenaceae); Dipmal (Leonotis nepetafolia (L.) W.Ait.) and Mentha longifolia (L.) Hudson. (Family Lamiaceae) were highly fungicidal against soybean fungal pathogens viz., Colletotrichum truncatum (Schwein) Andrus & Moore, Fusarium oxysporum Schl. ex Fr. and Macrophomina phaseolina (Tassi) Kadlag et al. / Journal of Basic Sciences, 2015, Special Issue on BioIPPF,

5 Page 132 Goid.(Mahapatra and Tewari, 1994, Qasem, 2004). Wang Ruhua, Baoli Zhou, Fengli Zhang and Qifa Zhang (2005).Allelopathic effects of root extracts of egg plants on Verticillium wilt (Verticillium dahliae) Allelopathy J Podolska G.,Bialy Z.,Jurzysta M. and G.R.Waller (2003)Effect of application of alfalfa root saponins aqueous solution on plant structure, yield and quality of winter wheat AllelopathyJournal11(2): Narwal, S.S. & Tauro. P.(1994)Allelopathy in Agriculture and forestry Scientific Publisher, Jodhpur (India). Chuihua et al. (2004) found out that Ageratum conyzoides L., a member of Asteraceae family contains allelopathins like 3-caryophyllene, p- bisabolene and p-farnescene that could exert synergistic inhibitory effect on test plants. It is herbicidal as well as fungicidal. 4 Conclusions Vernonia cinerea (L.) Less var. cinerea of Asteraceae family is a common weed. In the present work it was found out that the fresh leaf extracts of Vernonia of various concentrations inhibited significantly mycelial growth of a common saprophytic fungus Aspergillus niger Van Tiegh. The inhibition went on increasing with increase in leaf extract concentrations. It proved to have a fungicidal potential. It can be further used to find out its fungicidal potential against fungi that cause crop plant diseases. There are abundant species of Asteraceae family that could be tried to find out effect on not only saprophytic but phytopathogenic fungi that cause fungal diseases on agricultural crop plants. Instead of using nonbiodegradable synthetic agrochemicals to control fungal diseases of crops that also cause severe pollution why not to use plant originated biodegradable fungicides to control diseases of crop plants? Efforts are to be done to work out fungicidal properties of plant species of Asteraceae and other families of flowering plants. There is a wide scope for this type of work. Allelochemicals could be isolated and used in agriculture. Calculation of CD (critical difference): From summary table of single factor ANOVA, critical difference in between pairs of means is calculated by using following formula: estimated variance within groups/n CD= q* Where q i.e is the value from the studentized range (Table NO 7 from statistics book), n= df within groups). Tukeys HSD (Honestly significant Difference) test: The formula is: qs = (Y A - Y B )/SE (where Y A is the larger of the two means being compared and Y B is the smaller, of the two means being compared, SE= standard error of the data in question). Photographs were taken by using digital camera. 5 References 1 P Wang, Allelopathy Journal (2004), 13, 2, Almeida, M.R., Flora of Maharashtra, volume 3 (A) Orient Press, Mumbai (India) (2001). 3 Arora Charu, Kaushik R.D., Kumar K., Garg G.K., Allelopathy Journal, (2003), 11, 1, Chuihua Kong, Fei Hu, Wenju Liang,Wang Peng and Yong Jiang, 5 Mahapatra T. K. and S. N. Tewari, Allelopathy Journal. (1994), 1, 2, Molisch, Hans, The Influence of one plant on another". Allelopathy. Edited by Narwal S.S.,Translated by L.J.La Fleur & M.A. Bari Malik. (2001) Scientific Publisher, Jodhpur (India) (1937). 7 Narwal, S.S. & Tauro. P. Allelopathy in Agriculture and forestry Scientific Publisher, Jodhpur (India) (1994). Journal of Basic Sciences September 2015

6 Page 133 Print ISSN: E-ISSN Podolska G., Bialy Z., Jurzysta M. and G.R.Waller, Allelopathy Journal, (2003), 11, 2, Qasem J.R., Allelopathy Journal, (2004), 13, 2, Rice E.L. AllelopathyJournal (1984), 1, 2, Wang Ruhua, Baoli Zhou, Fengli Zhang and Qifa Zhang, Allelopathy Journal, (2005), 15, 1, Kadlag et al. / Journal of Basic Sciences, 2015, Special Issue on BioIPPF,