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Int.J.Curr.Microbiol.App.Sci (2020) 9(7): 346-354 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 9 Number 7 (2020) Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2020.907.037 In-Vitro Efficacy of Forms and Methods of Adhatoda vasica against Flacherie causing Bacteria of Silkworm, Bombyx mori L. K. V. Anitharani*, R. N. Bhaskar and H. G. Anusha Department of Sericulture, UAS GKVK, Bengaluru-560065 (Karnataka), India *Corresponding author ABSTRACT Keywords Adhatodavasica, bacterial flacherie, inhibition zone. Article Info Accepted: 05 June 2020 Available Online: 10 July 2020 The flacherie diseased silkworms may contain multiple species of bacteria viz., Streptococcus faecalis, Staphylococcus aureus, Serratia marcescens and Bacillus sp. The use of botanical formulations possessing antibacterial activity are preferred in silkworm rearing over the chemical agents which found to be effective on checking bacterial infection and enhance the growth of silkworm. Different forms and methods of application of Adhatoda vasica was found effective in the management of bacterial flacherie of silkworm. In the present study, three bacterial species (Bacillus sp., Staphylococcus sp. and Streptococcus sp.) were isolated from haemolymph of silkworm and used under invitro condition. The study revealed that, the aqueous, acetone and alcoholic extract of A.vasica at different concentrations (2, 4 and 6 %) were highly effective against three bacterial species. However, the maximum zone of inhibition was observed in alcoholic extract (8.00, 9.12 and 8.99, 9.62 mm)(7.42, 7.88 and 7.84, 8.63 mm) (8.25, 8.87 and 8.84, 9.21 mm) and minimum in aqueous extract(7.66, 8.46 and 8.25, 8.94 mm) (6.96 and 7.29., 7.35 and 7.79 mm) (6.46, 6.92 and 8.09, 8.46 mm) on 24 and 48 hours of incubationagainst10-5 and 10-7 spore dilution of Bacillus, Staphylococcus and Streptococcus species, respectively. microbial infections as reported in ancient ayurvedic compendium “Charaka Samhita” and Sushrita Samhita” (Chopra, 1982; Chatterjee and Pakrashi, 1991). Action of plant on microorganisms pathogenic to other animals and insects has been reported (Ray and Majumdar, 1974). Introduction Mulberry silkworm is being reared indoor, since they are highly susceptible to disease causing pathogens which lead to drastic reduction in cocoon production. Flacherieis a condition wherein the silkworms are infected by several species of bacteria viz., Streptococcus faecalis, Staphylococcus aureus, Serratia sp. and Bacillus sp. (Sugun, 2000). Recently, many attempts have been made on the use of plant extracts to combat This has commercially been exploited and utilized for management of pest and diseases of silkworm. Hence, an experiment was drawn in this line as to assess the efficacy of 346 Int.J.Curr.Microbiol.App.Sci (2020) 9(7): 346-354 application of Adhatoda vasica against bacterial pathogens causing flacherie in the mulberry silkworm, Bombyx mori L. The botanical extracted in aqueous, acetone and alcohol media were studied in-vitro. A.vasica were collected from „Sanjeevinivatika‟ (Herbal Garden) Division of Horticulture, University of Agricultural Sciences, Bengaluru. The required quantity of fresh leaves of A.vasica were harvested and surface sterilized with 70 % ethyl alcohol then washed with sterile distilled water and shade dried. The shade dried leaves were then powdered in electric blender at slow speed, sieved and kept stored in desiccators. Materials and Methods Silkworms affected by flacherie disease were spotted, 1 ml of haemolymph was collected and mixed with 9 ml distilled water to make the stock suspension from which serial dilutions(10-5 and 10-7) were prepared using 9 ml sterile water blanks (Siromani et al., 1994; Nataraju et al., 1999). Aqueous plant extract The required quantity of fresh leaves were harvested and surface sterilized with 70 % ethyl alcohol then washed with sterile distilled water. Later, they were taken in pestle and mortar separately and 10ml of sterile distilled water was added to 1 g of leaf for maceration. The extract was squeezed through double layered muslin cloth and used as stock solution. Further, the same was diluted by using sterile distilled water to achieve different concentrations (Karthikairaj et al., 2013). From the serial dilution prepared, 0.5 ml each was transferred to separate petridishes containing nutrient agar medium. The culture plates were incubated at 37°C and after 48 hours, the incubated bacterial culture was observed under microscope to know the type and nature of colony formed. Based on the shape, texture and colour, the colonies were selected and purified on nutrient agar plate. The purified bacterial isolates were identified as Bacillus sp., Staphylococcus sp.and Streptococcus sp. The pathogenicity of the individual bacterium was confirmed by the principle of Koch‟s postulates. These organisms were used for in-vitro studies (Figure- 1). Acetone herbal extract Preparation of herbal extracts Ten grams of herbal powder of A.vesica was kept in a conical flask soaked with acetone for 6 hours under air tight condition. The content was then stirred for an hour in magnetic stirrer and filtered through a filter paper. The residual extract was collected in a flask and the solvent is allowed to evaporate at room temperature. The extract was then stored at 4 ˚C until use. The resultant residue was made up to required volume using double distilled water and used for the study (Karthikairaj et al., 2013). Herbal powder Alcoholic herbal extract The extract of A.vasica (Adusoge) was prepared as per procedure adopted by Krishnaprasad et al., (1979). The leaves of Ten grams of botanical powder was packed separately in a burette column keeping glass wool at the bottom and filter paper rings on The botanical screened against Bacillus sp., Streptococcus sp. and Staphylococcus sp., bacteria isolated from silkworm haemolymph was leaf extract of A.vasica (common name: Adusoge, Family: Acanthaceae). 347 Int.J.Curr.Microbiol.App.Sci (2020) 9(7): 346-354 10-5and 10-7.Further, after 48 hours of incubation, Bacillus sp. of both the dilutions (10-5 and 10-7) exhibited the same trend of zone of inhibition viz., maximum in T3 (8.99 and 9.62 mm) and minimum of 7.66 and 8.46 mm., 8.25 and 8.94 mm (10-5 and 10-7)inT1 on 24 and 48 hours respectively (Table 1 and Figure 2). These results follow the same trend as that of (Karthikairaj et al., 2013) who reported maximum inhibition zone of (9.33, 11.27 and 12.13 mm) microbes involved in flacherie disease of silkworm for alcoholic plant extracts whereas comparatively less for their aqueous counterparts (8.83, 9.77 and 10.73 mm) at different concentrations of 50, 100 and 150 µl of Andrographis paniculata followed by Momardica charentia (7.83, 8.33, 9.43 and 6.77, 8.13 and 8.93 mm) and Ocimum sanctum (7.33, 7.67, 8.83 and 7.17, 7.33 and 8.30 mm). top. Alcoholic extract was collected from the leaf powder by pouring double distilled water and ethyl alcohol on top of the burette column. The collected extract was then concentrated in hot water bath and made up to 10 ml with distilled water and stored in the deep freezer. Required quantities of this extract was taken and diluted with distilled water to get the required concentrations (Karthikairaj et al., 2013). Placement of paper discs Sterilized whatman No.1 filter paper discs of 5mm diameter dipped in botanical extracts for 1 minute and drained by the edges of petriplate were placed at the centre of the petriplate. Four replications were maintained for each treatment, further control (distilled water+bacteria and distilled water) was used for comparison. The bacteria inoculated plates were incubated for 48 hours at room temperature. The diameter of the inhibition zone by different forms of botanicals against each bacterium was measured (mm) and recorded on 24 and 48 hours of incubation. Zone of inhibition (mm) observed in different concentrations (2 %, 4 % and 6 %) of Adhatoda vasica against Staphylococcus sp. The data pertaining to zone of inhibition at different concentrations of A.vasica against Staphylococcus sp., was found significant among forms, methods and concentration of botanical. However, as the concentration of botanicals increased (2, 4 and 6 %) at 24 hours of incubation in artificial media, it revealed increased zone of inhibition for inoculation dose of 10-7(5.35, 5.66 and 6.07 mm). The trend was same even at 10-5dilution (4.97, 5.38 and 5.72 mm) recorded for 2, 4 and 6 per cent botanical formulation and found significant. The forms of botanicals used after 24 and 48 hours for 10-5 and 10-7 inoculation doses revealed significant results, however maximum of 7.42 and 7.88., 7.84 and 8.63 mm zone of inhibition was recorded for T3 which was found comparatively more effective in inhibiting the growth of Staphylococcus. T1 – Aqueous extract T2 – Acetone extract T3 – Alcoholic extract T4 – Water Results and Discussion Zone of inhibition (mm) observed in different concentrations (2 %, 4 % and 6%) of Adhatoda vasica against Bacillus sp The aqueous, acetone and alcoholic extracts of A.vasica at different concentrations (2, 4 and 6 %) revealed significant results on zone of inhibition of Bacillus sp. with the dilutions of 10-5 and 10-7. The maximum zone of inhibition was recorded against T3 =Alcohol +Spraying (8.00 mm and 9.12 mm) after 24 hours of incubation with bacterial dilution of 348 Int.J.Curr.Microbiol.App.Sci (2020) 9(7): 346-354 Table.1 Zone of inhibition (mm) observed in different concentrations (2 %, 4 % and 6%) of Adhatoda vasica against Bacillus Dilution 24 hours -5 Conc. of botanical Forms of botanical T1 2% 10 4 % 6 % Mean 48 hours -7 2% 10 4 % 6 % Mean -5 -7 10 2% 4% 6% Mean 2% 10 4% 6% Mean 6.87 7.37 8.75 7.66 7.50 8.37 9.50 8.46 7.37 8.12 9.25 (2.71) (2.80) (3.04) (2.85) (2.82) (2.97) (3.16) (2.99) (2.80) (2.93) (3.12) 8.25 8.00 8.87 9.95 8.94 (2.95) (2.91) (3.06) (3.23) (3.07) T2 7.75 7.87 7.87 7.83 7.75 8.62 9.12 8.50 8.25 8.67 10.00 (2.87) (2.89) (2.89) (2.88) (2.87) (3.02) (3.10) (3.00) (2.95) (3.02) (3.24) 8.97 8.50 9.50 10.50 9.50 (3.07) (3.00) (3.16) (3.31) (3.16) T3 7.12 8.00 8.87 8.00 8.75 9.12 9.50 9.12 7.62 9.00 10.35 (2.76) (2.91) (3.06) (2.91) (3.04) (3.10) (3.16) (3.10) (2.84) (3.08) (3.29) 8.99 8.75 9.50 10.62 9.62 (3.08) (3.04) (3.16) (3.33) (3.18) T4 Mean F test S. Em ± CD 5% 0.00 (0.70) 0.00 (0.70) 0.00 (0.70) 0.00 (0.70) 0.00 (0.70) 0.00 (0.70) 5.44 5.81 6.37 5.87 6.00 6.53 7.03 6.52 5.81 6.45 7.40 (2.81) (2.43) (2.51) (2.62) (2.52) (2.54) (2.65) (2.74) (2.64) (2.51) (2.63) 0.00 (0.70) 0.00 (0.70) 6.31 6.97 7.77 7.02 6.55 (2.65) (2.61) (2.73) (2.87) (2.74) Dilutions Forms Concentrations Dilutions Forms Concentrations * 0.131 (0.022) 0.362 (0.060) * 0.278 (0.046) 0.768 (0.126) * 0.131 (0.022) 0.362 (0.060) * 0.091 (0.015) 0.251 (0.041) * 0.193 (0.031) 0.533 (0.086) * 0.091 (0.015) 0.251 (0.041) T1 : Aqueous form + Spraying, T2 : Acetone form + Spraying, T3 : Alcohol form + Spraying, T4 : Water; 349 * - Significant Int.J.Curr.Microbiol.App.Sci (2020) 9(7): 346-354 Table.2 Zone of inhibition (mm) observed in different concentrations (2 %, 4 % and 6%) of Adhatoda vasica against Staphylococcus sp. Dilution 24 hours -5 Conc. of botanical Forms of botanical T1 2% 10 4% 6% 48 hours -7 Mean 2% 10 4% 6% -5 Mean 2% -7 10 4% 6% Mean 2% 10 4% 6% Mean 6.62 6.88 7.38 6.96 6.88 7.25 7.75 7.29 6.88 7.38 7.8 7.35 7.25 7.63 8.5 7.79 (2.66) (2.71) (2.80) (2.73) (2.71) (2.78) (2.87) (2.79) (2.71) (2.80) (2.88) (2.80) (2.78) (2.85) (3.00) (2.87) T2 6.50 7.13 7.50 7.04 7.13 7.50 8.13 7.59 7.13 7.50 8.25 7.63 7.53 8.50 8.75 8.26 (2.64) (2.76) (2.82) (2.74) (2.76) (2.82) (2.93) (2.84) (2.76) (2.82) (2.95) (2.85) (2.83) (3.00) (3.04) (2.96) T3 6.75 7.50 8.00 7.42 7.38 7.88 8.38 7.88 7.13 7.63 8.75 7.84 8.13 8.13 9.62 8.63 (2.69) (2.82) (2.91) (2.81) (2.80) (2.89) (2.97) (2.89) (2.76) (2.85) (3.04) (2.88) (2.93) (2.93) (3.18) (3.02) T4 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 (0.70) (0.70) (0.70) (0.70) (0.70) (0.70) (0.70) (0.70) 4.97 5.38 5.72 5.36 5.35 5.66 6.07 5.69 5.29 5.63 6.20 5.70 5.73 6.06 6.72 6.17 (2.33) (2.42) (2.49) (2.41) (2.41) (2.48) (2.56) (2.48) (2.40) (2.47) (2.58) (2.49) (2.49) (2.56) (2.68) (2.58) Mean F test S. Em ± CD 5% Dilutions * 0.062 (0.011) Forms * 0.132 (0.024) Concentrations * 0.062 (0.011) Dilutions * 0.043 (0.007) Forms * 0.092 (0.016) Concentrations * 0.043 (0.007) 0.172 (0.031) 0.365 (0.066) 0.172 (0.031) 0.120 (0.020) 0.254 (0.043) 0.120 (0.020) T1 : Aqueous form + Spraying, T2 : Acetone form + Spraying, T3 : Alcohol form + Spraying, T4 : Water; * - Significant 350 Int.J.Curr.Microbiol.App.Sci (2020) 9(7): 346-354 Table.3 Zone of inhibition (mm) observed in different concentrations (2 %, 4 % and 6%) of Adhatoda vasica against Streptococcus sp. Dilution 24 hours -5 Conc. of botanical Forms of botanical T1 2% 10 4% 6% 48 hours -7 Mean 2% 10 4% 6% -5 Mean 2% -7 10 4% 6% Mean 2% 10 4% 6% Mean 5.75 6.62 7.00 6.46 6.50 7.00 7.25 6.92 7.63 8.00 8.63 8.09 7.75 8.38 9.25 8.46 (2.50) (2.66) (2.73) (2.63) (2.64) (2.73) (2.78) (2.73) (2.85) (2.91) (3.02) (2.93) (2.87) (2.98) (3.12) (2.99) T2 7.50 7.62 8.25 7.79 7.50 8.12 8.75 8.12 7.75 8.50 9.25 8.50 8.38 8.50 9.75 8.88 (2.82) (2.84) (2.95) (2.87) (2.82) (2.93) (3.04) (2.93) (2.87) (3.00) (3.12) (3.00) (2.97) (3.00) (3.20) (3.06) T3 7.62 8.37 8.75 8.25 8.00 8.62 10.00 8.87 8.00 8.63 9.88 8.84 8.50 8.88 10.25 9.21 (2.84) (2.97) (3.04) (2.95) (2.91) (3.01) (3.24) (3.06) (2.91) (3.02) (3.22) (3.05) (3.00) (3.06) (3.27) (3.11) T4 Mean F test S. Em ± CD 5% 0.00 (0.70) 0.00 (0.70) 0.00 (0.70) 0.00 (0.70) 0.00 (0.70) 0.00 (0.70) 0.00 (0.70) 0.00 (0.70) 5.22 5.65 6.00 5.62 5.50 5.94 6.50 5.98 5.85 6.28 6.94 6.36 6.16 6.44 7.31 6.64 (2.39) (2.48) (2.54) (2.47) (2.44) (2.53) (2.64) (2.54) (2.51) (2.60) (2.73) (2.61) (2.58) (2.63) (2.79) (2.67) Dilutions * 0.072 (0.013) Concentrations * 0.152 (0.027) Forms * 0.072 (0.013) Dilutions * 0.066 (0.011) Concentrations * 0.141 (0.023) Forms * 0.066 (0.011) 0.198 (0.035) 0.420 (0.073) 0.198 (0.035) 0.184 (0.030) 0.389 (0.064) 0.184 (0.030) T1 : Aqueous form + Spraying, T2 : Acetone form + Spraying, T3 : Alcohol form + Spraying, T4 : Water; * - Significant 351 Int.J.Curr.Microbiol.App.Sci (2020) 9(7): 346-354 Bacillus sp. Staphylococcus sp. Streptococcus sp. Fig.1 Bacterial species isolated from silkworm haemolymph 2 % 2 % 4 % 4 % 6 % Acetone form+Spraying Aqueous form+Spraying 2 % 6 % 4 % 6 % Alcoholic form+Spraying Water Fig.2 Inhibition zone of Bacillus sp. exhibited by Adhatoda vasica 352 Int.J.Curr.Microbiol.App.Sci (2020) 9(7): 346-354 The trend was observed similar with other liquid forms T2 (Acetone form + spraying) (7.04 and 7.59., 7.63 and 8.26 mm) and T1 (aqueous form + spraying) (6.96 and 7.29., 7.35 and 7.79 mm) and found significant in 10-5 and 10-7 dilution at 24 and 48 hours of incubation respectively compared to control batches (Table 2). viz., 2 (5.22 and 5.50 mm), 4 (5.65 and 5.50 mm) and 6 (6.00 and 6.50 mm) at both the bacterial dilutions of 10-5 and 10-7 respectively (Table 3). The zone of inhibition after 48 hours of incubation recorded significant difference between liquid forms (T1-8.09, T2-8.50 and T3-8.84 mm) and (T1-8.46, T2-8.88 and T39.21 mm) and the results were on par with each other against 10-5 dilution whereas at 107 dilutions all the forms and methods resulted in significant differences in inhibiting growth of bacteria (Table 3). These results were found on par with the findings of (Hossain et al., 2013), according to them, under in-vitro study the use of methanol, petroleum spirit, ethyl acetate and dichloromethane extracts of Tinospora cordifolia were found effective against Bacillus subtilis and Escherichia coli. The maximum zone of inhibition (11.33 and 8.33 mm) was recorded for E. coli in methanol and ethyl acetate, whereas zone of inhibition of 10.67 and 8.00 mm was observed for B. subtilis with petroleum spirit and dichloromethane extracts of Tinospora cordifolia. In addition, minimum zone (6.67 mm) was reported fordichloro-methane and ethyl acetate extracts used against E. coli and B. subtilis. It is further confirmed by (Samatha et al., 2013) that, at 1:1 concentration, the aqueous extract of Oroxylum indicum showed greater inhibition of 11.7 mm (Bacillus subtilis), 8.7 mm (Staphylococcus aureus), 6.6 mm(Staphylococcus albus) whereas at 1:2 concentration, maximum zone of inhibition(5.2 mm) was found in Bacillus cereus when compared to the control (6.3, 6.2, 4 and 3 mm). The in-vitro effect of Adhatoda vasica at different concentrations, methods and forms of application were revealed, significant difference and found effective against all the three bacterial species. To assess the effectiveness of these botanical formulations against Bacillus sp., Staphylococcus sp. and Streptococcus sp., the zone of inhibition was assessed. Among three bacterial species, the maximum zone of inhibition was observed in alcoholic extract on 24 and 48 hours of incubation against Bacillus sp. at 10-5 and 10-7 spore dilution. Zone of inhibition (mm) observed in different concentrations (2 %, 4 % and 6 %) of Adhatoda vasica against Streptococcus sp. The application of botanical extracts of A.vasica at different concentrations against Streptococcus sp. revealed significant differences pertaining to forms and methods of botanical application, different concentrations of botanicals and different bacterial dilutions at 24 hours of incubation. Further, significant differences were noticed among the treatments viz., T1 (6.46 and 6.92 mm), T2 (7.79 and 8.12 mm) and T3 (8.25 and 8.87 mm) over water control T4 (0.00 mm) after 24 hours of incubation. In addition, the significant difference was also noticed in between the per cent botanical concentration The trend was same in other bacterial species viz., Staphylococcus and Streptococcus. However the increased zone of inhibition was observed on 24 and 48 hours of incubation and on 72 hours onwards, the inhibition was found decreased due to the loss of 353 Int.J.Curr.Microbiol.App.Sci (2020) 9(7): 346-354 antibacterial components present in the botanical extracts might have decrease the property of botanical that leads to increase in all the three types of bacterial growth. Thatteroga in silkworm, BombyxmoriL. Indian J. Seric.,38. Pp: 149-151. Ray, P. G. and Majumdar, S. K.1974.Antimicrobial activity of some Indian plants. Economic Botany, 30: Pp: 317-320. SamathaTalari, Sampath, A., Sujatha, K. and RamaSwamyNanna.2013.Antibacterial Activity of Stem Bark Extracts of Oroxylumindicuman Endangered Ethnomedicinal Forest Tree. J. Pharmacy and Biol. Sci.,7(1): 24-28. ShamimHossain, TouhidaKhatun and Mehedi H. 2013.In-vitro bacterial effect of Tinospoacordifolia extracts against some selective bacterial pathogens. Int. J. Biosci., 3(7):156-161. Sironmani, A. T., Meena, P. and Vanitha Rani, R. 1994. Isolation and characterization of pathogenic bacterial species in the silkworm, Bombyxmori L. Sericologia,34: 97-102. Sugun, R. 2000.Status and management of Thatte disease of silkworm. Advances in Tropical Sericulture. Proc. Natl. Sem. Tropic. Seric.,UAS, GKVK, Bengaluru. Pp. 84-86. References Chatterjee, A. and Pakrashi, S. C. 1991.The treatise on Indian medicinal plants. I, XII-XX publications and information directorate, New Delhi: Pp:14-15. Chopra, R. N. 1982.Indigenous drugs of India, 2nd ed. Academic publishers, Calcutta.Pp: 72-74. Karthikairaj, K., Prasannakumar, K. andIsaiarasu, L. 2013. Use of plant extracts for the control of flacherie disease in silkworm, Bombyxmori L. (Lepidoptera: Bombicidae). Int. J. Microbiol. Res., 4(2): 158-161 Krishna Prasad, K. S., Siddaramaiah, A. L. andLingaraju, S. 1979. Possible control of muscardine disease of silkworms by using a few plant extracts. Curr. Res.,8(5): 79-80. Nataraju, B., Sivaprasad, V. andDatta, R. K. 1999. Studies on the cause of How to cite this article: Anitharani. K. V., R. N. Bhaskar and Anusha. H. G. 2020. In-Vitro Efficacy of Forms and Methods of Adhatoda vasica against Flacherie causing Bacteria of Silkworm, Bombyx mori L. Int.J.Curr.Microbiol.App.Sci. 9(07): 346-354. doi: https://doi.org/10.20546/ijcmas.2020.907.037 354