EJBR2022v12i1art37 ISSN 2449-8955 European Journal of Biological Research Research Article European Journal of Biological Research 2022; 12(1): 37-45 DOI: http://dx.doi.org/10.5281/zenodo.5906591 Efficacy of essential oils as antiseptics on the productive characteristics of the mulberry silkworm Bombyx mori L. Salwa S. Pasha *, Nagat H. Soliman Plant Protection Department, Faculty of Agriculture, Fayoum University, Egypt Corresponding author: E-mail: ssb01@fayoum.edu.eg; ORCID ID: 0000-0003-0854-3344 Received: 18 October 2021; Revised submission: 15 January 2022; Accepted: 21 January 2022 https://jbrodka.com/index.php/ejbr Copyright: © The Author(s) 2022. Licensee Joanna Bródka, Poland. This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/) ABSTRACT: The study aimed to test the efficacy of three essential oils (basil Ocimum basilicum L., lemon Citrus limon (L.) Osbeck, and thyme Thymus vulgaris L.) as disinfectants, including their positive and negative effects, on the biological and productive parameters of the silkworm Bombyx mori. Biological parameters: basil oil treatment at 2000 ppm the highest significant 5th instar larval weight and pupal weight were 2.226 g and 0.787 g. In addition, at the same concentration, recorded the lowest significant mortality percentage and 5th instar larval duration, were 0.787 g and 5.09% respectively. On the other hand, lemon and thyme oils at 4000 ppm come in the second place the same parameters, compared to the control and the chemical disinfectant. While it is equal to the concentration of 8000 ppm for the oils tested in all biological parameters with the control and chemical disinfectant. Economical parameters: basil oil at 2000 ppm and lemon and thyme oils at 4000 ppm had the highest significance for cocoon weights, cocoon shell weight, and silk productivity, which were 1.203 g, 0.220 g, 2.34 cg for basil oil, 1.139 g, 0.210 g, 2.367 cg for lemon oil and 1.265 g, 0.216 g, 2.397 cg for thyme oil, compared with control and disinfectant chemical groups (0.993 g, 0.157 g, 1.49 cg and 0.991 g, 0.160 g, 1.68 cg, respectively). The highest significant difference of cocoon percentages was seen with basil oil at 2000 ppm, compared to the other treatments. Keywords: Antiseptic; Bombyx mori; Nutritional enhancers; Essential oils. 1. INTRODUCTION Bombyx mori, also known as the silkworm, is one of the most commercially important insects in the world. It feeds on the leaves of the mulberry plant. It is the most unique cultivated silkworm genus and generates the best silk fiber among other genera [1]. The silkworm is subjected to several types of diseases; especially, the larval stage is the most critical, and it is necessary to take appropriate care during this stage. Diseases affecting B. mori cause heavy crop losses in sericulture, leading to crop failure and low harvest rates [2]. Different varieties of food plants are found to be effective economically on important characteristics of silk production [3]. Disease-free rearing practices such as bed disinfection and larval body decontamination are known to boost productivity [4]. The use of natural products has increased in the past 20 years. Recent studies have demonstrated the use of plant essential oils as pesticides and good plant protectants [5, 6]. Commercial products derived from essential oils are being developed for a variety of animal and human uses [7]. Aromatherapy, phytotherapy, Pasha & Soliman Essential oils as antiseptics on the productive characteristics of Bombyx mori 38 European Journal of Biological Research 2022; 12(1): 37-45 antibacterial and antifungal applications, and other pharmacological and therapeutic applications are a few uses of essential oils [8]. Some essential oils such as cinnamon, clove, and lavender oils exhibit inhibitory effects against Bacillus cereus and Proteus vulgaris (flacherie diseases), which are the associated pathogens infecting silkworms. In addition to its beneficial effects on biological and productive qualities, silk production has increased [9, 10]. Essential oils consist of several low-molecular-weight volatile compounds, terpenes, and phenolics that can be extracted from the major plant families Asteraceae, Lauraceae, Myrtaceae, and Lamiaceae. These compounds possess repellent, lethal, and growth-reduction effects on a variety of insects and microorganisms [11]. The chemical compounds found in essential oils have an effect on the growth of the silkworm. A high aldehyde concentration in essential oils has a better effect on silkworm development and cocoon quality, but a low acid concentration in the oils exerts a negative effect [12]. Furthermore, there is extensive research on the use of oils and plant extracts as food additives to improve the biological and productive characteristics of the silkworm [13]. This study was conducted to evaluate the effectiveness of some essential oils as a material disinfectant to protect silkworms from microbial infections, as well as to examine the extent of their safety on larvae and their effect on different productive traits. 2. MATERIALS AND METHODS 2.1. Source and rearing of B. mori The silkworm B. mori L. egg box (Egyptian hybrid) was purchased from the Sericulture Research Department., Plant Protection Research Institute, Agriculture Research Center. Dokki, Giza. In the spring, B. mori larvae were fed on fresh mulberry leaves (Morus alba var. indica) in the Plant Protection Department of Fayoum University's laboratory, at 27°C ± 2°C and 75% ± 5% RH [14]. 2.2. Source of essential oils The essential oils of basil (Ocimum basilicum L., Lamiaceae), lemon (Citrus limon (L.) Osbeck, Rutaceae), and thyme (Thymus vulgaris L., Lamiaceae) were purchased from the local market in Fayoum Governorate, Egypt, produced by the organic company for natural oils, cold-pressed and pure natural. Oils were used in the present study for the bioassay experiments on the larvae of B. mori as disinfectants and nutritional enhancers. A chemical bed disinfectant consisting of salicylic acid, paraformaldehyde, benzoic acid, and slaked lime was obtained from the Seric. Res. Dept., Plant Protec. Res. Inst, Agric. Res. Center. Dokki, Giza. 2.3. Bioassay technique First, the 2nd instar larvae of B. mori were divided into three treatment groups (in addition to the control and control sprinkled with the chemical disinfectant). Each treatment was separated into three concentrations, with three replicates for each concentration (each of 50 larvae). Each replicate was reared in a 30 × 15 × 4 cm carton tray. The larvae in each replicate were fed on treated or untreated mulberry leaves. The treated mulberry leaves were washed with water and immersed in three aqueous serial concentrations of 2000, 4000, and 8000 ppm of each essential oil containing one to three drops of Triton X 100 as the emulsifier. The untreated leaves Pasha & Soliman Essential oils as antiseptics on the productive characteristics of Bombyx mori 39 European Journal of Biological Research 2022; 12(1): 37-45 of the control group were dipped in distilled water containing one to three drops of Triton X-100 for 1 min. In addition, the untreated leaves in the control group with the chemical disinfectant were dipped in distilled water alone. The leaves were left to dry at room temperature before introducing to the larvae. The larvae are fed four times a day; the leaves treated with essential oils were introduced once a day to the silkworm larvae. On the other hand, in the control disinfectant treatment, the larvae were also sprinkled once a day, this is done daily from the 2nd instar larvae until cocoon formation. Serial concentrations of essential oils were prepared fresh for each treatment. In spring, the entire experiment was conducted twice in a row and the measurements averages of the two experiments were taken. 2.4. Biological parameters The larval duration was calculated at the end of the 5th instar larvae. The larval mortality percentage was calculated (for the 5th instars) as follows: Mortality % = Number of dead larvae / total number of larvae × 100 For recording the pupal weights, on the 7th day from cocoon spinning, the cocoons were carefully opened (using a fine cutter) and their pupae and cocoon shells were weighed after cleaning the exuviate to obtain pupal and cocoon shell weights. 2.5. Economical parameters The economical parameters included cocoon weight, cocoon shell weight, cocooning percentage, cocoon shell ratio, and silk productivity. Cocooning percentages were computed as described previously [15], as follows: Cocooning percentages = Number of cocoons formed / Total number of larvae kept × 100 The cocoon shell ratio was calculated using the following formula [16]: Cocoon shell ratio (%) = Fresh cocoon shell weight / Fresh cocoon weight × 100 Silk productivity was determined using the following formula [17]: Silk productivity per day (cg/day) = Cocoon shell weight / Fifth instar duration (day) × 100 Where, cg = centigram 2.6. Statistical analysis The results of the bioassays were analyzed using the Duncan test at the 0.05 probability level to determine the least significant differences. The mean ± standard errors (SEs) are represented for each value using the SPSS program software [18]. 3. RESULTS 3.1. Biological parameters Several studies on the influence and efficiency of essential oils on a variety of pests and diseases have been conducted in recent years. Renewable and distinct antibacterial, antifungal, and antiparasitic substances are derived from the biological and structural diversity of their constituents [19-21]. In the present study, we focused on three serial concentrations of the three essential oils of basil (O. basilium), lemon (C. limon), and thyme (T. vulgaris) as preventive agents to protect the silkworm B. mori from microbial infections. We also investigated the effect of these concentrations on the biological and productive characteristics of the silkworm. Pasha & Soliman Essential oils as antiseptics on the productive characteristics of Bombyx mori 40 European Journal of Biological Research 2022; 12(1): 37-45 The biological parameters (5th larval weight, 5th larval duration, 5th larval mortality and pupal weight) of the silkworm were recorded (Table 1 & Fig. 1). The highest significant 5th instar larval weight was 2.226 g with basil oil treatment at 2000 ppm, showing a considerable significant difference from that in the control and control disinfectant groups, where the larval weights were 1.995 and 1.993 g, respectively. The highest significant weights with lemon and thyme oil treatments ranged from 2.000 to 2.096 g at 2000 and 4000 ppm, respectively, whereas the lowest 5th instar larval weight was 1.859 g at 8000 ppm of basil essential oil concentration. Table 1. Means ± standard error (P≤0.05) of biological parameters for three essential oils efficiency on the 5th larval instar of silkworm Bombyx mori. Materials Conc. (ppm) 5th larval weight (g) 5th larval duration (day) 5th larval mortality % Pupal weight (g) Control 1.995bc ± 0.39 10.500ab ± 0.185 8.073ab ± 0.307 0.701 cd ± 0.005 Control + chemical disinfectant 1.993bc ± 0.39 9.500c ± 0.185 8.063ab ± 0.307 0.703cd ± 0.005 Basil Ocimum basilium 2000 2.226a ± 0.39 9.400c ± 0.185 5.090e ± 0.307 0.787a ± 0.005 4000 1.996bc ± 0.39 10.100b ± 0.185 7.203bc ± 0.307 0.753b ± 0.005 8000 1.859d ± 0.39 10.800 a ± 0.185 8.213a ± 0.307 0.705c ± 0.005 Lemon Citrus limon 2000 2.000bc ± 0.39 9.300c ± 0.185 6.300 cd ± 0.307 0.700 cd ± 0.005 4000 2.096b ± 0.39 9.200c ± 0.185 5.613de ± 0.307 0.775a ± 0.005 8000 1.997bc ± 0.39 10.200b ± 0.185 8.260a ± 0.307 0.688d ± 0.005 Thyme Thymus vulgaris 2000 2.026bc ± 0.39 10.067b ± 0.185 7.220bc ± 0.307 0.711c ± 0.005 4000 2.001bc ± 0.39 9.100c ± 0.185 6.093d ± 0.307 0.778a ± 0.005 8000 1.900cd ± 0.39 10.300ab ± 0.185 8.950a ± 0.307 .698cd ± 0.005 Duncan test ʺF-Testʺ 5.87** 9.90** 16.64** 60.97** (a,b,c,d) letters refer the means significant with the same column. ** refer to significant of ʺF-Testʺ with the same column. The lowest 5th instar larval duration of 9.1 and 9.2 days was observed for larvae fed on leaves treated with 4000 ppm concentration of thyme and lemon oils, respectively, followed by larvae fed on leaves treated with 2000 ppm of basil oil (9.4 days), whereas the highest larval duration of 10.8 days was observed at 8000 ppm of basil oil. Prolongation in the duration of the 5th larval stage was observed with an increase in the concentration of all oils, where the concentration of 8000 ppm had the highest duration of the 5th larval stage among all oils. The duration of the 5th larval stage was 9.5 and 10.5 days in the control and control + disinfectant groups, respectively. The mortality percentage was found to increase at high oil concentrations. Highly significant 5th larval mortality rates were 8.950%, 8.260%, and 8.213% under treatment with thyme, lemon, and basil essential oils at 8000 ppm, respectively. Moreover, the larval mortality rates in the control and control + disinfectant groups were 8.073% and 8.063%, respectively, which were not significant between high concentrations. The lowest mortality rate of 5.090% was recorded in larvae fed on leaves treated with basil oil at a concentration of 2000 ppm. Pasha & Soliman Essential oils as antiseptics on the productive characteristics of Bombyx mori 41 European Journal of Biological Research 2022; 12(1): 37-45 Figure 1. Means of biological parameters for three essential oils efficiency on the 5th larvae instar of silkworm Bombyx mori. There was no significant difference in mortality between larvae fed on leaves treated with high concentrations of all essential oils and larvae fed on untreated leaves. These mortality results indicated the possibility of using low concentrations of essential oils. Basil oil was found to be a safe disinfectant for the silkworm larvae as it resulted in lower mortality than that chemical disinfectants. Under all treatments of essential plant oils, highly significant pupal weights of 0.787, 0.778, and 0.775 g were observed with basil oil at 2000 and 4000 ppm and thyme and lemon oils, respectively. The lowest pupal weight of 0.698 g was recorded with thyme oil treatment at 8000 ppm. However, the mean weight of the pupa in the control group was 0.701 g. 3.2. Economical parameters Regarding the economical characteristics of the silkworm B. mori (Table 2 & Fig. 2), the highest significant cocoon weights of 1.265 and 1.203 g were observed with thyme and basil oils at 4000 and 2000 ppm, respectively, showing a considerable significant difference from that (0.993 g) in the control group. The lowest cocoon weight of 0.978 g was observed at 8000 ppm of basil oil. The lowest cocoon percentage of 88.457% was observed with thyme oil treatment at 8000 ppm, and the maximum significant cocoon percentage was 95.503% observed with basil oil treatment at 2000 ppm (Table 2 and Fig. 2). However, the cocoon percentages in the two control groups were 92.073% and 91.160%, respectively. Cocoon shell weights under treatment with all essential oils appeared to be significantly different, with the highest weight of 0.220 g recorded at 2000 ppm of basil oil, followed by 0.216 and 0.210 g with thyme and lemon oil treatments at 4000 ppm. Compared among all oils, the cocoon shell weight in the two control groups (0.157 and 0.160 g) appeared to be less significant. Pasha & Soliman Essential oils as antiseptics on the productive characteristics of Bombyx mori 42 European Journal of Biological Research 2022; 12(1): 37-45 Table 2. Means ± standard error (P≤0.05) of economical parameters for three essential oils efficiency on the silkworm Bombyx mori. Materials Conc. (ppm) Cocoon weight (g) Cocoon shell weight (g) Cocooning percentages % Cocoon shell ratio % Silk productivity cg/day Control 0.993b ± 0.057 0.157c ± 0.005 92.073abcd ± 1.229 15.807 a ± 0.830 1.49cd ± 0.065 Control + chemical disinfectant 0.991b ± 0.057 0.160c ± 0.005 91.160bcd ± 1.229 16.203 a ± 0.830 1.68c ± 0.065 Basil, Ocimum basilium 2000 1.203 a ± 0.057 0.220a ± 0.005 95.503 a ± 1.229 17.953 a ± 0.830 2.34 a ± 0.065 4000 1.001b ± 0.057 0.163c ± 0.005 90.173bcd ± 1.229 16.397 a ± 0.830 1.613c ± 0.065 8000 0.978 b ± 0.057 0.151c ± 0.005 88.050d ± 1.229 15.433 a ± 0.830 1.393d ± 0.065 Lemon Citrus limon 2000 1.092ab ± 0.057 0.186b ± 0.005 90.110bcd ± 1.229 17.027 a ± 0.830 2.00b ± 0.065 4000 1.139ab ± 0.057 0.210a ± 0.005 93.027 ab ± 1.229 18.133a ± 0.830 2.367 a ± 0.065 8000 1.000 b ± 0.057 0.167c ± 0.005 89.050bcd ± 1.229 16.650 a ± 0.830 1.63c ± 0.065 Thyme Thymus vulgaris 2000 1.119ab ± 0.057 0.190b ± 0.005 91.960abcd ± 1.229 17.140 a ± 0.830 1.883b ± 0.065 4000 1.265a ± 0.057 0.216a ± 0.005 92.397abc ± 1.229 16.133 a ± 0.830 2.397 a ± 0.065 8000 1.000 b ± 0.057 0.162c ± 0.005 88.457 cd ± 1.229 16.190 a ± 0.830 1.57cd ± 0.065 Duncan test ʺF-Testʺ 3.056* 23.90** 3.23** 1.047 32.67** (a,b,c,d) letters refer the means significant with the same column. ** refer to significant of ʺF-Testʺ with the same column. Figure 2. Means of economical parameters for three essential oils efficiency on the silkworm Bombyx mori. Among treatments with the three essential oils, there were no significant differences in the cocoon shell ratio. However, lemon oil treatment resulted in a greater cocoon shell ratio of 18.13% at 8000 ppm compared to 15.8% in the control group. The feeding efficiency of sericulture is generally determined by the production efficiency of cocoon shells. It was obvious that larvae fed on treated mulberry leaves dipped in the essential oils at various concentrations showed enhanced cocoon shell formation efficiency with all treatments and at higher rates than larvae fed on untreated mulberry leaves (control). Pasha & Soliman Essential oils as antiseptics on the productive characteristics of Bombyx mori 43 European Journal of Biological Research 2022; 12(1): 37-45 The silk productivity of larvae fed on oil-treated mulberry leaves was higher than that of larvae fed on untreated mulberry leaves, with highly significant differences of 2.397%, 2.367%, and 2.34% under treatment with thyme and lemon oils at 4000 ppm and basil oil at 2000 ppm, respectively, compared to 1.49% and 1.68% in the two control groups. 4. DISCUSSION To ensure successful rearing and higher cocoons production, silkworm larvae must be protected from infection by their associated microorganisms. Diseases appear clearly on the larvae in the 4th and 5th instars. As a result, we set out to protect the larvae by using oils tested since the beginning of the second instar larvae. The obtained results showed that the oils of basil, lemon, and thyme and reduced significantly the mortality of the 5th instar larvae than the control and chemical disinfectant. The essential oil of Ocimum sanctum is a broad fungitoxicant, with 1.0 μl/ml fumigation reducing the number of Aspergillus flavus isolates up to 74.01% [22]. Basil oil contains bioactive chemicals such as linalool (eugenol), methyl cinnamate, and estragole, which have been confirmed as antimicrobial activity [23, 24]. Moreover, was observed high antibacterial activity of essential oils Citrus limon, Ocimum basilicum and Thymus vulgaris [25]. In the present study, the cocoon percentage was increased by 95.5% in the larvae of B. mori treated with basil oil at 2000 ppm compared to 92.073% & 91.16% for control and chemical disinfectant respectively, which is consistent with a previous study [26], where the highest cocooning percentages for infected larvae (Bacillus thuringiensis and Beauveria bassiana) treated with 2% basil leaf extract were 97.3% and 94.0% respectively, compared to 92.0% in the control. In respect of silk productivity, the oils tested (basil, thyme, and lemon) outperformed the control and chemical disinfection. Another previous study [27] reported that Ocimum sanctum is highly efficient against bacterial flacherie and Staphylococcus sp., Bacillus sp., and Klebsiella cloacae, which are associated with silkworm larvae, and can be used to control microbial infections during silkworm rearing and improve silk productivity. Our results are also consistent with those which subjected B. mori larvae to artificial infestation with the conidial solution of B. bassiana and B. thuringiensis and treated them with basil oil concentrations [28]. Compared with control larvae infected with B. bassiana and B. thuringiensis, treatment with basil leaf extract increased the larval weight, pupal weight, cocoon weight, and cocoon shell weight and reduced the larval mortality rate. Consequently, the spread of bacterial and fungal infections was slowed. Furthermore, the biological and technological characteristics were enhanced, indicating that basil essential oil can be used in sericulture to improve the quality and quantity of silkworm cocoons. Our results also correspond with those which found that larval and pupal weights were increased significantly when fed with thyme extract [29]. On the other hand, with an increase in the oil concentrations, we detected decreases in larval and pupal weights, addition increases in mortality and larval duration, which are similar findings to those reported by Dewer and Mona [30]. 5. CONCLUSIONS The essential oils examined in this study may be used as disinfectants for silkworms at low and medium concentrations (2000 ppm & 4000 ppm) to protect them from their associated microbial infections. Pasha & Soliman Essential oils as antiseptics on the productive characteristics of Bombyx mori 44 European Journal of Biological Research 2022; 12(1): 37-45 Moreover, these oils had no negative impacts on the biological qualities of the larvae. The oil concentrations used in this study resulted in considerable and desirable increases in cocoon weight and silk productivity. Therefore, these essential oils are important and safe sources of antiseptics for the silkworm B. mori, as well as for improving silk productivity, especially basil oil followed by thyme and lemon oils. We recommend using basil essential oil at 2000 ppm concentration or 4000 ppm for lemon and thyme oils as an antiseptic and to increase silk productivity during rearing of the silkworm B. mori. Authors' Contributions: SP: designed the experiment chose the materials used in it, and prepared the tested concentrations. Collect the results in tables, analyzed them statistically, prepared drawings, interpreted data, wrote the manuscript and reviewed it. NS: carried out the experiment design on the larvae and recorded various outcomes and measurements. All authors read and approved the final manuscript. Conflict of Interest: The authors declare no conflict of interest. REFERENCES 1. Yilmaz O, Erturk YE, Coskun F, Ertugrul M. Biology of silkworm (Bombyx mori) in Turkey. Conference Pap, 2015. 2. Chopade P, Raghavendra CG, Mohana Kumar S, Bhaskar RN. Assessment of diseases in Bombyx mori silkworm – A survey. Global Trans Proceed. 2021; 2: 133-136. 3. Singh M, Singh DM. Effect on productivity of silk due to choice of different feeding plants (Tarminalia Arjuna – Tasar silkworm, mulberry plant –Bombyx mori) in Ambikapur District Surguja Chhattisgarh. Int J Dev Res. 2020; 10(4): 34952-34959. 4. Greiss H, Petkov N, Yungen M. Optimized rearing conditions for silkworm Bombyx mori L. egg production under Egyptian conditions. Philippine J Sci. 2002; 131(2): 137-141. 5. Isman MB. Bioinsecticides based on plant essential oils: A short overview. Z Naturforsch. 2020; 75(7-8)c: 179-182. 6. Mukarram M, Khan MMA, Zehra A, Choudhary S, Aftab T, Naeem M. Biosynthesis of lemongrass essential oil and the underlying mechanism for its insecticidal activity. Med Arom Plants. 2021; 429-443. 7. Tripathi AK, Upadhyay S, Bhuiyan M, Bhattacharya PR. A review on prospects of essential oils as bio pesticide in insect-pest management. J Pharmacogn Phytother. 2009; 1(5): 52-63. 8. Hamid AA, Aiyelaagbe O, Usman LA. Essential oils: Its medicinal and pharmacological uses. Int J Curr Res. 2011; 3(2): 86-98. 9. Omar AS, Fathy M. Dina. Evaluation of controlling silkworm bacterial diseases using propolis extract and cinnamon oil. J Agric Chem Biotechnol. 2016; 7(7): 213-218. 10. Sampath A, Sujatha K, Suryam A, Singaracharya MA. Antibacterial activity of phyto essential oils on Flaccherie causing bacteria in the mulberry silk worm, B. mori. L. J Pharm Biolog Sci. 2015; 10(5): 29-33. 11. Regnault-Roger C, Vincent C, Arnason JT. Essential oils in insect control: Low-risk products in a high-stakes world. Annu Rev Entomol. 2012; 57: 405-424. 12. Choudhury SN, Vajczikova I. Variations in the essential oil composition of Persea bombycina (King ex Hook. f.) Kost and its effect on muga silkworm (Antheraea assama Ww) - A new report. Indian J Chem. 2003; 42B: 640-647. 13. Singh P, Prasad S, Upadhyay VB. Effect of the essential oil Aloe vera on the cocoon traits of multivoltine mulberry silkworm Bombyx mori Linn. Eur J Biol Sci. 2014; 6(1): 7-12. 14. Krishnaswami S. New technology of silkworm rearing II. Central Sericulture Research and Training Institute, 1978. Pasha & Soliman Essential oils as antiseptics on the productive characteristics of Bombyx mori 45 European Journal of Biological Research 2022; 12(1): 37-45 15. Goudar KS, Kaliwal BB. Effect of cortisone and hydrocortisone on the biochemical changes in the fat body and haemolymph of the silkworm, Bombix mori L. Int J Industr Entomol. 2001; 2(2):181-184. 16. Krishnaswami S, Narasimhanna MN, Suryanarayan SK, Kumararaj S. Manual on sericulture: Silkworm Rearing. FAO Agricultural Services Bulletin. 1973; 15(2): 42-43. 17. Chattopadhyay C. Wilt Complex of Castor (Ricinus communis L.): Role of reniform (Rotylenchulus reniformis Linford and Oliveira) nematode. J Oilseeds Res. 1995; 12(2): 203-207. 18. Snedecor GW, Cochran WGS. Statistical methods applied to experiments in agriculture and biology. 6th edn. Ames, Iowa: Iowa State University Press, 1967. 19. Marwa NM, Eman MH. Efficiency of some therapeutic essential oils as antibacterial and antioxidants on some biochemical parameters of infected silkworm Bombyx mori L. Egypt Acad J Biol Sci. 2017; 10: 91-98. 20. Pachiappan P, Aruchamy MC, Ramanna SK. Evaluation of antibacterial efficacy of certain botanicals against bacterial pathogen Bacillus sp. of silkworm, Bombyx mori L. Int J Industr Entomol. 2009; 18: 49-52. 21. Sakkas H, Papadopoulou C. Antimicrobial activity of basil, oregano, and thyme essential oils. J Microbiol Biotechnol. 2017; 27(3): 429-438. 22. Kumar A, Dubey NK, Srivastava S. Antifungal evaluation of Ocimum sanctum essential oil against fungal deterioration of raw materials of Rauvolfia serpentina during storage. Indust Crops Prod. 2013; 45: 30-35. 23. Falowo AB, Mukumbo FE, Idamokoro EM, Afolayan AJ, Muchenje V. Phytochemical constituents and antioxidant activity of sweet basil (Ocimum basilicum L.) essential oil on ground beef from Boran and Nguni cattle. Int J Food Sci. 2019: 2019: 1-8. 24. Carrasco H, Raimondi M, Svetaz L, Liberto MD, Rodriguez MV, Espinoza L, et al. Antifungal activity of eugenol analogues. Influence of different substituents and studies on mechanism of action. Molecules. 2012; 17: 1002-1024. 25. Soković M, Glamočlija J, Marin PD, Brkić D, van Griensven LJLD. Antibacterial effects of the essential oils of commonly consumed medicinal herbs using an in vitro model. Molecules. 2010; 15: 7532-7546. 26. Helaly W, Elyamani E, Saad M. Influence of some plant extracts as antiseptics to control bacterial and fungal diseases of silkworms, Bombyx mori L. Zagazig J Agric Res. 2020; 47(3): 307-317. 27. Karthikairaj K, Isaiarasu L, Sakthivel N. Efficacy of some herbal extracts on microbes causing flacherie disease in mulberry silkworm, Bombyx mori L. J Biopest. 2014; 7: 89-93. 28. Saad MSI, Elyamani EMY, Helaly WMM. Controlling of bacterial and fungal diseases that contaminating mulberry silkworm, Bombyx mori by using some plant extracts. Bull Nat Res Centre. 2019; 43: 172. 29. Mona M Mahmoud. Effect of certain evaluated plant crude extracts on the productivity of silkworm (Bombyx mori L.). J Adv Agric Res. 2014; 19(1): 50-59. 30. Dewer Y, Mona MM. Effectiveness and safety of some essential oils of aromatic plants on the growth and silk production of the silkworm Bombyx mori L. J Entomol Zool Stud. 2014; 2(2): 81-86.