8 © 2023 by the authors; licensee Asian Online Journal Publishing Group Agriculture and Food Sciences Research Vol. 10, No. 2, 8-13, 2023 ISSN(E) 2411-6653/ ISSN(P) 2518-0193 DOI: 10.20448/aesr.v10i2.5186 © 2023 by the authors; licensee Asian Online Journal Publishing Group Invitro analysis of antifungal effects of botanicals on sclerotinia sclerotiorum causing white mold disease Prashant Gyanwali1 Renuka Khanal2 Netra Prasad Pokharel3 Bhuwan Tharu4 Rajan Koirala5 Sandesh Paudel6 Rajan Paudel7 ( Corresponding Author) 1,2,3,4,5,6,7Institute of Agriculture and Animal Science, Paklihawa Campus, Tribhuwan University, Nepal. 1Email: Prashantgyawali7@gmail.com 2Email: Renukhanal57@gmail.com 3Email: netrapokharel73@gmail.com 4Email: tharubhuwan44@gmail.com 5Email: rajankoirala567@gmail.com 6Email: paudels761@gmail.com 7Email: rajan@iaas.edu.np Abstract White mold, Sclerotinia sclerotiorum, is a devastating fungal plant pathogen that has affected many crop species worldwide. Using chemicals to control the disease has been practiced over the years, whose prolonged application has negatively impacted the environment, thus finding an organic solution is crucial. The analysis quantifies the effect of 5 different local plants that have been proven to have fungicidal properties; Artemisia vulgaris L., Azadirachta indica L., Zingiber officinale Roscoe, Allium sativum L., and Lantana camara L. Poisoned food technique was used to study the inhibition effect, carried out by inoculating and growing the fungus on PDA media infused with botanical extracts. The data of mycelial mat diameter was recorded till the control plates were fully occupied. The growth-inhibiting capacity was found as 100% by Allium sativum, 28% & 43% by Azadirachta indica, 22.44% & 44% by Zingiber officinale, and 16.33% & 8.55% by Lantana camara at 10% and 20% concentrations respectively. Only a slight difference between the overall inhibition effect of the two concentrations was found with 37.22% inhibition by 20% concentration and 33.38% inhibition by 10% concentration. No inhibition effect was observed from Artemisia vulgaris which could be due to heat neutralization of the active constituent during sterilization. Further research needs to be conducted using the botanical with different sterilization techniques. This in-vitro study identified garlic as a critical antifungal alternative to conventional fungicides. Field experiments need to be done to prove its effectiveness. Keywords: Fungal-growth inhibition, Invitro analysis, Phytochemicals, Phytopathology, Plant extracts, Plant protection, White mold. Citation | Gyanwali, P., Khanal, R., Pokharel, N. P., Tharu, B., Koirala, R., Paudel, S., & Paudel, R. (2023). Invitro analysis of antifungal effects of botanicals on sclerotinia sclerotiorum causing white mold disease. Agriculture and Food Sciences Research, 10(2), 8– 13. 10.20448/aesr.v10i2.5186. History: Received: 1 September 2023 Revised: 27 October 2023 Accepted: 13 November 2023 Published: 30 November 2023 Licensed: This work is licensed under a Creative Commons Attribution 4.0 License Publisher: Asian Online Journal Publishing Group Funding: This study received no specific financial support. Institutional Review Board Statement: The Ethical Committee of the Institute of Agriculture and Animal Science, Paklihawa, Tribhuvan University, Nepal has granted approval for this study on 24 October 2022. Transparency: The authors state that the manuscript is honest, truthful, and transparent, that no key aspects of the investigation have been omitted, and that any differences from the study as planned have been clarified. This study followed all writing ethics. Competing Interests: The authors declare that they have no competing interests. Authors’ Contributions: All authors contributed equally to the conception and design of the study. All authors have read and agreed to the published version of the manuscript. Contents 1. Introduction ......................................................................................................................................................................................... 9 2. Materials and Methodology .............................................................................................................................................................. 9 3. Result .................................................................................................................................................................................................. 11 4. Discussion .......................................................................................................................................................................................... 11 5. Conclusion ......................................................................................................................................................................................... 12 References .............................................................................................................................................................................................. 12 mailto:Prashantgyawali7@gmail.com mailto:Renukhanal57@gmail.com mailto:netrapokharel73@gmail.com mailto:tharubhuwan44@gmail.com mailto:rajankoirala567@gmail.com mailto:paudels761@gmail.com mailto:rajan@iaas.edu.np https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ https://www.doi.org/10.20448/aesr.v10i2.5186 https://orcid.org/0000-0001-6428-491X https://orcid.org/0009-0001-6929-4284 https://orcid.org/0009-0001-9237-3992 https://orcid.org/0009-0006-0783-4245 https://orcid.org/0009-0004-0663-2380 https://orcid.org/0009-0006-9293-2762 https://orcid.org/0000-0001-5108-6113 Agriculture and Food Sciences Research, 2023, 10(2): 8-13 9 © 2023 by the authors; licensee Asian Online Journal Publishing Group Contribution of this paper to the literature The research provides information that differs in botanicals used and their preparation from other similar research and is a unique study against white mold. The paper adds the knowledge of how different botanicals fare against white mold and guides further research regarding its organic control. 1. Introduction Sclerotinia sclerotiorum (Lib.) de Bary (Helotiales: Sclerotiniaceae) is known by multiple names but its most common name recognized worldwide is ‘White Mold’ as it is characterized by cotton-like fluffy white mycelial growth seen on infected plants’ stems. The pathogen exhibits its symptoms by creating water-soaked lesions on leaves that move downwards to the stem where it develops into necrotic tissues before developing its characteristic cottony mycelium [1]. Sclerotinia is a polyphagous, necrotrophic, homothallic pathogen that is rampant worldwide, especially in temperate regions [2]. It is a devastating plant pathogen that has infected over 75 families, 278 genera, 408 species, and 42 subspecies of plants. A more pronounced effect of Sclerotinia is seen in dicot plants like peas, soybean, sunflower, rapeseed, and occasionally in monocot crops and grasses [3]. S. sclerotiorum can cause varying degrees of yield losses to different crops, ranging from 20 to 50% loss in canola production [4] and 10 to 50% loss in lettuce production [5]. Plant pests have been interfering with and preventing crops from reaching their full potential yield since the beginning of agriculture. Their damage to plant parts, systems, physiological processes, and ultimately yield has been one of the contributing factors to food scarcity in the world [6, 7]. Almost 40% loss of crop production and 20% loss in post-harvest is incurred every year due to insect pests, weeds, and diseases in the world [8]. To combat the losses, many chemical pesticides have been developed over the years which on one hand have helped reduce the pests’ effect while on the other hand have developed a host of problems like resistant pests, persistent pollutants, biological life hazards, and loss of biodiversity due to death of non-target organisms [9, 10]. So, there have been many attempts at developing alternatives to chemical pesticides like biopesticides, botanical extracts, and microbial pesticides. These alternatives have the benefit of being biodegradable, extremely efficient and accurate, and less harmful to the health of humans and the environment [11]. Many studies have been done on controlling Sclerotinia using fungicides [12, 13] from which numerous negative effects arise due to their prolonged use [14]. Therefore many studies are being conducted to measure the efficacy of different biopesticides in the control of fungi [15]. So, farmers are in dire need of appropriate and sustainable approaches to the management of such pests and pathogens [16]. The purpose of this research is to evaluate the efficacy of some locally available plants of Nepal with fungicidal effects against white mold in-vitro, which could be later incorporated into the farmers’ fields with further processing and development. 2. Materials and Methodology 2.1. Plant Extract Preparation The plant extracts were produced through a simple crude aqueous extraction similar to the process used by Timila and Manandhar [17]. First, the fresh botanicals were washed to clean any dirt or external chemicals present in them. Then after shade drying, 100 g each of fresh plant parts were taken and ground into paste separately using a mixer. Each resultant paste was mixed with 100 ml of distilled water. Extra care was taken to create a constant weight-to-volume ratio of botanicals to water so that their concentration would be the same throughout each extract. The mixture was then filtered through a muslin cloth and we obtained the botanical extracts in the form of filtrate as shown in Figure 1. The aqueous extracts kept in separate flasks were autoclaved at 121 °C @ 15 psi for 20 minutes to eliminate any microorganisms present within. Figure 1. Aqueous botanical extracts of (from left) A. vulgaris, A. sativum, A. indica, Z. officinale and L. camara. 2.2. Media Preparation The media of choice for the analysis was PDA (Potato Dextrose Agar) prepared by mixing ready-made PDA powder with distilled water. The analysis was carried out using the “Poisoned Food Technique” as described by Balouiri, et al. [18] where two concentrations (10% and 20%) of each plant extract were infused with PDA, creating a total of 10 unique growth media. To create the two concentrations of 10% and 20%, 10 ml and 20 ml botanical extracts were mixed with 90 ml and 80 ml molten PDA respectively. Unaltered PDA media was kept separately as control and four replications for each unique media and control were made by pouring molten PDA 20 ml each in Petri dishes. Agriculture and Food Sciences Research, 2023, 10(2): 8-13 10 © 2023 by the authors; licensee Asian Online Journal Publishing Group 2.3. Fungal Culture Preparation Sclerotinia for the analysis was isolated from the Garden pea (Pisum sativum) pod shown in Figure 2. The PDA media was inoculated with threads of mycelia initially and it was allowed to grow at 24 °C for a week in an incubator. The inoculating processes were repeated two more times from the obtained growth to ensure the exclusion of any other contaminants and obtain a pure culture. The final pure culture of 7 days was then used to inoculate all the different media and control with the help of a cork borer sterilized in an open flame. Mycelium discs of 5 mm diameter were punched out from the pure culture and placed on the media at the center of each petri dish. The discs were placed with the mycelial side facing down to assist the pathogen in establishing on the new media. The whole inoculation process was done under aseptic conditions inside the laminar air-flow cabinet, and the inoculated dishes were incubated at 24 °C in dark. Figure 2. Infection of S. sclerotiorum on garden pea. 2.4. Experimental Design and Antifungal Activity Assay The experiments were conducted in Completely Randomized Design (CRD) with 5 treatments of 2 concentrations viz: 10% and 20%. Four replications each of treatments and control were kept resulting in a total of 44 Petri plates. The fungicidal effects of botanicals were recorded by daily (24 hours) observation and measurement of the radial growth of mycelium, till the control plates were completely covered by the mycelial mat. For the antifungal activity assay, the percentage of mycelium growth inhibition was calculated by using the formula mentioned by Al-Samarrai, et al. [19]; Bekker, et al. [20] and Javed, et al. [21]. Percentage Inhibition = (𝐶−𝑇) 𝐶 × 100% Where, C = colony diameter (cm) of the control. T = colony diameter (cm) of the test plate. The measurement of mycelial diameter was taken in cm and an average of 4 replications was taken for each treatment. The inhibition percentage was calculated for both concentrations of all the botanical extracts for 3 days until the control plate was fully occupied by the mycelial mat and the results are presented in Table 1. 2.5. Statistical Analysis The data were recorded in MS Excel 2016, along with calculation of mean and standard error of mean. The Analysis of Variation (ANOVA) was done through R-stat version 4.2.2. Fisher-LSD test was used to compare means at 0.05 significance level. Table 1. Growth inhibition of S. sclerotiorum by the use of botanicals. Treatments 10% concentration 20% concentration Mean growth inhibition % Av. diameter (cm)* Inhibition %* Av. diameter(cm)* Inhibition %* A. indica 6.48 28 5.13 43 35.5 A. vulgaris 9 0 8.88 1.33 0.67 A. sativum 0 100 0 100 100 Z. officinale 6.98 22.44 6.03 33 27.72 L. camara 7.53 16.33 8.23 8.55 12.44 Control 9 --------- 9 --------- -------- LSD (0.05) 9.80 3.34 SEm 1.45 0.494975 CV % 19.47 5.95 Grand mean 33.38 37.22 Note: * Significant difference at 0.05 probability level; LSD: Least significant difference; SEm: Standard error of mean; CV: Coefficient of variation. Agriculture and Food Sciences Research, 2023, 10(2): 8-13 11 © 2023 by the authors; licensee Asian Online Journal Publishing Group 3. Result After 72 hours of inoculation, the control Petri plate was fully covered by the mycelia while both concentrations of A. sativum did not have any growth, keeping their inhibition at 100%. A. indica and Z. officinale inhibited the growth by 28% & 22.44% in 10% concentration and 43% & 33% in 20% concentration respectively. L. camara also showed its antifungal property by inhibiting the growth by 16.33% & 8.55% in 10% & 20 % concentrations respectively. But A. vulgaris only suppressed the growth by 1.33% in the 20% concentration while no effect was seen in the 10% concentration. There was little difference between the mean inhibition effect of the two concentrations. 20% concentration was only slightly better with 37.22% mean inhibition compared to 33.38% of 10% concentration. Overall, Allium sativum performed the best among all the botanicals, completely inhibiting the growth of Sclerotinia sclerotiorum, with A. indica, Z. officinale, and L. camara showing mild to low levels of inhibition against the pathogen with 35.5%, 27.72%, and 12.44% mean inhibition across the two concentrations respectively. The inhibition levels are graphically shown in Figure 3 and the petri plates after 72 hours of inoculation can be seen in Figure 4. Figure 3. Percentage of inhibition of S. sclerotiorum by the use of botanicals (72 hours). Figure 4. Mycelial growth after 72 hours in (A): A. indica, (B): A. vulgaris, (C): A. sativum, (D): Z. officinale, I: L. camara, (F): Control, where (i): 10% concentration and (ii): 20% concentration. 4. Discussion The world’s increasing population demands greater food production every year. This arises a challenge to protect the crops and reduce food loss, especially from pests and pathogens. Globally 30% of crop products are lost due to fungal plant pathogens, and the toxins produced by the fungal growth and changes in color, odor, and taste, degrades food on a large scale [22]. Fungicide use can decrease such losses of food, but in the wake of organic food Agriculture and Food Sciences Research, 2023, 10(2): 8-13 12 © 2023 by the authors; licensee Asian Online Journal Publishing Group in recent years, their usage has been limited [23]. Therefore, many attempts have been made in finding organic solutions to the fungal problem, one of which is the use of plant botanicals. The botanicals selected for the analysis have potential antifungal properties which have been shown by various studies throughout the years; Zingiber officinale [24] Azadirachta indica [25, 26] Artemisia vulgaris [27] Allium sativum [28] Lantana camara [29]. These plants have been used for our plant pathological needs and purposes through multiple generations spanning thousands of years [30]. In that course of time, their foundation, and the key constituents responsible for their beneficial uses have been documented. The compounds responsible for the antifungal nature of the botanicals are presented in Table 2. The concentration, amount, and quality of these phytochemicals depend vastly on the genotype, environmental condition, soil condition, plant parts harvested, harvesting season, and the stage at which they are harvested [31]. The plant parts which have been used for this analysis have all been collected in the locality of Rupandehi, Nepal, and so these parameters are likely to change along with the efficacy against Sclerotinia in comparison to native plants of any other region. Table 2. Chemical constituents of the botanicals used for the analysis. Plant Family Key Constituent References Artemisia vulgaris (Mugwort leaf) Asteraceae α-thujone, Ascaridole β-thujone 1,8-cineole Satyal, et al. [32]; Blagojević, et al. [33] and Cetin, et al. [34] Azadirachta indica (Neem leaves) Meliaceae Quercetin Azadirachtin Sithisarn and Gritsanapan [35] and Mahmoud, et al. [36] Zingiber officinale (Ginger rhizome) Zingiberaceae Gingerols Terpenes Park, et al. [37] and Yeh, et al. [38] Allium sativum (Garlic cloves) Amaryllidaceae Allicin Ajoenes Leontiev, et al. [39] and El-Saber, et al. [40] Lantana camara (Leaves) Verbenaceae Germacrene-D, E-caryophyllene Bicyclogermacrene, isocaryophyllene Passos, et al. [41] and Sousa, et al. [42] The most effective among all the botanicals used was found to be Allium sativum, followed by Azadirachta indica, Zingiber officinale, and Lantana camara. Similar results have been obtained in many experiments done worldwide [15, 17, 43]. A. vulgaris did not show any significant effect and failed to inhibit fungal growth. The antifungal property of Artemisia species, just like the other botanicals, is governed by its secondary metabolites. One of the secondary metabolites present in Artemisia is a compound Ascaridole [32, 44]. It is a thermolabile compound [45] so it decomposes when subject to extreme heat. This could be the reason for the negative inhibition shown by A. vulgaris as the compound could have been destroyed when it was being autoclaved for sterilization. 5. Conclusion The world is battling with food insecurity and the use of chemicals. While the short-term immediate benefits are favorable, continued chemical usage can be devastating for our food supply in the future and the use of bio- rational compounds is imperative for sustainable agriculture. So, it is important in identifying the best organic alternative to some of the major diseases of crops. These identified botanicals can be then used as a source to isolate the specific chemical constituent and develop a much more effective remedy for agricultural diseases. Such analysis can help identify hidden control measures of our locality and further aid in developing new medicines for diseases of plants. The obtained result of the antifungal nature of the botanicals used here, needs to be further confirmed in the field condition. Many other local botanicals can be examined for their efficacy against Sclerotinia sclerotiorum and efforts can be made to isolate the chemical compounds. Further experimentations with different extraction and application methods need to be conducted before ruling out any potential plant. References [1] M. D. Bolton, B. P. Thomma, and B. D. Nelson, "Sclerotinia sclerotiorum (Lib.) de Bary: Biology and molecular traits of a cosmopolitan pathogen," Molecular Plant Pathology, vol. 7, no. 1, pp. 1-16, 2006. https://doi.org/10.1111/j.1364- 3703.2005.00316.x [2] G. S. Saharan and N. Mehta, Sclerotinia diseases of crop plants: Biology, ecology and disease management. The Netherlands: Springer Science & Business Media, 2008. [3] G. Boland and R. Hall, "Index of plant hosts of Sclerotinia sclerotiorum," Canadian Journal of Plant Pathology, vol. 16, no. 2, pp. 93- 108, 1994. https://doi.org/10.1080/07060669409500766 [4] C. A. O’Sullivan, K. Belt, and L. F. Thatcher, "Tackling control of a cosmopolitan phytopathogen: Sclerotinia," Frontiers in Plant Science, vol. 12, p. 707509, 2021. https://doi.org/10.3389/fpls.2021.707509 [5] C. Young, J. P. Clarkson, J. Smith, M. Watling, K. Phelps, and J. Whipps, "Environmental conditions influencing Sclerotinia sclerotiorum infection and disease development in lettuce," Plant Pathology, vol. 53, no. 4, pp. 387-397, 2004. [6] E. C. Oerke, "Crop losses to pests," The Journal of Agricultural Science, vol. 144, no. 1, pp. 31-43, 2006. https://doi.org/10.1017/S0021859605005708 [7] P. H. Schwartz and W. Klassen, "Pests," The Antioch Review, vol. 38, no. 4, pp. 493-515, 1980. [8] D. Pimentel and R. Peshin, Integrated pest management: Pesticide problems (no. 15300). Netherlands: Springer Science & Business Media, 2014. [9] G. M. Lengai, J. W. Muthomi, and E. R. Mbega, "Phytochemical activity and role of botanical pesticides in pest management for sustainable agricultural crop production," Scientific African, vol. 7, p. e00239, 2020. https://doi.org/10.1016/j.sciaf.2019.e00239 [10] I. Mahmood, S. R. Imadi, K. Shazadi, A. Gul, and K. R. Hakeem, Effects of pesticides on environment. In: Hakeem KR, Akhtar MS, Abdullah SNA, eds. Plant, soil and microbes. Switzerland: Springer, 2016, pp. 253–269. [11] L. Chengala and N. Singh, "Botanical pesticides—A major alternative to chemical pesticides: A review," International Journal Life Sciences, vol. 5, no. 4, pp. 722-729, 2017. [12] D. Mueller et al., "Efficacy of fungicides on Sclerotinia sclerotiorum and their potential for control of Sclerotinia stem rot on soybean," Plant Disease, vol. 86, no. 1, pp. 26-31, 2002. https://doi.org/10.1094/PDIS.2002.86.1.26 [13] C. Bradley et al., "Efficacy of fungicides for control of Sclerotinia stem rot of canola," Plant Disease, vol. 90, no. 9, pp. 1129-1134, 2006. https://doi.org/10.1094/pd-90-1129 https://doi.org/10.1111/j.1364-3703.2005.00316.x https://doi.org/10.1111/j.1364-3703.2005.00316.x https://doi.org/10.1080/07060669409500766 https://doi.org/10.3389/fpls.2021.707509 https://doi.org/10.1017/S0021859605005708 https://doi.org/10.1016/j.sciaf.2019.e00239 https://doi.org/10.1094/PDIS.2002.86.1.26 https://doi.org/10.1094/pd-90-1129 Agriculture and Food Sciences Research, 2023, 10(2): 8-13 13 © 2023 by the authors; licensee Asian Online Journal Publishing Group [14] W. Riah, K. Laval, E. Laroche-Ajzenberg, C. Mougin, X. Latour, and I. Trinsoutrot-Gattin, "Effects of pesticides on soil enzymes: A review," Environmental Chemistry Letters, vol. 12, pp. 257-273, 2014. https://doi.org/10.1007/s10311-014-0458-2 [15] P. Upadhyay and A. Tewari, "Evaluation of botanical extracts, animal wastes, organic and inorganic salts, micronutrients and bio- agents against Sclerotinia sclerotiorum (Lib) de Bary a cause of Sclerotinia rot of rapeseed-mustard under field conditions," Bulletin of Environment, Pharmacology and Life Sciences, vol. 8, pp. 60-65, 2019. [16] M. Pandey et al., "An overview on possible management strategies for coffee white stem borer Xylotrechus quadripes Chevrolat (Coleoptera: Cerambycidae) in Nepal," Heliyon, vol. 8, no. 9, p. e10445, 2022. https://doi.org/10.1016/j.heliyon.2022.e10445 [17] R. D. Timila and S. Manandhar, "In vitro bio-assay of some botanicals against some important soil-borne fungi," Journal of the Plant Protection Society, vol. 5, pp. 87-95, 2018. [18] M. Balouiri, M. Sadiki, and S. K. Ibnsouda, "Methods for in vitro evaluating antimicrobial activity: A review," Journal of Pharmaceutical Analysis, vol. 6, no. 2, pp. 71-79, 2016. https://doi.org/10.1016/j.jpha.2015.11.005 [19] G. Al-Samarrai, H. Singh, and M. Syarhabil, "Evaluating eco-friendly botanicals (natural plant extracts) as alternatives to synthetic fungicides," Annals of Agricultural and Environmental Medicine, vol. 19, no. 4, pp. 673-676, 2012. [20] T. Bekker, C. Kaiser, R. vd Merwe, and N. Labuschagne, "In-vitro inhibition of mycelial growth of several phytopathogenic fungi by soluble potassium silicate," South African Journal of Plant and Soil, vol. 23, no. 3, pp. 169-172, 2006. https://doi.org/10.1080/02571862.2006.10634750 [21] S. Javed, S. Mushtaq, I. Khokhar, R. Ahmad, and M. S. Haider, "Comparative antimicrobial activity of clove and fennel essential oils against food borne pathogenic fungi and food spoilage bacteria," African Journal of Biotechnology, vol. 11, no. 94, pp. 16065-16070, 2012. https://doi.org/10.5897/ajb11.3058 [22] S. V. Avery, I. Singleton, N. Magan, and G. H. Goldman, "The fungal threat to global food security," Fungal Biology, vol. 123, no. 8, pp. 555-557, 2019. https://doi.org/10.1016/j.funbio.2019.03.006 [23] R. Hansmann, I. Baur, and C. R. Binder, "Increasing organic food consumption: An integrating model of drivers and barriers," Journal of Cleaner Production, vol. 275, p. 123058, 2020. https://doi.org/10.1016/j.jclepro.2020.123058 [24] K. K. Mishra et al., Medicinal plants having antifungal properties. In Medicinal Plants-Use in Prevention and Treatment of Diseases. IntechOpen. https://doi.org/10.5772/intechopen.90674, 2020. [25] T. Govindachari, G. Suresh, G. Gopalakrishnan, B. Banumathy, and S. Masilamani, "Identification of antifungal compounds from the seed oil of Azadirachta indica," Phytoparasitica, vol. 26, no. 2, pp. 109-116, 1998. https://doi.org/10.1007/bf02980677 [26] U. Singh, H. Singh, and R. Singh, "The fungicidal effect of neem (Azadirachta indica) extracts on some soil-borne pathogens of gram (Cicer arietinum)," Mycologia, vol. 72, no. 6, pp. 1077-1093, 1980. https://doi.org/10.2307/3759561 [27] H. Ekiert, J. Pajor, P. Klin, A. Rzepiela, H. Ślesak, and A. Szopa, "Significance of Artemisia vulgaris L.(Common Mugwort) in the history of medicine and its possible contemporary applications substantiated by phytochemical and pharmacological studies," Molecules, vol. 25, no. 19, p. 4415, 2020. https://doi.org/10.3390/molecules25194415 [28] S. Hayat, Z. Cheng, H. Ahmad, M. Ali, X. Chen, and M. Wang, "Garlic, from remedy to stimulant: Evaluation of antifungal potential reveals diversity in phytoalexin allicin content among garlic cultivars; allicin containing aqueous garlic extracts trigger antioxidants in cucumber," Frontiers in Plant Science, vol. 7, p. 1235, 2016. https://doi.org/10.3389/fpls.2016.01235 [29] M. Fayaz, M. Bhat, M. Fayaz, A. Kumar, and A. Jain, "Antifungal activity of Lantana camara L. leaf extracts in different solvents against some pathogenic fungal strains," Pharmacologia, vol. 8, no. 3, pp. 105-112, 2017. https://doi.org/10.5567/pharmacologia.2017.105.112 [30] K. G. Ramawat, S. Dass, and M. Mathur, Herbal drugs: Ethnomedicine to modern medicine. Berlin, Heidelberg: Springer, 2009. [31] M. J. Abad, L. M. Bedoya, L. Apaza, and P. Bermejo, "The Artemisia L. genus: A review of bioactive essential oils," Molecules, vol. 17, no. 3, pp. 2542-2566, 2012. https://doi.org/10.3390/molecules17032542 [32] P. Satyal et al., "Bioactivities of volatile components from Nepalese Artemisia species," Natural Product Communications, vol. 7, no. 12, pp. 1651–1658, 2012. https://doi.org/10.1177/1934578x1200701228 [33] P. Blagojević, N. Radulović, R. Palić, and G. Stojanović, "Chemical composition of the essential oils of Serbian wild-growing Artemisia absinthium and Artemisia vulgaris," Journal of Agricultural and Food Chemistry, vol. 54, no. 13, pp. 4780-4789, 2006. https://doi.org/10.1021/jf060123o [34] B. Cetin et al., "Chemical composition of hydrodistilled essential oil of Artemisia incana (L.) Druce and antimicrobial activity against foodborne microorganisms," Chemistry & Biodiversity, vol. 6, no. 12, pp. 2302-2310, 2009. https://doi.org/10.1002/cbdv.200800317 [35] P. Sithisarn and W. Gritsanapan, "Variability of antioxidative quercetin content in Siamese neem tree leaves in Thailand by TLC- densitometry," Acta Horticulturae, vol. 786, pp. 161–168, 2008. https://doi.org/10.17660/actahortic.2008.786.17 [36] D. Mahmoud, N. Hassanein, K. Youssef, and M. Abou Zeid, "Das bau-SOLL im tunnelbau wo liegen die missverständnisse wo liegen die größten irrtümer," Brazilian Journal of Microbiology: Publication of the Brazilian Society for Microbiology, vol. 42, no. 3, pp. 1007-1016, 2011. [37] M. Park, J. Bae, and D. S. Lee, "Antibacterial activity of [10]‐gingerol and [12]‐gingerol isolated from ginger rhizome against periodontal bacteria," Phytotherapy Research: An International Journal Devoted to Pharmacological and Toxicological Evaluation of Natural Product Derivatives, vol. 22, no. 11, pp. 1446-1449, 2008. https://doi.org/10.1002/ptr.2473 [38] H.-Y. Yeh, C.-H. Chuang, H.-C. Chen, C.-J. Wan, T.-l. Chen, and L.-Y. Lin, "Bioactive components analysis of two various gingers (Zingiber officinale Roscoe) and antioxidant effect of ginger extracts," LWT-Food Science and Technology, vol. 55, no. 1, pp. 329- 334, 2014. https://doi.org/10.1016/j.lwt.2013.08.003 [39] R. Leontiev, N. Hohaus, C. Jacob, M. C. Gruhlke, and A. J. Slusarenko, "A comparison of the antibacterial and antifungal activities of thiosulfinate analogues of allicin," Scientific Reports, vol. 8, no. 1, p. 6763, 2018. https://doi.org/10.1038/s41598-018-25154-9 [40] B. G. El-Saber et al., "Chemical constituents and pharmacological activities of garlic (Allium sativum L.): A review," Nutrients, vol. 12, no. 3, p. 872, 2020. https://doi.org/10.3390/nu12030872 [41] J. L. Passos, L. C. A. Barbosa, A. J. Demuner, E. S. Alvarenga, C. M. Da Silva, and R. W. Barreto, "Chemical characterization of volatile compounds of Lantana camara L. and L. radula Sw. and their antifungal activity," Molecules, vol. 17, no. 10, pp. 11447- 11455, 2012. https://doi.org/10.3390/molecules171011447 [42] E. O. Sousa, F. S. Barreto, F. F. Rodrigues, A. R. Campos, and J. G. Costa, "Chemical composition of the essential oils of Lantana camara L. and Lantana montevidensis Briq. and their synergistic antibiotic effects on aminoglycosides," Journal of Essential Oil Research, vol. 24, no. 5, pp. 447-452, 2012. https://doi.org/10.1080/10412905.2012.703494 [43] P. Fatehpuria, R. Sasode, D. Chobe, G. Veerendra, and S. Reeti, "Studies on field evaluation of Brassica germplasm/varieties against Sclerotinia sclerotiorum under inoculated condition," Flora and Fauna (Jhansi), vol. 23, no. 1, pp. 87-89, 2017. https://doi.org/10.33451/florafauna.v23i1pp87-89 [44] V. M. Dembitsky, "Bioactive peroxides as potential therapeutic agents," European Journal of Medicinal Chemistry, vol. 43, no. 2, pp. 223-251, 2008. https://doi.org/10.1016/j.ejmech.2007.04.019 [45] J. F. Cavalli, F. Tomi, A. F. Bernardini, and J. Casanova, "Combined analysis of the essential oil of Chenopodium ambrosioides by GC, GC‐MS and 13C‐NMR spectroscopy: Quantitative determination of ascaridole, a heat‐sensitive compound," Phytochemical Analysis: An International Journal of Plant Chemical and Biochemical Techniques, vol. 15, no. 5, pp. 275-279, 2004. https://doi.org/10.1002/pca.761 Asian Online Journal Publishing Group is not responsible or answerable for any loss, damage or liability, etc. caused in relation to/arising out of the use of the content. Any queries should be directed to the corresponding author of the article. https://doi.org/10.1007/s10311-014-0458-2 https://doi.org/10.1016/j.heliyon.2022.e10445 https://doi.org/10.1016/j.jpha.2015.11.005 https://doi.org/10.1080/02571862.2006.10634750 https://doi.org/10.5897/ajb11.3058 https://doi.org/10.1016/j.funbio.2019.03.006 https://doi.org/10.1016/j.jclepro.2020.123058 https://doi.org/10.5772/intechopen.90674 https://doi.org/10.1007/bf02980677 https://doi.org/10.2307/3759561 https://doi.org/10.3390/molecules25194415 https://doi.org/10.3389/fpls.2016.01235 https://doi.org/10.5567/pharmacologia.2017.105.112 https://doi.org/10.3390/molecules17032542 https://doi.org/10.1177/1934578x1200701228 https://doi.org/10.1021/jf060123o https://doi.org/10.1002/cbdv.200800317 https://doi.org/10.17660/actahortic.2008.786.17 https://doi.org/10.1002/ptr.2473 https://doi.org/10.1016/j.lwt.2013.08.003 https://doi.org/10.1038/s41598-018-25154-9 https://doi.org/10.3390/nu12030872 https://doi.org/10.3390/molecules171011447 https://doi.org/10.1080/10412905.2012.703494 https://doi.org/10.33451/florafauna.v23i1pp87-89 https://doi.org/10.1016/j.ejmech.2007.04.019 https://doi.org/10.1002/pca.761