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© 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


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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. 

 
 



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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 

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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 



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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.  
 

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