1

 
 

In vitro Antifungal Activity of Ethanolic Leaf Extracts of Artemisia vulgaris

(Damong Maria) against Fusarium oxysporum Causing
Fusarium Wilt Disease on Banana (Musa spp.)

Department of Education
Region IV-A CALABARZON
Division of Cavite
Trece Martires City National High School

An Entry to the Division Science and Technology Fair

(Science Investigatory Project)

Irish Kyle Angeles

Hannz Christian Fiel
Jedu Emmanuel V. Panit
Proponents

April B. Flores
Research Adviser

 
 
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Ii
 

Table of Contents

Contents Page

ACKNOWLEDGEMENT iii

ABSTRACT iv

LIST OF FIGURES v

LIST OF TABLES vi

LIST OF PLATES vii

THE PROBLEM AND ITS BACKGROUND 1

A.   Introduction 1

B.   Statement of the Problem 6

C.   Objectives of the Study 7

D.   Significance of the Study 8

E.   Scope and Limitations of the Study 9

F.   Definition of Terms 10

REVIEW OF RELATED LITERATURE 11

METHODOLOGY 23

A.   Collection of Artemisia vulgaris 23

B.   Preparation of the Pure and Ethanolic Extracts 23

 
 
     ii

C.   Measuring the Zones of Inhibition of the Ethanolic Extracts Using 24

Cup-Cylinder method

D.   Statistical Analysis 24

RESULTS AND DISCUSSION 25

SUMMARY, CONCLUSION AND RECOMMENDATION 34

Summary 34

Conclusion 35

Recommendation 35

BIBLIOGRAPHY 36

APPENDICES 47

A.   Report of Antifungal analysis 48

 
 
     iii

ACKNOWLEDGEMENT

The researchers would like to extend sincere gratitude and appreciation to all the

people and institutes behind the accomplishment of this work.

To all the researchers’ families, especially parents for their understanding, moral

support , encouragement and financial assistance amidst the pandemic;

To Mrs. April Flores, their research adviser, for her undying support, guidance,

insightful suggestions, criticisms and comments in writing the research paper for the

success of this research project;

To Mrs. Irene A. Papa, the university researcher of Biotech Laboratory at the

University of the Philippines Los Baños, for assisting and helping the researchers in the

conduct of this study;

Above all, the researchers want to offer this endeavor to God, the author of

knowledge and wisdom, for his blessing and opportunities throughout the process. For

giving knowledge, strength, persistence, safety and guidance until the finalization of the

study. For He has always brought us up and for an endless encouragement to accomplish

this work.

Irish Kyle Angeles

Hannz Christian Fiel

Jedu Emmanuel Panit

 
 
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ABSTRACT

Fusarium oxysporum, a soil-borne fungus is the causative agent of the global

plant disease Fusarium wilt, which has been severely infesting the banana industry in the

country for almost two decades. Small and medium farmers have been struggling to

mitigate the spread of this devastating disease through several control methods including

the use of chemical fungicides but unfortunately, some strains have already developed

resistance. This study evaluated the in vitro antifungal effect of the ethanolic leaf extracts

of Artemisia vulgaris on Fusarium oxysporum. The pure and ethanol leaf extracts (25%,

50% and 75% concentrations), obtained through maceration, were tested against the

pathogen using the cup cylinder method. The diameter of the zone of inhibition (ZOI)

were measured and compared to the commercial antifungal. All the four concentrations

of the plant material exhibited strong to moderate antifungal activity against the test

organism. The maximum efficacy is at 75% concentration, with a mean ZOI of 16.5mm,

followed by the 50%, 25% and 100% concentration with mean diameter ZOI of 13.0mm,

8.9mm and 7.8mm respectively. Result of t-test between the ZOI of 75% ethanol leaf

extracts and the commercial antifungal showed significant difference in their antifungal

effect, with the 75% concentration surpassing the commercial drug (p-value=0.00009). T-

test between the ZOI of 50% ethanolic leaf extracts and the commercial antifungal

generated similar favorable results (p-value= 0.006) for the 50% concentration. Findings

of this study reveals how Artemisia vulgaris can be a potent fungicidal agent to help the

banana growers in the country control the population of the plant pathogen.

 
 
     v
 

LIST OF FIGURES

Figures Page

Figure 1 Zones of Inhibition (ZOI) Measurement of 26

Replicates in each Treatment

 
 
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LIST OF TABLES

Tables Page
TABLE 1 Mean diameter of ZOI of ethanolic leaf extracts of 26

Artemisia vulgaris on Fusarium oxysporum

TABLE 2.1 Results of One-way ANOVA on the ZOI of treatments 27

TABLE 2.2 Summary of descriptive statistics for Table 2.1 28  

TABLE 3.1 T-test between T3 and Positive Control 28

TABLE 3.2 T-test between T1 and Positive Control 29

TABLE 3.3 T-test between T1 and Negative Control 30

TABLE 3.4 T-test between T2 and Positive Control 31

TABLE 3.5 T-test between T4 and Positive Control 32

TABLE 3.6 T-test between T1 and T4 33

 
 
     vii

LIST OF PLATES

Photographs Page

Plate 1 Purchase of Artemisia vulgaris (Damong Maria) 51

plant sample, first batch

Plate 2 Dried Artemisia vulgaris (Damong Maria) 52

plant sample (first batch)

Plate 3 Separating leaves and stem of Artemisia vulgaris plant 53

sample and cutting of dried leaves

Plate 4 Purchase of fresh plant sample, second batch 54

Plate 5 Fusarium oxysporum grown on Potato Dextrose Agar (PDA) 55

Plate 6 Materials used for cup cylinder method 56

Plate 7 Assay Plate 57

 
 
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Chapter I

THE PROBLEM AND ITS BACKGROUND

Introduction

In the Philippines, there is a high risk and a major concern of mycotoxin

contamination, due to the tropical, warm and humid conditions that are favorable to the

growth of pathogenic fungi. These pathogenic fungi contain mycotoxins that cause

diseases and fungal infections to plants which results in a heavy loss in production of

agricultural crops in the Philippines and significanly contribute to the decline in the

quality and quantity of agricultural commodities (Balendres, Karlovsky and Cumagun

2019).

It is reported that seven mycotoxigenic Aspergillus species, four Fusarium

species, and one Penicillium species have been isolated from various agricultural crop

commodities in the country. Despite the increase in the understanding of mycotoxigenic

fungi and mycotoxins in the country, very limited knowledge exists on practices and

measures that control both the fungi and the toxins, thus, putting a risk in the context of

food security and safety (Pangga, Salvacion, and Cumagun 2015).

Fusarium oxysporum is a plant pathogen that causes serious damages in

agricultural crops, both in the field and during postharvest storage (Thrane 2014, 76 –

81). It can survive in the air for a long period of time causing financial problems to the

farmers and an increase in agricultural cost in markets. The pathogen is ranked 5 in a list
th

 
 
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of top 10 fungal plant pathogens based on Scientific and Economic Importance (Kang,

Demers, Jimenez and Rep 2012). It can spread within plantations, wheels of farm

machineries and shoes of farm workers and once farm soil is contaminated, susceptible

cultivars can only be grown with great difficulty and with much crop loss (Centre for

Agriculture and Bioscience International 2019).

On top of that, it is well-known for causing a disease called Fusarium wilt which

is fatal to plants. This devastating disease has been a major threat in banana production

for more than a century. According to the Planet Natural Research Center in 2020, the

disease starts when Fusarium oxysporum infects the soil and attacks the root of the plant.

As the infection spreads up into the stems and leaves, it restricts water flow and blocks

nutrients causing the stems to eventually turn black and leaves to turn yellow and wilt. As

the disease progresses, the younger leaves will also be affected, and the plant eventually

dies. The disease is difficult to get rid of, and eventually results in loss of rotation of

crops.

According to the Food and Agriculture Organization (FAO) of the United Nations

in 2014, bananas are the 5th most important food crop in the world and the 4th most

important food crop among the world’s least-developed countries. It is a vital source of

income, employment and export revenues for exporting countries, which are mostly

developing countries in Latin America, Southeast Asia and Africa. The Philippines is

ranked 5th among the top producing countries of bananas. It is one of the country’s top

export crops, thus considered the most important fruit crop in the country in terms of

 
 
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volume of production and export earnings. It is projected that favorable areas for

Fusarium wilt in the Philippines will increase from 21 percent under baseline climate

conditions to 27 percent, covering 91.2 percent and 28.5 percent of the highly and

moderately suitable banana areas of the country, respectively.

The FAO of the United Nations is warning the countries to step up in monitoring,

reporting and prevention of Fusarium wilt, as one of the world’s most destructive banana

diseases which recently spread throughout the world (Cumagun, de la Cueva and Aguilar

2019). In the Philippines, according to plantation owners in Mindanao, the dreaded

Fusarium wilt, locally known as Panama disease, may have already ravaged more than a

quarter of all the banana plantations in the region. Large areas of banana farms have

already been abandoned particularly in Davao City, due to the spread of Fusarium wilt

(Mindanao Banana Farmers Exporters Association 2020).

In 2016, as reported by Visperas in The Philippine Star, a total of 15,500 hectares

of banana plantations have been affected by the deadly Fusarium wilt disease in Southern

Mindanao, the region where most of the country’s export-quality bananas are cultivated.

This banana wilt is posing a serious threat to food security and economic welfare.

According to Virgilio Gutierrez, focal person of the Department of Agriculture Davao

Region, there are at least 700 farmers who have been affected by the Fusarium wilt

disease. They placed appropriate measures to help minimize the spread of the disease by

just paying those who burn the infected plants.

 
 
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Fusarium oxysporum can also be harmful to humans, since it has strains that are

pathogenic to the human body. Its mycotoxins cause the diseases fungal keratitis,

onychomycosis, and hyalohyphomycosis. All of these cases pertain to injuries such as

burn wounds, cell death due to blood deprivation or lack in oxygen of the brain, extensive

contamination, and ischemic skin graft (Annaissie and Nucci 2007).

The spread of multidrug-resistant strains of fungus and the reduced number of

fungicides available make it necessary to discover new classes of antifungals from natural

products including medicinal plants (Koushlesh et al. 2019), as for the synthetic

fungicides impact negatively on food and plant safety and damage the quality of

agricultural crop commodities. In the Philippines, Fusarium species have been detected

and were found to contaminate major crop commodities (Karlovsky, 2019). Chemical

fungicides are used to control diseases caused by Fusarium oxysporum such as Fusarium

wilt or Panama disease. Unfortunately, few fungi have developed resistance to them, and

the use of natural products like plant extracts for the management of fungal diseases in

plants is the substitute to those synthetic fungicides due to their fewer negative impacts or

side effects and implications on humans and environment.

Upon reviewing the literature on recommended medicinal herbs as a source of

natural products, it can be concluded that Artemisia vulgaris, which is widely cultivated

in the Philippines, exhibit a lot of useful bioactivities. It is used to treat stomach problems

and a powerful remedy for intestinal worms (Dela Cruz, Bacani, Dayrit and Paglinawan

2017). It has many records and uses in traditional Philippine medicine including

 
 
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treatment for painful menstruation, late menstruation, trouble urinating, diarrhea, etc.

(Philippine Institute of Traditional and Alternative Health Care 2017). Moreover, the

plant extracts also contain different medicinal activities which are proven to be used as

insecticide, herbicides spray and fungicides which can impact pathogenic fungi found in

the soil and within the plant foliage. The plant also produces numerous chemicals widely

used in the application of anti-bacterial, antifungal, and insecticidal agents (Rusconi and

Hooks 2019).

The study of Devi and Chettry (2013) investigated the in vitro antifungal activity

of aqueous extracts from locally available plants, including Artemisia vulgaris, that are

known for their medicinal benefits against Drechslera oryzae, an organism of brown leaf

spot of rice. The plant extracts were tested against the organism at different

concentrations using poisoned food technique. Results revealed that Artemisia vulgaris

was second to the most effective fungicides to control the disease at 20% concentration.

The fungicidal activity of the locally selected plants used was attributed to the presence

of antifungal compounds (Devi and Chhetry 2013, vol. 3, 1-3). Similarly, anti-pathogenic

effects of the ethanol and chloroform extracts of the leaves of Artemisia vulgaris on

Aspergillus niger and Candida albicans species were also reported in the recent study of

Lalitha et al. (2020).

Previous studies provide sufficient evidence to support the antipathogenic effects

of Artemisia vulgaris. To the best of researchers’ knowledge, no work had been

published on its antifungal activity against Fusarium oxysporum, the causative agent of

 
 
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the lethal fungal disease of banana, the Fusarium wilt. With the current battle of the

banana growers in the Philippines against the Fusarium wilt disease, this study is timely

and relevant as it provides a sustainable solution to reducing the Fusarium oxysporum

population in banana plantations. This study investigated the in vitro antifungal activity

of ethanolic leaf extracts of Artemisia vulgaris against the pathogen. Results of this study

can be used to develop effective fungicides at low cost and minimize the negative

impacts of using synthetic fungicides to humans and environment. This will also help

improve crop yields, generating more income for the banana growers and contributing to

the growth of the Philippine economy in general.

Statement of the Problem

Fusarium wilt has been infecting banana plantations, especially in the region of

Mindanao, where most of the Philippines’ export quality bananas are cultivated. The

country is affected by this as bananas are one of the main export crops in the country, and

the infestation is a threat to food security. This disease, caused by the fungus Fusarium

oxysporum, may also induce human diseases such as fungal keratitis, onychomycosis and

hyalohyphomycosis. While farmers use chemical fungicides, some strains of fungi have

become resistant, thus reducing the number of fungicides that are effective and

accessible. With this scenario, this study aims to create a natural, affordable, sustainable

and environment-friendly fungicide from Artemisia vulgaris leaf extract to be the

substitute for synthetic fungicides to lessen their negative impacts to humans and the

environment.

 
 
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Objectives of the Study

This study determined the fungicidal activity of Artemisia vulgaris ethanolic leaf

extract against Fusarium oxysporum.

Specifically, this study aimed to:

1.   evaluate the fungicidal activity of each concentration of Artemisia vulgaris

ethanolic (25%, 50% and 75%) and pure leaf extract against Fusarium oxysporum

based on the mean diameter of zone of inhibition (ZOI);

2.   determine if there will be significant difference among the fungicidal activity of

ethanolic (25%, 50% and 75%) and pure leaf extracts of Artemisia vulgaris and

the control treatments against Fusarium oxysporum in terms of the mean diameter

of zone of inhibition (ZOI);

3.   determine if there will be significant difference between the fungicidal activity of

each ethanolic (25%, 50%, 75%) and pure leaf extracts of Artemisia vulgaris and

the commercial antifungal against Fusarium oxysporum based on the mean

diameter of zone of inhibition (ZOI);

4.   determine the most effective concentration (25%, 50%, 75% and 100%) of

Artemisia vulgaris leaf extract against Fusarium oxysporum based on the mean

diameter of zone of inhibition (ZOI)

 
 
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Significance of the Study

Studies indicate that Fusarium oxysporum causes severe damage to the plant,

particularly in the banana plant. For more than a century, the devastating disease called

Fusarium wilt has been a major threat to banana production (Smith, Smedley and Lennon

2011). It is recognized as among the most destructive diseases of the banana worldwide

causing mass die-offs. Banana growers in the Philippines have been quietly battling a

plague against this disease as it continues to worsen. In addition, Fusarium oxysporum

with its mycotoxins can also be harmful to humans as they cause burn wounds, cell death

due to blood deprivation or lack in oxygen of the brain, extensive contamination, and

ischemic skin graft (Annaissie and Nucci 2007).

This study aimed to determine the fungicidal activity of the ethanolic leaf extract

of Artemisia vulgaris against Fusarium oxysporum. As the disease continues to spread

and grow in different areas worldwide, banana production will invariably be hampered by

viral and pest infestation, thus leading to soil fertility decrease, low economic welfare and

putting food security at risk.

The results of the study will also help in developing an eco-friendly way of

eliminating the pathogen by producing an alternative to chemical fungicides. Moreover,

the results of the study can help in the development of a fungicide from the plant extract

protecting human as consumers from the threat of synthetic pesticide. Above all, this will

improve the livelihood of banana growers by improving their crop yields at low cost.

 
 
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Scope and Limitation of the Study

The study primarily focused on determining the fungicidal activity of Artemisia

vulgaris leaf extract against Fusarium oxysporum. Artemisia vulgaris were collected

from General Trias, Cavite and the ethanolic leaf extracts (25%, 50% and 75%) were

obtained through maceration using 95% ethanol as solvent. All samples were transported

to the Biotech Laboratory of the University of the Philippines Los Baños (UPLB) where

the antifungal assay was conducted using Fusarium oxysporum as the test organism. The

cup-cylinder method was performed to test the antifungal activity of the samples. The

zones of inhibition (ZOI) of the ethanolic and pure leaf extracts were measured and

compared to the commercial antifungal. The study was conducted from November to

December 2020.

 
 
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Definition of Terms

The following terms are defined based on context or how they were used in the

study:

Artemisia vulgaris. Artemisia vulgaris, locally known as Damong Maria, is the

plant material used in this study to test the fungicidal activity of its leaf extract against the

fungus Fusarium oxysporum.

Ethanolic Leaf Extracts. This refers to the leaf extracts of Artemisia vulgaris

where 95% ethanol was used as solvent for extraction. The ethanolic leaf extracts were

obtained through maceration at 25%, 50% and 75% concentrations.

Fungicidal Activity. Fungicidal activity refers to the ability of the Artemisia

vulgaris ethanolic leaf extract to inhibit the growth of Fusarium oxysporum. The

fungicidal activity of the leaf extract was evaluated using cup cylinder method where the

zones of inhibition of the ethanolic extracts at different concentrations on the pathogen

were measured.

Fusarium oxysporum. This is the fungus where the ethanolic leaf extracts of

Artemisia vulgaris was tested. This pathogen is responsible for the Fusarium wilt or

Panama disease in bananas. A 7-day old culture of Fusarium oxysporum (Foc) used in the

study was provided by the Biotech Laboratory of the University of the Philippines Los

Banos (UPLB).

 
 
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Chapter II

REVIEW OF RELATED LITERATURE

This chapter presents the information collected from different reliable sources

about the problem and the variables of the study, specifically the target pest, its impacts

on agriculture, and the plant material tested against the pest. Findings of previous studies

about the antipathogenic effects of Artemisia vulgaris are summarized and presented here

as they were used as bases and foundation of this current study.

Banana Cultivation and Production in the Philippines

Banana (Musa spp.) is one of the world’s most important fruit crops that is widely

cultivated in tropical and subtropical countries for its valuable applications in the food

industry and economy. Its enormous by-products are an excellent source of highly

valuable raw materials for other industries by recycling agricultural waste. This prevents

an ultimate loss of a huge amount of untapped biomass and environmental issues (Zhang

et al. 2005).

In the Philippines, primarily in Mindanao, bananas are among the most important

fruit crops in terms of both production volume and export earnings. Almost six million

farm households depend on banana crops for their livelihood because approximately 75

percent of bananas that are being produced in the Philippines originate from Mindanao

(Havemann and Rosenthal 2015).

 
 
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Davao Region has the highest banana production with 36.9 percent of total

production followed by Northern Mindanao and South-Central Mindanao with 22.5

percent and 12.2 percent, respectively. The production of banana from July to September

2020 is approximately 2.36 million metric tons and it dropped by -1.3 percent from its

2019 same period level of 2.39 million metric tons (Philippine Statistics Authority, 2013)

Fusarium Wilt Disease and its Impact on Banana Production in the Philippines

The Philippines is one of the top five countries producing bananas in the world,

with the majority of its production concentrated in the Davao region. Due to pests and

diseases as well as nutritional issues, banana production, particularly Cavendish, is

declining. The crop is a heavy feeder and for its optimum development, requires a lot of

nutrients. However, the productivity of the crop is declining, despite daily fertilization

(Salibay and Salomez 2009).

In four banana-growing areas in SOCSARGEN (South Cotabato, Sarangani

Province, General Santos City) Mindanao, on-site insect pest surveys and interviews with

banana farmers were conducted to determine the incidence of insect pests, as well as the

views and perceptions of farmers regarding integrated pest management as a pest control

system. Results of the survey showed that banana production is severely hampered by

viral and pest infestation, soil fertility decreasing, wilt of Fusarium oxysporum, and other

problems. While in their farms, all farmers use a wide range of methods, a significant

 
 
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number believe that these are insufficient to bring down populations of pests. The use of

synthetic inputs as a viable farming alternative is seriously considered by a few (Aguilar,

Lasalita-Zapico, Namocatcat, Fortich and Bojadores 2014).

Fusarium oxysporum is ranked fifth in a list of top 10 fungal plant pathogens

based on scientific and economic importance (Kang, Demers, Jimenez and Rep 2012). It

can be dispersed from area to area and from country to country and is also considered as

invasive. It can also spread within plantations, wheels of farm machinery, shoes of farm

workers and once farm soil is contaminated, susceptible cultivars can only be grown with

great difficulty and with much crop loss (CABI 2019).

The plant pathogen has many different formae species which are each selectively

pathogenic on a limited number of crops. Even within one crop, different formae

speciales may occur and cause different symptoms. Fusarium oxysporum is the

only Fusarium that actually grows inside the vessel system of the host plant and spreads

upwards inside the plants. This fungus causes vascular wilt, root rot, stem rot, leaf

lesions, fruit rot and post-harvest decay. Fusarium oxysporum will make the whole plant

turn yellow and wilts (Koppert, 2021).

Fusarium oxysporum is the causal agent of the disease Fusarium wilt which is a

major threat not only to Cavendish banana production, but to more than a hundred

species of plants including important banana crops (Berrocal-Lobo and Molina 2007).

The spread of the pathogen can be controlled by cleaning seeds and removing infected

 
 
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plant tissues from the area, but crop rotation is somehow ineffective due to its longevity

(Britannica 2017).

According to the study of Planet Natural Research Center 2018, Fusarium wilt or

the so-called Panama disease, develops during hot weather and low soil moisture

encourages this plant disease. This fungal disease can survive for years in soil and can

spread easily by water, insects and garden equipment. Moreover, Plant Health Australia

(2021) stated that Fusarium oxysporum can wilt over a short period of time. Root to root

contact, water movement, contaminated tools and soil contaminants can be the cause of

how the disease can rapidly spread. Once the fungus is established, it persists in the soil

for many years.

Fusarium wilt might have already damaged numerous plantations in Mindanao, as

claimed by the owners in the region. In 2015, plant pathologists from the Department of

Agriculture (DA) identified that Fusarium wilt infected 30,000 hectares and the area that

is infected can no longer be planted with banana and other soft- stemmed crops.

According to the farmers, rapid spread of the disease is expected due to the lack of

appropriate biosafety measures in farms (Cayon, 2020).

Studies of Venkataramana et al. 2014, Divakara et al. 2014, Kalagatur et al. 2015

and Kumar et al. 2016 reported that Fusarium toxins are the most abundant natural

contaminants of diets containing cereals and other grains and suspected to be implicated

in numerous diseases among mammals and other living beings. The fumonisins, which

belong to the family of food-borne carcinogenic mycotoxins, have reports of toxic

 
 
     15

activity of Fusarium oxysporum strains isolated from various products and exhibited

different degrees of toxicity to experimental animals (Venkataramana et al., 2012).

The study of Salvacion et al. (2019) examined the suitability of bananas and the

possible distribution of Fusarium wilt in the Philippines using fuzzy logic and maximum

entropy strategy under current and future climate conditions. The study concluded that

climate change is projected to have a direct and indirect effect on crop production due to

changes in crop suitability, a decline in productivity, and a higher occurrence of pests and

diseases. In the case of bananas, changes in the suitability and distribution of Fusarium

wilt may pose a significant threat to its production system due to climate change. These

risks, being a big dollar earner for the Philippines, can significantly affect the economic

and food production system of the country. The influence of climate change on the

suitability of bananas in the area may be minimal, based on the results. But the expected

changes in future rainfall may increase the areas that are favorable for the incidence of

Fusarium wilt. It is projected that favorable areas for Fusarium wilt in the Philippines will

increase from 21 percent under baseline climate conditions to 27 percent, covering 91.2

percent and 28.5 percent of the highly and moderately suitable banana areas of the

country, respectively. Such coverage represents approximately 67 percent of the total

banana harvested area of the country.

 
 
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Chemical Fungicides: Adverse Effects and Development of Pests Resistance

Fusarium oxysporum causes major economic losses by inducing necrosis and

wilting symptoms in many plant crops. Management of Fusarium wilt is achieved mainly

by the use of chemical fungicides which affect the soil health and their efficiency is often

limited by pathogenic variability. Hence, understanding the nature of interaction between

pathogen and host may help to select and improve better cultivars (Swarupa, Ravishankar

and Planta 2014).

Some fungicides can cause skin and eye irritation, throat irritation and coughing

when inhaled. The study published in the Proceedings of the National Academy of

Sciences in 2007 showed that fungicides may permanently silence or reprogram normal

genes, which can last for several generations (Singleton, 2015).

According to a 2012 study published in the Ecology Letters, the fungicide

chlorothalonil, the most commonly used synthetic fungicide in the United States, is toxic

for aquatic animals such as tadpoles, oysters and fish, when chemical run-off from plants

contaminates nearby water or groundwater. Crop seeds treated by mercury-based

fungicides poisoned livestock. Fungicides that include copper sulphate are toxic to bees

and wild birds (Singleton, 2015).

According to Bendre and Barhate (1998), the management strategies should

include modified cultural practices, resistant varieties, beneficial biocontrol agents and

minimum use of chemicals. Chemical fungicides are used to control diseases caused by

Fusarium oxysporum such as Fusarium wilt. Unfortunately, few fungi have developed

 
 
     17

resistance to them. When plants develop a resistance to a common biochemical marker in

a fungicide, they will be resistant to all other products within that same chemical family.

Recommendation in having proper pest control management is producing natural and

environment-friendly fungicides. The use of natural products like plant extracts for the

management of fungal diseases in plants is considered to be the substitute to those

synthetic fungicides due to their less negative impacts or side effects and implications on

humans and environment.

These indicate that the botanical extracts could be a good alternative in

developing a potent plant-based fungicides which can be used in organic farming for the

management of Fusarium oxysporum or other fungus.

Studies on the Use of Plant Extracts Against Fusarium oxysporum

Several plant extracts were used in different studies to investigate their inhibition

activity on the growth of Fusarium oxysporum. In the study of Bindu Sharma and Padma

Kumar (2009), antifungal potential of three weed plant extracts, namely Capparis

decidua, Lantana camara and Tridax procumbers were tested to determine if the three

weed plants have the eco-friendly compounds that will be responsible for controlling

plant diseases caused by Fusarium oxysporum. The spore germination/ spore counting

technique was followed for the evaluation of the antifungal properties of the extracts at

three different concentrations. Results showed that only Tridax procumbers and Capparis

decidua have the necessary ecofriendly compounds for inhibiting Fusarium oxysporum.

Results revealed that the free flavonoids and sterols of T. procumbens (flower) and bound

 
 
     18

flavonoids of C. decidua (fruit and stem) totally inhibited spore germination of the fungi

(100%).

Anil Kumar Ramaiah and Raj Kumar Garampalli (2015) utilized the food poison

technique for the evaluation of antifungal activity of fifteen different extracts, at four

different concentrations (10%, 20%, 40% and 60%) each, against Fusarium oxyporum.

Using the in vitro fungal assay, results revealed that out of all fifteen plant extracts used,

only three plant extracts were proved to be potential in inhibiting Fusarium oxysporum,

namely Solanum indicum (78.33%), Azadirachta indica (75.00%) and Oxalis latifolia

(70.33%).

Study of Siva, Ganesan, Banumathy and Muthuchelian (2008) also used poison

food technique by in vitro study using water, ethanol and acetone as a solvent to

determine the toxicity and antifungal activity of 20 medicinal different plant crude

extracts. It was found out that all of the plant extracts used at 50% concentrations were

effective in reducing the mycelial growth of Fusarium oxysporum in egg plants.

However, higher inhibition was noticed in four medicinal plant extracts namely,

Adhatoda vasica, Jatropha curcas, Sapindus emarginatus and Vitex negundo. Those four

plants were selected for further concentration changes (10%, 20%, 30% and 40%).

Results showed that only Adhatoda vasica at 40% concentration recorded 100%

inhibition against Fusarium oxysporum while the remaining three plants produced similar

inhibitory effect with 50% concentration.

 
 
     19

Previous studies conducted have proven that plant extracts possess antifungal

compounds with high inhibition rate against Fusarium oxysporum. Hence, can be used to

develop alternative and novel fungicides against this pathogen.

Artemisia vulgaris as Potential Antifungal Agent against Fusarium oxysporum

Artemisia vulgaris, also known as mugwort, has a long history of use in herbal

medicine in terms of digestive system, menstrual cramps and worms. All parts of the

plants are considered as anthelmintic, antiseptic and antispasmodic and its leaves have an

antibacterial action (Plants For A Future, n.d.). It is also used to repel insects and can

prevent infestations and development of larvae. Just like many other members of the

Artemisia family, it is also an effective medicine for worms in the human body and often

given as treats to animals (Combs 2014).

In the study of Singh (2012), there has been an observation that the antimicrobial

activity of Artemisia vulgaris can be explored because it contains numerous essential oils

that have an effect against microorganism. The study of antimicrobial activity of

Artemisia vulgaris essential oil tested in various microbes and pathogens revealed that all

microbes under the study were sensitive to the oil extracted from the plant. Analysis of

the results revealed that the oil extracts may contain considerable antifungal and anti-

bacillary activity that can be enhanced in medicinal uses.

Along with the pathogens' valuable health care effects, medicinal plants are used

for the treatment of several microbial and non-microbial diseases. They have become
 
 
     20

popular and appropriate because of the affordability, reliability, availability, and low

toxicity for therapeutic use. Anti-pathogenic effects of the crude extract of Artemisia

vulgaris leaf extract on Aspergillus niger and Candida albicans species have been carried

out in the present study of Lalitha et al. (2020). The plant species are known Artemisia in

the family Asteraceae, exhibit a plethora of bioactivities, such as anthelmintic, antiseptic

and for its anti-inflammatory properties that is also commonly used. Antifungal activity

was carried out in the case of ethanol and chloroform extracts of Artemisia vulgaris

leaves, and the inhibition zone of the extracts at different concentrations were

determined.

Another study, Manandhar, Luitel and Dahal (2019), investigated the extracts

from Artemisia vulgaris in terms of antimicrobial properties through agar well diffusion

method. The methanolic extracts from the plant showed antimicrobial activity against

various microorganisms. The study confirmed the efficacy of the plant extracts as natural

antimicrobials and hence can be used in the medical field to treat infectious diseases

caused by this microorganism.

In the study of Hiremath, Kolume and Muddapur (2011), wherein the comparison

of Artemisia vulgaris with standard antibiotics were observed, the extracts of Artemisia

vulgaris were tested for their in vitro agar diffusion method of antimicrobial activity.

Using Escherichia coli and Staphylococcus aureus as test species, the antimicrobial

activity of the aqueous, alcoholic, petroleum ether and benzene extract leaves of the plant

was examined. Against all the two microorganisms, all the extracts were successful. The

 
 
     21

outcome indicates that, relative to standard antibiotics, plant extracts have maximum

inhibitory activity against Gram negative and Gram positive species. Major activity

against Escherichia coli and Staphylococcus aureus have been demonstrated in petroleum

ether and plant benzene extracts. Similarly, Artemisia vulgaris has demonstrated strong

inhibitory activity in the aqueous and alcohol extract of the plant, suggesting that the

plant can effectively combat these species.

It can be concluded from the results of the above-mentioned studies that plant leaf

extracts of Artemisia vulgaris demonstrate antimicrobial activity against different test

species used. Some of the extracts (aqueous and alcohol) were more effective than

commercial antibiotics in eliminating the pathogenic microorganisms.

In terms of the plant’s antifungal activity, the study of Devi and Chettry (2013)

investigated the in vitro antifungal activity of aqueous extracts from locally available

plants that is known for their medicinal benefits including Artemisia vulgaris against

Drechslera oryzae, an organism of brown leaf spot of rice. The plant extracts were tested

against the organism at different concentrations using poisoned food technique. Results

showed that Artemisia vulgaris was second to the most effective fungicides to control the

disease at highest 20% concentration. The fungicidal activity of the locally selected

plants was attributed to the presence of antifungal compounds (Devi and Chhetry 2013,

vol. 3, 1-3).

The results of Soares et al. (2015) study reported that the Artemisia vulgaris

essential oil is an inhibitor of growth against the mycotoxigenic fungi Aspergillus flavus
 
 
     22

and Dermanyssus gallinae, glimpsing the possibility that this plant species could be used

in control of these two pests and other pathogenic fungi from both human and plants.

Reviewing the literature, information presented clearly showed the recent

alarming impacts of the Fusarium wilt disease, caused by the fungus, Fusarium

oxysporum, in agriculture particularly in banana plantations. Supported with the findings

of the previous studies on the antimicrobial properties and activities of Artemisia

vulgaris, the researchers decided to evaluate the antifungal activity of its leaf extracts

against Fusarium oxysporum, since no studies have yet been conducted on this area. The

researchers aimed to provide sufficient scientific evidence to support the idea that this

plant material can be a potential source of fungicide against Fusarium oxysporum so that

it can be used to help the banana growers in the Philippines on their efforts to at least

reduce, if not totally eliminate, the impacts of Fusarium wilt disease on banana

plantations.

 
 
     23

Chapter III

METHODOLOGY

This section presents the materials and standard procedures followed in evaluating

the antifungal activity of Artemisia vulgaris leaf extracts against Fusarium oxysporum.

Collection of Artemisia vulgaris

Artemisia vulgaris plants were purchased from Lolita Pacio’s Garden in General

Trias, Cavite. The leaves were washed then air-dried for seven days to remove moisture.

One hundred grams of dried leaves were cut into smaller pieces before extraction.

Preparation of the Pure and Ethanolic Extracts

Dried leaf samples were extracted through maceration using 95% ethanol as the

solvent material. The ethanolic leaf extracts were prepared by submerging the dry

samples in ethanol for 48 hours, following the 25%, 50% and 75% concentrations. The

extracts were filtered, and the supernatant were collected in vials and stored at 4°C until

testing (Hastuti et al. 2017). To prepare the 100% extract, the fresh leaf samples were

placed inside a cheesecloth, then pound and squeezed until a 5mL extract was obtained.

All samples were transported to the University of the Philippines Los Banos

(UPLB) BIOTECH where the antifungal assay was performed.

 
 
     24

Measuring the Zones of Inhibition of the Ethanolic Extracts Using

Cup-Cylinder method

A 7-day old culture of Fusarium oxysporum (Foc) was grown on Potato Dextrose

Agar slants (PDA) and served as test organism.

Ten (10) ml of PDA was poured on a plate as base agar and 5ml of PDA seeded

with Foc was overlayed onto the solidified base agar as top agar. Sterile cup cylinders

were then deposited onto seeded top agar. An aliquot of 0.1 ml of the ethanolic extracts

of Artemisia vulgaris were deposited into the cup cylinders. The plates were incubated at

30˚C for 4 days after which the diameter of zones of inhibition (ZOI) were measured

using digital Vernier caliper (Raymundo et al. 2015). Nystatin, a commercial antifungal,

served as positive control.

Statistical Analysis

The experiments were performed in triplicates. The mean zone of inhibition (ZOI)

of each concentration of Artemisia vulgaris on Fusarium oxysporum was computed.

Differences among the ZOI of the ethanolic, pure extracts and the positive control were

analyzed using One-way Analysis of Variance (ANOVA), the level of significance was

set at 0.05. T-test for independent samples was also used to compare the antifungal

activity between the treatments used.

 
 
     25

Chapter IV

RESULTS AND DISCUSSION

This chapter presents the results on the evaluation of the antifungal activity of

Artemisia vulgaris leaf extracts against Fusarium oxysporum in terms of the zone of

inhibition (ZOI). All the concentrations of the leaf extract of the plant material (25%,

50%, 75% and 100%) exhibited antifungal activity against the pathogen.

In vitro antifungal activity of Artemisia vulgaris pure and ethanolic leaf extracts

against Fusarium oxysporum

The antifungal activity of different concentrations of ethanol leaf extract of

Artemisia vulgaris is described in Table 1 and Figure 1. All the four concentrations (23%,

50%, 75% and 100%) exhibited antifungal activity against the pathogen. Treatment 3,

which is 75% concentration, exhibited maximum efficacy against Fusarium oxysporum

with an average of 16.513mm diameters of zone of inhibition (ZOI). The antifungal

activity of the Artemisia vulgaris is synonymous with the results of similar studies in the

past, and it is due to the presence of several phytochemical compounds and metabolites in

the leaves such as flavonoids, alkaloids, tannins, saponins, acetylenes, phenolic acids,

organic acids and among others (Francisco et al. 2019).

 
 
     26

TABLE 1. Mean diameter of ZOI of ethanolic leaf extracts of Artemisia vulgaris on

Fusarium oxysporum

Treatment and Concentration Count of Average ZOI (mm)

Replicate

T1 (25%) 3 7.84

T2 (50%) 3 12.95

T3 (75%) 3 16.51

T4 (100% pure) 3 8.88

Nystatin (+ control) 3 10.57

Ethyl Alcohol (- control) 3 7.32

Zones  of  Inhibition  (ZOI)  Measurement  of  Replicates  in  each  

Treatment
20
18
16
14
ZOI  (mm)

12
10
8
6
4
2
0
Positive   Negative  
T1 T2 T3 T4
control control
R1 7.45 13.92 15.91 8.69 10.07 7.35
R2 7.49 12.39 16.46 8.09 10.73 7.3
R3 8.57 12.53 17.17 9.85 10.92 7.32
TREATMENTS

Figure 1. ZOI of each treatment groups. (T1: 25% ethanolic leaf extract; T2: 50% ethanolic
leaf extract; T3: 75% ethanolic leaf extract; T4: 100% pure leaf extract; Positive
control (Nystatin); Negative control: (ethyl alcohol)

 
 
     27

Comparison of the In vitro Antifungal Activity of Pure and Ethanolic Leaf Extracts

of Artemisia vulgaris on Fusarium oxysporum

Further analysis using One-away ANOVA showed that there is a significant

difference (p-value < 0.05) among the fungicidal activity of the treatments in terms of the

ZOI on the growth of the pathogen (Table 2.1). Based on the descriptive statistics,

Treatment 3 (75% concentration) exhibited the highest antifungal activity among all the

treatments (Table 2.2).

TABLE 2.1. Results of One-way ANOVA on the ZOI of treatments

Source of Interpretation
Variation SS df MS F P-value F crit
Between Reject Ho
Groups 185.34 5 37.07 88.31 0.000000005 3.11

Within
Groups 5.03 12 0.44

Total 190.38 17
α = 0.05

 
 
     28

TABLE 2.2. Summary of descriptive statistics for Table 2.1.

Groups Count Average Variance

1 3 7.84 0.40

2 3 12.95 0.72

3 3 16.51 0.40

4 3 8.88 0.80

5 3 10.57 0.20

6 3 7.3 0.0006

Comparison Between the Antifungal Activity of Ethanolic Leaf Extract to the

Commercial Antifungal

The results of t-test between the mean diameter of the ZOI of Treatment 3 (75%

ethanolic leaf extracts) and the commercial antifungal showed that there is significant

difference (p-value < 0.05) on their antifungal activity against Fusarium oxysporum.

Based on the descriptive statistics, the 75% ethanolic leaf extracts has surpassed the

antifungal activity of the commercial antifungal used in the experiment (Table 3.1).

TABLE 3.1. T-test between the mean ZOI of T3 and Positive Control

T3 (75% ethanolic Positive Control

extract) (Nystatin)
Mean 16.51 10.57
Variance 0.40 0.20
Observations 3 3
Pooled Variance 0.30
Hypothesized Mean
Difference 0

 
 
     29

df 4
t Stat 13.30
P(T<=t) one-tail 0.00009
t Critical one-tail 2.13
P(T<=t) two-tail 0.0002
t Critical two-tail 2.78

Comparing the antifungal activity of Treatment 1 (25% ethanolic leaf extracts)

and the commercial antifungal against the test organism, result of t-test revealed that the

mean diameter of their ZOI are statistically different at 5% level of significance (p-value

< 0.05). Based on the mean, 25% concentration has lower antifungal activity compared

with the commercial antifungal (Table 3.2) Nevertheless, the 25% ethanolic leaf extract

still exhibited antifungal activity against Fusarium oxysporum even at a lower

concentration.

TABLE 3.2. T-test between the mean ZOI of T1 and Positive Control

T1 (25% ethanolic Positive Control

extract) (Nystatin)
Mean 7.83 10.57
Variance 0.40 0.20
Observations 3 3
Pooled Variance 0.30
Hypothesized Mean
Difference 0
df 4
t Stat -6.11
P(T<=t) one-tail 0.002
t Critical one-tail 2.13
P(T<=t) two-tail 0.004
t Critical two-tail 2.78

 
 
     30

T-test between the T1 (25% concentration) and the negative control (ethyl

alcohol) revealed that there is no significant difference (p-value > 0.05) on their mean

diameter of ZOI on Fusarium oxysporum (Table 3.3). Since the mean diameter of ZOI of

the 25% concentration is 7.83, it has still exhibited antifungal effect on the pathogen.

However, its effect based on ZOI is statistically the same with that of the ethyl alcohol.

This suggests that increasing the concentration of the ethanolic leaf extracts will further

enhance the fungicidal activity of the plant material against the test organism.

TABLE 3.3. T-test between the mean ZOI of T1 and Negative Control

T1 (25% Negative Control

extract) (Ethyl alcohol)
Mean 7.4 7.32
Variance 0.40 0.0006
Observations 3 3
Pooled Variance 0.20
Hypothesized Mean Difference 0
df 4
t Stat 1.40
P(T<=t) one-tail 0.12
t Critical one-tail 2.13
P(T<=t) two-tail 0.23
t Critical two-tail 2.78

α = 0.05

The ZOI of ethanolic leaf extract at 50% concentration is statistically different (p-

value < 0.05) from the ZOI of the commercial antifungal used against Fusarium

oxysporum (Table 3.4). The table shows that based on the mean diameter of ZOI, 50%

ethanolic leaf extracts has higher antifungal activity than the commercial antifungal used.

 
 
     31

This result also reveals that the 50% concentration is ranked second to 75% ethanolic

extracts in terms of the fungicidal effect against Fusarium oxysporum.

TABLE 3.4. T-test between the mean ZOI of T2 and Positive Control

T2 (50% ethanolic Positive Control

extract) (Nystatin)
Mean 12.95 10.57
Variance 0.72 0.20
Observations 3 3
Pooled Variance 0.46
Hypothesized Mean
Difference 0
df 4
t Stat 4.3
P(T<=t) one-tail 0.006
t Critical one-tail 2.13
P(T<=t) two-tail 0.01
t Critical two-tail 2.77
α = 0.05

When the antifungal activity of the 100% pure leaf extracts of Artemisia vulgaris

was compared to the commercial antifungal in terms of the ZOI, t-test reveals that their

mean diameter of ZOI are statistically different at 5% level of significance (p-value <

0.05) (Table 3.5). The lower antifungal activity of the pure leaf extracts compared to the

mean of other treatments suggests the importance of using solvent, such as ethanol, to

better extract the compounds present in the leaves where the fungicidal effect of the plant

material is attributed to by the past studies.

 
 
     32

TABLE 3.5. T-test between the mean ZOI of T4 and Positive Control

Positive Control
T4 (100% extract) (Nystatin)
Mean 8.88 10.57
Variance 0.80 0.20
Observations 3 3
Pooled Variance 0.50
Hypothesized Mean
Difference 0
df 4
t Stat -2.94
P(T<=t) one-tail 0.02
t Critical one-tail 2.13
P(T<=t) two-tail 0.04
t Critical two-tail 2.78
α = 0.05

Table 3.6 shows the results of t-test between the diameters of ZOI of Treatment 1

(25% ethanolic leaf extract) and the 100% pure leaf extracts. No significant difference

exists between their antifungal activity (p-value > 0.05), with mean diameter of ZOI of

7.84mm and 7.88mm respectively. Despite a big difference on their concentrations, both

exhibited equal antifungal effect on Fusarium oxysporum, suggesting the significant

effect of the ethanolic extraction on the fungicidal activity of the leaves of Artemisia

vulgaris against the pathogen. This implies that the ethanolic extraction can contribute to

the efficacy of the plant material as antipathogenic agent since it helps to better extract

the phytochemical compounds present in the plant that will further enhance the plant’s

bioactivities. Many solvents, including methanol, ethanol, acetone, and water, have been

used for extracting bioactive compounds from the plant material (Truong et al. 2019).

 
 
     33

TABLE 3.6. T-test between the mean ZOI of T1 and T4

T1 T4
(25% ethanolic extract) (100% extract)
Mean 7.84 8.88
Variance 0.40 0.80
Observations 3 3
Pooled Variance 0.60
Hypothesized Mean
Difference 0
df 4
t Stat -1.64
P(T<=t) one-tail 0.09
t Critical one-tail 2.13
P(T<=t) two-tail 0.18
t Critical two-tail 2.78
α = 0.05

Summarizing the results of the statistical analysis used in comparing the

antifungal activity of the pure and ethanolic leaf extracts of Artemisia vulgaris, it clearly

reveals that the 75% ethanolic extract exhibited the highest antifungal activity against

Fusarium oxysporum, followed by the 50% concentration, while both the 25% and 100%

concentration have the least but equal antifungal effect on the pathogen. The ethanolic

extraction indeed help enhanced the fungicidal effect of the plant material against the

pathogen. This finding is consistent with the results reported by Hiremath, Kolume and

Muddapur (2011) on their study investigating the aqueous and ethanolic extracts of

Artemisia vulgaris against test organisms and their comparisons to standard antibiotics.

All ethanolic extracts of the plant exhibited strong inhibitory activity against the selected

pathogens.

 
 
     34

Chapter V

SUMMARY, CONCLUSIONS AND RECOMMENDATIONS

Summary

The study was conducted to determine the in vitro antifungal activity of ethanolic

leaf extracts of Artemisia vulgaris against Fusarium oxysporum. Four concentrations

(25%, 50%, 75% and 100%) of leaf extracts were tested against the pathogen using the

cup cylinder method and the zones of inhibition (ZOI) were compared to determine

which concentration has the highest antifungal effect against the pathogen. All the four

concentrations tested exhibited antifungal activity against Fusarium oxysporum. Results

reveal that the ethanolic extracts has the maximum efficacy at 75% concentration, having

a mean diameter of 16.5mm ZOI, then followed by the 50%, 25% and 100%

concentration with mean diameter of 13.0mm, 8.9mm and 7.8mm respectively.

T-test between the ZOI of 75% ethanolic leaf extracts and the commercial

antifungal used showed that the 75% concentration of Artemisia vulgaris has surpassed

the antifungal effect of the commercial drug against Fusarium oxysporum (p-

value=0.00009). Similar result was also obtained when the antifungal activity of the 50%

ethanolic leaf extracts was compared with the commercial antifungal used (p-value=

0.006). Meanwhile, the 25% and 100% concentration have exhibited statistically equal

fungicidal effect on the fungus (p-value>0.05).

 
 
     35

Conclusion

All the ethanol extracts of Artemisia vulgaris exhibited strong to moderate

antifungal activity against Fusarium oxysporum. Among the three concentrations of

ethanol extracts tested, Treatment 3 with 75% concentration has the highest inhibitory

activity against the pathogen, even surpassing the efficacy of the commercial antifungal.

From these findings, it can be concluded that Artemisia vulgaris can be a promising

source of antifungal compounds, which can be isolated, to develop effective yet low-cost

fungicide against Fusarium oxysporum.

Recommendation

This study suggests the isolation of bioactive compounds from Artemisia vulgaris

to be further investigated especially the impact on toxicology and safety of these

compounds before they can be developed as bio fungicide. Since the ethanol extracts of

the leaves of the plant were proven to have antifungal effect on the plant pathogen, it is

recommended th at future studies consider investigating the fungicidal activity of other

ethanolic concentrations of the plant material. Different solvents and extraction methods

are also recommended to further evaluate the efficacy of the plant as a fungicide.

 
 
     36

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     47

APPENDICES

 
 
     48

APPENDIX A

Report of Antifungal Analysis

 
 
     49

 
 
     50

PLATES

 
 
     51

Plate 1. Purchase of Damong Maria (Artemisa vulgaris)

plant sample, first batch

 
 
     52

Plate 2. Dried Damong Maria (Artemisia vulgaris),

plant sample (first batch)

 
 
     53

Plate 3. Separating leaves and stem of Dried Damong Maria plant sample and
cutting of Dried Damong Maria leaves into small pieces

 
 
     54

Plate 4. Purchase of Fresh Damong Maria Plant Sample, Second Batch

 
 
     55

Plate 5. Fusarium oxysporum grown on Potato Dextrose Agar (PDA)

 
 
     56

Plate 6. Materials used for cup cylinder method

 
 
     57

Plate 7. Assay Plate