Inhibition of Fusarium oxysporum f. sp.

cubense Using Decocted Guava

(Psidium guajava) Leaves Extracts

A Research
Presented to the Faculty of Los Baños Senior High School
San Antonio, Los Baños, Laguna

In Partial Fulfillment of the Requirements for the Subject

Inquiries, Investigation, and Immersion (3I’s)

Alforja, Nikie
Anzano, Ronel
Mana-ay, Jerika
Marquez, Claude
Otero, Venus Grace
Rivera, Ommel
Sadio, Aeron Earl
Secretario, John Paul
Taran, Kevin
Vargas, Marielle Janine

February 2020
 Acknowledgement

The researchers would like to extend their deepest gratitude to all the

people that contribute to the creation of the study from the concepts to the

experimentation and to the entire process in conducting the study.

The researchers would really like to thank and praise God for His kindness

and support to the entire team while conducting the research study.

The entire team of the researchers of the study would also like to extend

their gratitude to Mrs. Eden Taran for giving support and providing materials in

the decoction process of guava leaves extract and for the making of the research

paper.

To Ma’am Mylene D. Rizare and to her whole team in their department in

Department of Science and Technology - Forest Products, Research and

Developent Institute for aiding the researchers with their experiment and served

as their research consultant.

To Dr. Michael William V. Puna III Ed. D. for allowing the researcher to

conduct their research outside the school and go to their institute and to be

excused from their classes.

To the research advisers; Ma’am Karen Bismonte and Ma’am Rowena

Caganan for being their consultant and for aiding the entire study.

ii
 Abstract

The use of decocted plant extracts, in many studies and traditions, had

prove its antiseptic and antimicrobial properties. Inhibition of pathological fungi

would help to lessen environmental and commercial concerns since soil-borne

diseases like fungi prevent a plant to grow and produce quality fruits. One of

these fungi that is soil-borne and causes the plant, specifically the tree of the

banana, to wilt is the Fusarium oxysporum f. sp. cubense and this causes the

disease called Fusarium wilt which is commonly known as Panaman disease.

This fungus was used as the test organism since the study aims to inhibit the

growth of Fusarium oxysporum f. sp. cubense which could help to reduce

environmental concerns and reduce losses in banana production industry. In

order to resolve the issue about this fungus, decocted guava leaves extract was

used. The guava leaves undergone decoction process where they were boiled

with a 100% distilled water where 50 grams of guava leaves were soaked and

boiled with 500 milliliters of distilled water. This decocted extract from guava

leaves was used to test its anti-fungal property against Fusarium oxysporum f.

sp. cubense and if it can inhibit the growth of the fungus. 6 (six) concentrations

were prepared (10%, 20%, 30%, 40%, 50%, and 100%) with 2 (two) control

variebles; one standard control which is a commercial fungicide (propiconazole)

and one control with no treatment added. Using the Disk Diffusion Method to

measure the inhibition of the fungus from the treatments used, the set-ups were

observed for their first 24 hours and 48 hours after the treatments were applied.

After the first 24 hours and 48 hours, there was no inhibition observed from the

iii
six concentrations of decocted guava leaves extract while only the standard

control showed inhibition of Fusarium oxysporum f. sp. cubense which was

categorized of having a very slight to slight inhibition. The study failed to inhibit

the growth of the Fusarium oxysporum f. sp. cubense. The findings of the study

might still help to improve further studies about Panama disease especially to the

Fusarium oxysporum f. sp. cubense.

 Table of Contents

Title Page……………………………………………………………………..………i
iv
Acknowledgement…………………………………………………..………………ii

Abstract………………………………………………………………..……………..iii

Table of Contents…………………………………………..……………………….v

List of Figures...…………………………………………………………..…………vii

List of Tables…………………………………………………………………..…….viii

I. The Problem and Its Background…………………………………………..…1

Introduction…………………………………………………….…1

Background of the Study……………………………….………2

Conceptual Framework…………………………………………3

Statement of the Problem………………………………………4

Hypothesis………………………………………….……………..4

Scope and Limitations……………………………………..……5

Significance of the Study…………………………………….…5

Definition of Terms………………………………………………6

II. Related Literature and Studies…………………………………………..……8

Related Literature……………………………………………..…8

Related Studies……………………………………………..……9

III. Methodology……………………………………………………………………..13

Research Design…………………………………………………13

Research Materials and Data Gathering Procedures…...…14

 Statistical Tool……………………………………………………17

IV. Results and Discussions………………………………………………………18

V. Summary, Conclusion, and Recommendations……………………………22

Summary of Findings……………………………………………22

Conclusion……………………………………………………...…22

Recommendations…………………………………………….…23

References…………………………………………………………………………...24

Appendices…………………………………………………………………………..

vi
 List of Figures

Figure Title Page

1 Input, Process, Output (IPO) Diagram

2 Research Procedure Flow Chart

3 Visual Representation of the Measurements of

Fungal Inhibition for the Different Concentrations
of the Guava Leaves Extract (GLE) after 24 hours
from the Date of Application

4 Visual Representation of the Measurements of

Fungal Inhibition for the Different Concentrations
of the Guava Leaves Extract (GLE) after 48 hours
from the Date of Application

vii
 List of Tables
Table Title Page

1 Measurements of Fungal Inhibition for the Different

Concentrations of the Guava Leaves Extract (GLE) After
24 Hours from the Date of Application

2 Measurements of Fungal Inhibition for the Different

Concentrations of the Guava Leaves Extract (GLE) After
48 Hours from the Date of Application

viii
 CHAPTER 1
The Problem and Its Background
Introduction

The Fusarium wilt, or more commonly known as Panama disease, is a

disease on bananas caused by the fungus Fusarium oxysporum f. sp. cubense.

The fungus is soil-borne and attacks the plant from its root and invades the

xylem. This results into the blockage of the passage of water and nutrients in the

plant, which eventually leads to the death of the plant. This disease was first

discovered in 1890 in Panama, earning it its infamous name. Although it was first

discovered in 1890, the disease was rampant in the 1950s and ‘60’s when it

devastated the Gros Michel, a cultivar of banana that was dominating the global

banana industry at that time (The Editors of Encyclopaedia Britannica, 2019).

After the downfall of the Gros Michel, the Cavendish cultivar rose in fame as a

substitute. But soon after, in 1987, it is discovered that the cultivar is also

susceptible to the disease. The Cavendish cultivar is the currently leading cultivar

of banana in the world market, accounting for 51% of the global production of

bananas in 2016 (FruiTop, 2016). The Philippines, being the second top exporter

of bananas worldwide, relies heavily in the production of these bananas. With the

devastating effects that the disease could do to the banana industry, this must be

resolve and the study aims to inhibit the fungi using the decocted extract of

guava leaves.

Guava (Psidium guajava) trees are one of the most abundant trees in the

Philippines. Its leaves have been used as a traditional medicine for curing

1
several diseases and is even famous for serving as an antiseptic for men that

undergo circumcision in the country. The decoction or boiling of fresh guava

leaves is used often to clean wounds to prevent infections and other cleaning

purposes on the body and helps to aid diarrhea. It is believed and tested through

several in vitro and in vivo assays that guava leaves contain active components

that have anti-fungal or antimicrobial effects.

Background of the Study

The researchers came up with the idea that soil-borne diseases which

attack the plant and cause production loss must be resolve with a simple but

effective material or tool that could benefit not only the researchers but also the

banana industry and the environment. It is a major concern since products made

out of banana are one of the major exporting products and produce a very huge

amount of revenue which contribute to the growth of the Philippine economy. To

address the Panama disease problem, this study will utilize an experiment to test

the effectiveness of decocted guava leaves extract in treating Fusarium

oxysporum f. sp. cubense, the causal fungus of the Panama disease. Since the

use of guava and many of its parts especially its leaves have many known

records of traditions where people use it as an antiseptic solution and many

studies proved it right with its antimicrobial property. It is believed that guava

leaves contain active components that are responsible for its antifungal activity.

This antifungal properties of the extracts will be tested via disk diffusion method.
Conceptual Framework
2

Materials:
- Guava Leaves
- Fusarium oxysporum f. sp. cubense
INPUT

- Decoction of Guava Leaves

- Conducting disk diffusion testing to examine the antifungal properties of
the Extracts Against Fusarium oxysporum f. sp. cubense
PROCESS

- A research paper tackling the problems about Panama disease and

providing a feasible solution

OUTPUT

Figure 1. Input, Process, Output (IPO) Diagram

Statement of the Problem

3
This study used disk diffusion method to test the antifungal properties that

the guava leaves extracts have on Fusarium oxysporum f. sp. cubense.

Specifically, it answers the following:

1. What effects does the guava leaves extracts have on the growth of the

Fusarium oxysporum f. sp. cubense?

2. How large is the zone of inhibition in the assays treated with the guava leaves

extracts?

3. What difference is there on the zone of inhibition does different levels of

concentration for the guava leaves extracts have?

Null Hypotheses

The following are the hypotheses of the study:

The guava leaves extracts do not have any significant effect on the inhibition of

Fusarium oxysporum f. sp. cubense.

There are no significant differences between the different levels of concentration

of the extracts on the measurements of the zone of inhibition.

Scope and Limitations
4
The study used the extracts from guava leaves to inhibit the propagation

of Fusarium oxysporum f.sp. cubense. It covered all the processes involved,

including everything from the extraction from the guava leaves up until the testing

process. In order to get the extracts from the guava leaves, the leaves were

boiled using distilled water. The study utilized in vitro testing to determine the

antifungal properties of the guava leaves extract against Fusarium oxysporum

f.sp. cubense. Five different concentrations of guava leaves extracts (10%, 20%,

30% ,40% and 50%) were used together with two control groups (a standard

fungicide and no treatment).

This study, however, does not cover the application of the extracts directly

on the trees of the banana infected with the fungi. It also does not include

analysis of the constituents that are present in the extracts.

Significance of the Study

This study tested the antifungal properties of guava leaves extracts

against Fusarium oxysporum f.sp. cubense. This study benefits the following

sectors:
 Banana Farmers. Farmers can benefit by having the knowledge to

combat the disease that causes them losses and having an available solution to

their problems.

Banana Industry. Through finding possible solutions for the diseases of

bananas, the banana industry can lessen its losses.

5 the losses of banana due to the Panama

Community. By helping lessen

disease, the community can enjoy good quality of bananas as well as no decline

in the supply in the market for bananas.

Environment. Since the decocted guava leaves extract might inhibit the

growth of the fungus that will be tested, this could help to lessen eenviironmental

concerns and this extract has nutrients present that could help to the

nourishment of the soil and could help to the growth of the plant.

Future Researchers. This study will serve as a reference for future

researches and for further improvements of the study.

Definition of Terms

Anti-fungal Activity. It refers to the biological activity of either inhibiting

the growth of fungi or the process of killing off fungal cells. The use of decocted

guava leaves extract to inhibit fungi will be measured by this property.

Banana. A plant that is usually tall and commonly mistaken as a tree but

it is just a plant with sturdy pseudostem. In this study, it is the tree which is
vulnerable to the Panama disease caused by the fungi Fusarium oxysporum f.

sp. cubense.

Decoction. A process by which a part of a plant was boiled with water to

extract the substances present in it. It is usually done for medicinal purposes.

This is the process that will be used to get the extract of guava leaves using

distilled water.
6

Fusarium oxysporum f. sp. cubense. A species of fungi that is soil-

borne which attacks the plant from its roots and invades the xylem that will result

in Panama disease. It is the fungi that will undergo inhibition in the study.

Fusarium wilt. Commonly known as Panama disease, is a fungal disease

of bananas caused by Fusarium oxysporum f. sp. cubense. Relating to the study,

it is the disease that will be treated by inhibiting its growth using essential oils.

Guava leaves. Leaves from the species of Psidium guajava which have

been used as a traditional medicine for treating several diseases and infections

due to its antiseptic properties. In this study, it is the part of the plant that will be

used to undergo decoction to its extracts on the inhibition of the Fusarium

oxysporum f. sp. cubense.

Inhibition. The act of preventing or slowing the activity or occurrence of

something. Relating in the study, it is the process that will be used treat to fungus

Fusarium oxysporum f. sp. cubense.

In Vitro Analysis. A process where biological properties are observed

outside of actual living orgamisms and instead on controlled, external

environments such as test tubes and Petri dishes. It is the process that will be

used to test the inhibition properties of the essential oil.

CHAPTER 2
Review of Related Literature and Studies
7
This chapter presents different literatures and studies related to Fusarium

wilt/Panama disease, Fusarium oxysporum, and guava to provide evidence that

this study is feasible and to serve as guidelines towards the utilization of the

materials needed for the testing of the anti-fungal activity of the guava leaves

against Fusarium oxysporum.

RELATED LITERATURE:

Fusarium oxysporum f. sp. cubense

The Foc or Fusarium oxysporum f. sp. cubense is a member of the F.

oxysporum species complex and contains nonpathogens and plant and animal

pathogens. There are four phototypes (known as ‘races’) of Foc recognized in

banana namely 1) race 1, which attacked ‘Gros Michel’ bananas; 2) race 2,

which affects ABB cooking bananas; 3) subtropical race 4 (SR4), which affects

Cavendish cultivars in the subtropics; and 4) tropical race 4 (TR4), which affects

the same cultivars (Phytoparasitica, 2015).

Oxysporum f. sp. cubense is one of the special forms of Fusarium

oxysporum species intricate of pathogenic and non-pathogenic with the structure

similar to filamentous fungi. This special form is consisting of pathogenic strains

that cause fusarium wilt in bananas (retrieved from http://www.promusa.org/,

January 08, 2020).

Guava
8

The guava is kind of fruit bearing tree that can naturally grow in the tropic

regions. It can be found near houses and in the backyards. It normally grows to a

height of 4 meters and its leaves grows in pairs or opposite to each other. The

plant also bears diminutive white hued flowers that possess several stamens

maturing into smoothed or pear-shaped, yellowish fruits containing numerous

seeds. As part of being a common tree here in the Philippines, the most

important thing that the fruit has, is that it is very rich in nutritional value like

Vitamin C. (retrieved from https://elmaskincare.com/herbs/herbs_guava.htm,

January 08, 2020)

RELATED STUDIES:

Fusarium wilt/Panama disease

Panama disease also known as Fusarium wilt of banana is studied to be a

“polycyclic” disease according to (Ploetz 2015). In addition to this, it is known that

even just a small amount of pathogen infects the field and may result to massive

losses. And Philippines and China are the first countries that suffered the

outbreaks (Buddenhagen, 2009)

Daly, A., et.al. (2006) explained that the internal symptoms of panama

disease attack the xylem vessels of the plant that results to discoloration in a
circular pattern. The infection proceeds into the pseudo-stem, causing the plant

to wilt. But there are no cases that the infection affects the fruit.

Ploetz, R.C. (2015) stated that fusarium wilt, also referred as “Panama

Disease” is considered to be one of the most destructive disease on banana.

Prevention to this disease is deficient in a lot of breeds and cultivars. Seeking

9
better resistance to this and other diseases is necessary.

The re-emergence of the Panama disease or Fusarium Wilt among

bananas were observed during the mid-twentieth century when a soil-borne

fungus identified as Fusarium oxysporum f. sp. cubense or Foc devastated

industries in Central America and the Caribbean. It greatly threatened the global

banana production and so people shifted to resistant Cavendish cultivars in order

to recover from the effects of the said fungi (Foc Race 1). Eventually, a new

strain of Foc called Tropical Race 4 (TR4) were able to invade the Cavendish

cultivars. This seriously threatened and affected wider range of banana varieties

including those locally grown by smallhold farmers. Infections among bananas

led to a big loss in banana production (Vicente et al., 2014).

As stated by Dita, M., et. al. (2018), banana is the world's most important

fruit in terms of production, volume and trade (FAOSTAT, 2017). It is also one of

the world's top 10 staple food. The production of Banana is greatly affected by a

disease caused by a soil-borne pathogen (Fusarium oxysporum f. sp. cubense)

with an extremely long residence time in soil that infects the xylem, induces wilt

and kills the Banana plant which is commonly known as Fusarium wilt. The
disease was considered as one of the most devastating Banana diseases in the

history (Stover and Simmonds, 1987).

Fusarium oxysporum f. sp. cubense

Fusarium oxysporum formae specialis

10 cubense (Foc) is a fungus

transmitted by soil that is the cause of Fusarium wilt, which is considered as a

disease that makes the most damage to bananas. It is believed that the fungus

developed with its host causing it to spread and infect other banana-growing

countries more specifically Asian countries and some other tropical countries.

(Mostert, D., et. al., 2017).

According to Damodaran, T., et. al. (2019), bananas that are infected by

Fusarium wilt caused by Fusarium oxysporum f. sp cubense has a symptom of

yellowing of mature leaves which is infecting the younger leaves. The petiole of

the leaves bend and collapse slowly. The laminae of the leaves decrease making

it shrivels and the psuedostem splits vertically and shows discoloration.

Guava

Ferdinand, N. et. al. (2014) stated that the guava (Psidium guajava) are

domestic from South Africa and are growing in the tropics. It is usually used in

treating diseases such as cancer, gastrointestinal and respiratory disorders, and

many other diseases. All the studies related on the extracts and essential oil that
they contain proves that they have anti-inflammatory, antibiotics, analgesics,

hepato protective and antioxidant activities.

Biswas, B., et. al. (2013) stated that guava (Psidium guajava) is a plant

used as a traditional medicine because it is believed to contain active

components that is helpful in treating different kind of diseases. All of its parts

have been used in controlling different conditions such as malaria, dysentery,

vomiting, etc. They also stated that this plant helps in managing conditions like

hypertension and diabetes. 11

Out of the numerous biological activities, the Psidium genus is known for

having antifungal effect and is proven through in vitro and in vivo assays.

Psidium guajava L. is used for the treatment of the different cases and also the

symptoms related to the infections cause by Candida spp because of its

therapeutic versatility. (Bezarra, C. F., et. al. 2018)

According to Mushtaq, M., et. al. (2014), the leaves of the Guava are

known for its properties such as anti-inflammatory property for treating different

cases like various lung problems. Guava leaves also help in treating wounds,

ulcer, various bacterial infections and cleansing the blood because it is proven

that it has strong antimicrobial properties.

As stated by Das, M., and Subhagata Goswami, (2019), with the help of

pytochemical analysis, it appears that guava leaves have a wide range of

polyphenolic compounds. Also, Guava leaves have a strong antimicrobial

property (antifungal and antibacterial). As polyphenolic property becomes one

with the antimicrobial property, it may be the cause of the antibacterial and

antifungal property of the guava.

CHAPTER 3
Methodology
12

This chapter presents the methods and processes on how the study will

be conducted. It states what the appropriate design should be used, the locale,

the materials for the study, the data-gathering process, and the statistical tool for

the data gathered.

Research Design

This study utilized experimental research design. Experimental research

design, as stated by Key, J., 1997, is a plan of procedures that helps the

researchers validate his hypothesis using conclusions observed from the

relationships between his independent and dependent variables. It serves as the

framework on how the experiment is conducted.

The study consisted of six (6) experimental groups and two (2) control

groups. Experimental research was utilized in order to test if the independent

variable (IV), which is the different concentrations of the decocted guava leaves
extract, have significant effects on the dependent variable (DV), the anti-fungal

activity of the extracts. The experimental groups were subjected into a treatment

of six different concentrations of the guava leaves extracts (IV), while one control

group was subjected to a standard control and the other, none. After 24 hours,

the zone of inhibition was measured for all groups to determine if there is a

significant anti-fungal activity (DV) exhibited by the extracts. After another 24

hours, the final measurements for all groups were measured.

Research Instrument

The study used observation logs as its research instrument. The data from

the experiments were observed and logged in a data sheet. The zone of

inhibition, which is the dependent variable in the experiment, for each setup were

measured 24 hours and 48 hours after the initial set-up.

Research Procedures

The procedures done in the study is represented by the diagram below:

Collection of Extraction from

Materials Guava Leaves

Measurement Preparation of
of Zone of Setups (Paper
Inhibition Disk Diffusion)

Figure 2. Research Procedure Flow Chart

 The diagram is further explained by the interpretations below:

A. Collection of Materials

The first step in the process is the collection of all the necessary materials

for the experiment. The study only utilized one raw material, which is the guava

leaves. The leaves from the guava were the ones used to obtain the extracts

which acted as the independent variable for the study. The leaves were collected

fresh from the tree and then air dried for 1 week for the extraction process.

B. Extraction Process

14 used is a conventional way of extraction

The extraction method that was

from the leaves. The leaves that were dried for a week were pulverized in a

blender. 50g of the pulverized leaves were then mixed with 500mL of distilled

water. The ratio of the leaves to the water is 1g:10mL. The mixture was then

boiled for 15 minutes. After cooling down, the extracts were then filtered using a

filter paper. The extracts were refrigerated until further use.

C. Preparation of Setups (Disk Diffusion)

For the testing of the anti-fungal activity of the guava leaves extracts, the

disk diffusion method was used. The first step in the process was the preparation

of the growth media for the Fusarium oxysporum to grow on. Malt extract agar

was prepared by mixing 500mL of distilled water with 12.5g of malt extract and

9g of agar in a 1000mL Erlenmeyer flask. Two of this solution were prepared and

were set to rest. After 2 hours, the malt extract agar (MEA) solutions were poured

in to 40 petri dishes. The plates were then stored to condition the growth media.
While waiting for the growth media to condition, the inoculum of the Fusarium

oxysporum was prepared. The spores of the fungi were mixed with 10mL of

sterilized distilled water. 1mL were extracted from the mixture and then mixed

with 100mL of sterilized distilled water in a flask. The mixture was then

transferred into a sterile sprayer. After the plates containing the growth media

were conditioned, the mixture was sprayed once to every plate. The plates were

then stored once again. These were the culture media. The preparation for the

different concentrations of the guava leaves extracts came after. Five

concentrations were prepared for the extracts: 10%,20%,30%,40%, and 50%.

The extracts were diluted in distilled water to achieve these concentrations, such

that the following ratios were used: 1.5mL extracts:13.5mL distilled water for the

10% concentration; 3mL extracts:12mL distilled water for the 20% concentration;

4.5mL extracts:10.5mL distilled water for the 30% concentration; 6mL

extracts:9mL distilled water for the 40% concentration; and 7.5mL extracts:7.5mL

distilled water for the 50% concentration. These solutions were then mixed using

a vortex mixer for 10 seconds each. After the preparation of the different

concentrations of the guava leaves extracts, the solutions were then poured into

sterile petri plates. Filter papers (18mm in diameter) were submerged into each

solution for 1 min. There were 5 replicates of the culture media for every

concentration of the guava leaves extracts. For each replicate, a filter paper that

was submerged from the replicate’s corresponding concentration group was

placed in one side of the plate. This was done until all the plates were inserted a

filter paper from their concentration group. For the control groups, 5 replicates
were also allotted for each group. Filter papers submerged into a propiconazole

solution, made by mixing 2mL of propiconazole with 100mL of distilled water,

was used for the standard control group, while the other control group received

no treatment. All the plates were labeled in accordance to their group.

D. Measurement of Zone of Inhibition

For the final process, the zone of inhibition in each plate were measured

24 hours and 48 hours after all the setups were prepared. All the data from the

observation and measurements were recorded in the data sheet. After that, the

data were finally analyzed and interpreted.

16
Statistical Tool

This study utilized One-Way ANOVA as its statistical tool. One-Way

ANOVA is a statistical tool that is used to compare the means of two or more

groups. Its purpose is to find if there is a significant difference between the mean

scores of the groups. It is used when only one independent variable is present for

all the groups involved.

The study tested if having different concentrations of the guava leaf

extracts (IV) have any difference in measuring the fungal-inhibition properties of

the extracts (DV) by getting the zone of inhibition of every replicate for every

concentration level. The mean scores of the groups were compared to see if

there is a significant difference between different concentration levels on treating

the fungi.
 CHAPTER
17 4

Results and Discussion

This chapter presents the results obtained from the experiment done using

the methodologies in the previous chapter. It also includes the interpretation and

the analysis of the data obtained.

Table 1. Measurements of Fungal Inhibition for the Different Concentrations of

the Guava Leaves Extract (GLE) After 24 Hours from the Date of Application
Treatment Mean of the Classification of Zone
Measurements of of Inhibition
Fungal Inhibition (%)
10% of guava leaves 0 No Inhibition
extract (GLE)
20% of guava leaves 0 No Inhibition
extract (GLE)
30% of guava leaves 0 No Inhibition
extract (GLE)
40% of guava leaves 0 No Inhibition
extract (GLE)
50% of guava leaves 0 No Inhibition
extract (GLE)
100% of guava leaves 0 No Inhibition
extract (GLE)
 Standard Control 13.3 Very Slight Inhibition
(Propiconazole)
Control 0 No Inhibition

As shown in Table 1, all concentrations of the guava leaves extracts

(GLE) showed no inhibition against Fusarium oxysporum f. sp. cubense. Only

the standard control, which is the propiconazole, showed a very slight inhibition

with 13.3% fungal inhibition. This means that on the first 24 hours of treatment,

all concentrations of the guava leaves extracts proved to be ineffective against

inhibiting the growth of the fungi.

18
Fungal Inhibition (%)
14 13.3
12
10
8
6
4
2
0 0 0 0 0 0 0
0

Fungal Inhibition (%)

Figure 3. Visual Representation of the Measurements of Fungal Inhibition for the

Different Concentrations of the Guava Leaves Extract (GLE) after 24 hours from the Date
of Application
 As seen in Figure 3, all of the treatments except for the standard control,

which is the propiconazole, showed no signs of inhibition against Fusarium

oxysporum f. sp. cubense. The standard control showed 13.3% inhibition which

means that it has a very slight inhibitory activity against Fusarium oxysporum f.

sp. cubense.

Table 2. Measurements of Fungal Inhibition for the Different Concentrations of

the Guava Leaves Extract (GLE) After1948 Hours from the Date of Application
Treatment Mean of the Classification of Zone
Measurements of of Inhibition
Fungal Inhibition (%)
10% of guava leaves 0 No Inhibition
extract (GLE)
20% of guava leaves 0 No Inhibition
extract (GLE)
30% of guava leaves 0 No Inhibition
extract (GLE)
40% of guava leaves 0 No Inhibition
extract (GLE)
50% of guava leaves 0 No Inhibition
extract (GLE)
100% of guava leaves 0 No Inhibition
extract (GLE)
Standard Control 28.7 Slight Inhibition
(Propiconazole)
Control 0 No Inhibition
 According to Table 2, all concentrations of the guava leaves extracts still

showed no sign of inhibition against Fusarium oxysporum f. sp. cubense. The

standard control (Propiconazole) on the other hand, showed an increase of

fungal inhibition by 15.4%, from 13.3% to 28.7% after 48 hours.

Fungal Inhibition (%)

35
30 28.7
25
20
15
10
5
0 0 0 0 0 0 0
0

20

Fungal Inhibition (%)

Figure 4. Visual Representation of the Measurements of Fungal Inhibition for the
Different Concentrations of the Guava Leaves Extract (GLE) After 48 hours from the Date
of Application

Figure 4 shows that 48 hours after the treatment of the assays, there is

still no fungal inhibition for all the concentrations of the guava leaves extracts.

The only treatment that showed a difference from the first measurements is the

standard control with 28.7% fungal inhibition instead of the 13.3%.

Overall, the data showed that the guava leaves extracts showed no

inhibition against Fusarium oxysporum f. sp. cubense. It is also shown that there
is no significant difference between the means of the different concentrations of

the extracts, meaning that the null hypothesis is true and is accepted.

CHAPTER 5
21
Summary, Conclusions, and Recommendations

This chapter presents the summary of the findings of the whole research,

as well as the conclusions for the study and recommendations for further

improvements or uses of the study.

Summary

The anti-fungal properties of different concentrations of guava leaves

extracts were tested against Fusarium oxysporum f. sp. cubense. In order to test

the effectivity of the extracts, the disk diffusion method was used. Six

concentrations of the extracts were used: 10%, 20%, 30%, 40%, 50%, and

100%, along with a standard control, the propiconazole, and a control with no

treatments. The zone of inhibition was measured for 24 hours and 48 hours to

see the percentage of fungal inhibition against Fusarium oxysporum f. sp.

cubense. The results of the test showed that all the concentrations of the guava

leaves extracts showed a 0% fungal inhibition. This means that the extracts were

not able to inhibit the growth of Fusarium oxysporum f. sp. cubense. Only the

standard control showed a sign of inhibition with 13.3% or a very slight inhibition
24 hours after the treatment and 28.7% or a slight inhibition 48 hours after the

treatment.

Conclusion

Based on the results, it can be inferred that the guava leaves extracts are

not an effective treatment against Fusarium oxysporum f. sp. cubense. The

extracts showed no sign of inhibition and has no effects against the fungi.

Furthermore, the results also show that there is no significant difference between
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the concentrations of the guava leaves extracts since all the values of their mean

are the same. This means that the null hypothesis is true, and therefore is

accepted.

Recommendations

For further improvements of the study, the following recommendations are

given to those that will conduct similar researches. The first is to change the

solvent of the extracts. The solvent used for the extracts in the study is only

distilled water which has a little to no efficacy in inhibiting the growth of a fungus,

ethanol or methanol solutions have better efficacy against water and both can

dissolve the components of the leaves better than distilled water. The next is to

try different raw materials for the extracts. There are many plant extracts that

have anti-fungal properties, and may prove to be effective against Fusarium

oxysporum f. sp. cubense. Another is to try the extracts on different microbes.

The extracts may prove to be ineffective against Fusarium oxysporum f. sp.

cubense, but it does not mean that it can’t be effective against other microbes
like another species of fungi or even bacteria. Last is to do an in-vivo testing

against Fusarium oxysporum f. sp. cubense. The actual application of a

treatment to the fungi is the final goal of the study, and it is needed to provide an

answer to the actual problem at hand.

References
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 Appendices

Inhibition of Fusarium oxsyporum f. sp. cubense Using Guava Leaves Extract

24HRS AVE INHI 48 HRS AVE INHI

1 2 3 1 2 3
T1 10% GE 1 90 90 90 90 0 90 90 90 90 0
T1 10% GE 2 90 90 90 90 0 90 90 90 90 0
T1 10% GE 3 90 90 90 90 0 90 90 90 90 0
T1 10% GE 4 90 90 90 90 0 90 90 90 90 0
T1 10% GE 5 90 90 90 90 0 90 90 90 90 0
Average 90 90

T2 20% GE 1 90 90 90 90 0 90 90 90 90 0
T2 20% GE 2 90 90 90 90 0 90 90 90 90 0
T2 20% GE 3 90 90 90 90 0 90 90 90 90 0
T2 20% GE 4 90 90 90 90 0 90 90 90 90 0
T2 20% GE 5 90 90 90 90 0 90 90 90 90 0
Average 90 90

T3 30% GE 1 90 902 903 904 0 905 906 907 908 Column1

T3 30% GE 2 90 90 90 90 0 90 90 90 90 0
T3 30% GE 3 90 90 90 90 0 90 90 90 90 0
T3 30% GE 4 90 90 90 90 0 90 90 90 90 0
T3 30% GE 5 90 90 90 90 0 90 90 90 90 0
Average 90 90
T4 40% GE 1 90 90 90 90 0 90 90 90 90 0
T4 40% GE 2 90 90 90 90 0 90 90 90 90 0
T4 40% GE 3 90 90 90 90 0 90 90 90 90 0
T4 40% GE 4 90 90 90 90 0 90 90 90 90 0
T4 40% GE 5 90 90 90 90 0 90 90 90 90 0
Average 90 90
T5 50% GE 1 90 90 90 90 0 90 90 90 90 0
T5 50% GE 2 90 90 90 90 0 90 90 90 90 0
T5 50% GE 3 90 90 90 90 0 90 90 90 90 0
T5 50% GE 4 90 90 90 90 0 90 90 90 90 0
T5 50% GE 5 90 90 90 90 0 90 90 90 90 0
Average 90 90
T6 100% GE 1 90 90 90 90 0 90 90 90 90 0
T6 100% GE 2 90 90 90 90 0 90 90 90 90 0
T6 100% GE 3 90 90 90 90 0 90 90 90 90 0
T6 100% GE 4 90 90 90 90 0 90 90 90 90 0
T6 100% GE 5 90 90 90 90 0 90 90 90 90 0
Average 90 90
T7 STD 1 79 83 80 80.7 10.4 62 59 66 62.3 30.7
T7 STD 2 76 79 80 78.3 13.0 63 57 63 61.0 32.2
T7 STD 3 78 80 79 79.0 12.2 73 65 69 69.0 23.3
T7 STD 4 74 78 77 76.3 15.2 64 63 64 63.7 29.3
T7 STD 5 76 73 79 76.0 15.6 63 65 66 64.7 28.1
Average 78.1 13.3 64.1 28.7
CONTROL 1 90 90 90 90 0 90 90 90 90 0
CONTROL 2 90 90 90 90 0 90 90 90 90 0
CONTROL 3 90 90 90 90 0 90 90 90 90 0
CONTROL 4 90 90 90 90 0 90 90 90 90 0
CONTROL 5 90 90 90 90 0 90 90 90 90 0
Average 90 90

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 Inhibition
TREATMENT Fusarium oxysporum f. sp.
cubense
24hrs 48hrs
T1 10% of guava leaves extract 0 NI 0 NI
T2 20% of guava leaves extract 0 NI 0 NI
T3 30% of guava leaves extract 0 NI 0 NI
T4 40% of guava leaves extract 0 NI 0 NI
T5 50% of guava leaves extract 0 NI 0 NI
T6 100% of guava leaves extract 0 NI 0 NI
T7 STD. Propiconazole 13.3 VSI 28.7 SI
Control 0 0
Legend:

% Fungal Growth % Fungal Inhibition Classification of Degree of Inhibition

0-20 80-100 Very High Inhibition (VH)
21-40 60-79 High Inhibition (H)
41-60 40-59 Moderate Inhibition (MI)
61-80 20-39 Slight Inhibition (SI)
81-100 1-19 Very Slight Inhibition (VSI)
0 No Inhibition (NI)

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