Botong (Barringtonia Asiatica) Seed Extract as an Anti-fungal Agent for

Powdery Mildew in Eggplant (Solanum melongena)

A Science Investigatory Project Proposal

Presented to the Faculty of
Basud National High School
In Partial Fulfilment of the Requirement
for Science Research II

Researchers:

Johnbel Abad
Jan Anthony Vela
John Martin Sayno

Mischelle Hidalgo
Research Adviser

Region V-Bicol
Division of Camarines Norte
BASUD NATIONAL HIGH SCHOOL
SY 2014-2015

1
 ABSTRACT

The constant effort of the government to increase food production resulted to the

utilization of synthetic chemicals. Indeed, this has improved crop yield over time.

However, continuous reliance to chemicals has depleted the quality of our agricultural

soil. Effects in nutrient content, acidity, and even the presence of pathogens are some

of the pressing impacts to the environment. Hence, the promotion of organic farming.

Executive Order (EO) 481 calls for the “Promotion and Development of Organic

Agriculture in the Philippines” to further guarantee food and environmental safety

towards sustainable farming.

Thus, the researchers conducted a study focusing on the anti-fungal property of

Botong seed extract against White Powdery Mildew on eggplant leaves. Effectiveness

was measured in terms of area of infection and the number of days of disinfection.

Different levels of concentrations were tested; T1, 50% Botong seed extract and 50%

water; T2, 75% Botong seed extract and 25% water; T3, 100% Botong seed extract.

Significant difference through One-Way ANOVA was utilized to determine the most

effective level of concentration.

Analysis of data revealed that Treatment 3 is the most effective treatments

among the different mixtures used in terms of area of infection and the number of days

it disinfected. From the identified infected area of 75mm, after treatment it was reduced

to 18.33 mm, followed by T2, 20mm and T1, 21.66mm respectively. Further, at 1% level

of significance, the computed f-value,15.2 is greater than the tabular value, 8.02. This

validates the alternative hypothesis of the study.

2
 RESEARCH PLAN

RATIONALE

Over the years, farmers yielded on the use of synthetic chemicals to improve

crop production. Ironically, this has led to the depletion of soil quality and the

emergence of many pathogens due to the biological alterations and chemical

components of the soil. Consequently, these chemicals are absorbed by the plants

which were passed on to us, humans. In the principle of biological magnification,

continuous in-take of chemically intoxicated plants will result to the increase of harmful

chemicals to human body. Thereby causing the widespread of different kinds of serious

diseases. Hence the saying, “We become what we eat.” The same substances pose

threats to our environment- both terrestrial and aquatic ecosystem.

Thus, the formulation of the study aimed at producing effective natural-based

fungicide using Botong (Baringtonia, asiatica) seed extract against White Powdery

Mildew on eggplants.

A. QUESTIONS OR PROBLEMS BEING ADDRESSED

This study will be conducted to determine the effectiveness of Barringtonia

asiatica seed extract as an antifungal agents against Powdery Mildew in Eggplant

(Solanum melongena).

Specifically, it will answer the following questions:

1. How effective is the Barringtonia asiatica seed extract in disinfecting the eggplant

from Powdery Mildew in terms of:

3
 A. Area of infection

B. Number of days to eliminate Powdery Mildew in eggplant (Solanum

melongena)

2. What is the most effective concentration of Barringtonia asiatica seed extract

needed to disinfect the eggplant (Solanum melongena)?

3. Is there any significant difference among the three treatments in terms of area of

infection and number of days to eliminate powdery mildews?

A. GOALS/EXPECTED OUTCOMES/HYPOTHESES

Formulation of effective yet environment-friendly fungicide against white powdery

mildew.

Hypothesis Alternative

There is a significant difference between the effects of different concentrations of

Barringtonia asiatica seed extract as an antifungal agent against White Powdery Mildew

in eggplants (Solanum melongena).

Null Hypothesis

There is no significant difference between the effects of different concentrations

of Barringtonia asiatica seed extract as an antifungal agent against White Powdery

Mildew in eggplants (Solanum melongena).

4
B. METHODOLOGY

1. RESEARCH DESIGN

The Single Group Design will be utilized in this study wherein three (3) treatment

will be used with 4 replicates.

T1 R1 T1 R2 T1 R3 T1 R4

T2 R2 T2 R1 T2 R3 T2 R4

T3 R3 T3 R2 T3 R1 T3 R4

Figure 1: Experimental Layout Design

Wherein:

Treatment 1 (T1) = 50% Botong (Barringtonia Assiatica) and 50% Water

Treatment 2 (T2) = 75% Botong (Barringtonia Assiatica) and 25% Water

Treatment 3 (T3) = 100% Botong (Barringtonia Assiatica

5
2. Materials

This study will use the following materials:

15 pcs of Botong Sterilized white cloth

Sterilized Surgical Gloves 500ml Beaker
Eggplants- 16pcs. Knife
Spray Bottle Distilled Water
Mortar and pestle Grinder

3. Methods and Procedure

1. Collection of Materials

1.1 Preparation of Laboratory Apparatus

All the laboratory equipment/apparatus will be washed in free flowing water. After

the equipment are washed, they will be placed in a clean disinfected tray with cover.

1.2 Collection of Botong (Barringtonia assiatica)

Botong (Barringtonia assiatica) fruit will be collected from Brgy. Pambuhan,

Mercedes Camarines Norte by the researchers. The seedlings will be randomly

selected by the Department of Agriculture with the help of the qualified scientist.

1.3 Planting and Caring of the Eggplant

The area of plantation is selected by the Department of Agriculture with the help

of the qualified scientist, the area will selected based from the temperature (21-30 C).

6
 The seedlings are randomly selected. After the selection, 12 seedlings will be

planted into 16 x18 size of seed bags and will be placed in the Department of

Agriculture to avoid contamination of other microorganism.

Daily watering of the eggplant will be applied and conducted to sustain the

moisture of soil.

1.4 Collection of Powdery Mildew

The Powdery Mildew will be collected from the Department of Agriculture with the

help of the qualified scientist.

Powdery Mildew will be inoculated in the leaves of each plant by putting the

leaves of powdery mildew infected eggplant at the top of uninfected eggplant.

The area of infection of powdery mildew will be monitored and measured by the

researchers every 5 days within 15 days.

2. Extraction of the Seed Extract

The seed will be grounded and pounded using blender and mortar and pestle. The

extract will be squeezed out using the sterilized cloth and will be stored in refrigerator to

avoid spoilage.

3. Preparation of Treatments

- Treatment 1 (50% Botong Seed Extract, 50% Water) fifty (50) ml of Botong seed

will be extracted and stored in a tightly sealed sterilized bottle. After that

7
 botong seed extract will be mixed in 50 ml of water, creating a 100 ml solution

that is 50% water and 50% botong seed extract

- Treatment 2 (75% Botong Seed Extract, 25% Water) seventy-five (75) ml of

Botong seed will be extracted stored in a tightly sealed sterilized bottle. After

that botong seed is extracted, it will be mixed in 25 ml of water, creating a 100

ml solution that is 25% water and 75% botong seed extract.

- Treatment 3 (100% Botong Seed Extract) one-hundread (100) ml of Botong

seed extract will be stored in a tightly sealed sterilized bottle. Water will no

longer be mixed in this treatment to create a solution that is 100% botong seed

extract

4. Application of Treatments to Uncontrolled Area

After 12 days of fungi infestation, treatment will be applied every 2 days directly to

the infected parts of the plants and will be monitored daily by the researchers. The

decrease in the area of infection will be monitored by the researchers daily.

5. Testing

The antifungal activity of Botong (Barringtonia assiatica) seed extract will be tested

against powdery mildew by applying the Botong seed extract in the inoculated pathogen

at an interval of three up to 15th day time interval. This will be done three times with

different concentration of Botong (Barringtonia assiatica). To determine which

concentration is the most effective. The researchers will observe and compute the

percent inhibition, disease incidence and the statistical analysis.

8
 Flow Chart

Collection of Planting of the

Materials Eggplant

Extraction of Seed Collection and

Extract culturing of
powdery mildew

Application of Testing
Treatments to
Uncontrolled Area

Statistical
Analysis Of Data

Figure 5: Flow Chart of Experimentation

9
4. Statistical Analysis

The data obtained will be analyzed using One-Way Analysis of Variance

(ANOVA), to test the effectiveness of Botong Seed Extract as antifungal agent against

Powdery Mildew and if there any significant difference between the different treatments

against Powdery Mildew in eggplants.

The following formula were used:

" Lx2
Tss = L.X2 -
N

1
Ssb = r ∑(sum of each column) 2 - CLX)2

Ssw = Tss – Ssb

Dft = N -1

Dfb = no. of column - 1

Mssb = Ssb
Dfb

Mssw= Ssw
Dfw

F = Mssb
Mssw

10
 INTRODUCTION

Food production through farming technology has been the subject of many

researches, particularly in the Philippines nowadays. Our country basically relies on its

agricultural resources. The constant efforts to increase highly nutritional crop yield

resulted to discovery and adoption of chemical pest control like fungicide.

Indeed, the tremendous increase in crop yields associated with the ‘green’

revolution would not have been achieved without the contribution of these synthetic

compounds. (Dayan, Franck et.al, 2009) However, concerns over the potential impact of

pesticides on the environment has now become more pressing and more stringent due

to its harmful effects.

In fact, from 1977 to 1987 pesticide use in our country, including fungicide,

increased by 325%, however, crops like rice yield increased by only 30% during this

time. Pesticide imports have continued to increase, while rice and corn yields have

increased at a much smaller rate (Tirado, Reyes & David, Bedoya, 2008). This data

revealed that synthetic fungicide and other pesticides can slightly contribute to increase

crop productions only.

Meanwhile, soil nutrients degradation is linked to the application of synthetic

fungicides and other chemicals. The N fertilization, the soil moisture, soil temperature,

and also the presence of pesticide residues in the agricultural environment may disturb

the natural degradation processes. Some substrates, including pesticides, may inhibit,

but also stimulate the microbial activity. Thus, disturbances of the microbial activity may

influence the important biogeochemical processes of the entire soil environment

(Stefani, Angelo, et.al 2012).

11
 In addition, the use of such fungicides may cause adverse effects to terrestrial

and aquatic ecosystems if fungicide residues persist in soil, or if they migrate off-site to

surface and ground waters (Wightwick, Adam et.al 2010).

Likewise, among the most common chronic effects from pesticides exposure are

eye problems (mainly chronic irritation), dermatological problems, respiratory problems

(mainly asthma), neurologic disorders and kidney and gastrointestinal abnormalities

(Tirado, Reyes & David, Bedoya, 2008).

Agricultural researchers believed that there are proven alternatives to this

expensive and harmful agriculture system: farmers are already fertilizing soils and

protecting crops with organic and sustainable techniques that work with nature, not

against it, and which can provide food for all. (Pretty & Badgley, 2003)

Actually, organic agriculture started long before the promulgation of a state policy

relating to it. But it was in 2005 that was considered a milestone year for the

government in stimulating organic agriculture through Executive Order (EO) 481 calling

for the “Promotion and Development of Organic Agriculture in the Philippines”. It aims to

promote and develop organic agriculture as a farming scheme that would enhance

global competitiveness, forge effective networking and collaboration among

stakeholders, and guarantee food and environmental safety. Furthermore, it covers the

integrity of organic products through the approved organic certification procedures and

organic production, handling and processing standards. (Organic Agriculture Act

Towards a Greener Philippines, 2013)

12
 Thus, innovations on organic or natural-based fungicides are being discovered

and developed to eradicate fungi infestation among agricultural crops and at the same

time eliminate harmful effects to the environment and mankind.

Plants are good source of antifungal agents. A large number of plant compounds

have been reported to have antifungal activity. Well known examples include;

flavonoids, lactones, proteins, sulfur compounds, cyanogenic glycosides and

glucosinolates and essential oils (Sokovic, Marina D. 2013).

According to the chemical analysis conducted by the Department of Science and

Technology, Botong (Barringtonia asiatica) seeds have a high concentration of

saponins, tannins, triterpenes and traces of alkaloids and glycosides which is a great

antifungal agent.

On the other hand, powdery mildew causes diseases to many agricultural crops.

A wide variety of vegetable crops are affected by powdery mildews, including artichoke,

beans, beets, carrot, cucumber, eggplant, lettuce, melons, parsnips, peas, peppers,

pumpkins, radicchio, radishes, squash, tomatillo, tomatoes, and turnips. Severely

infected plants may have reduced yields, shortened production times, and fruit that has

little flavor. (Davis, RM, 2008).

With these in mind, the researchers conceptualized this study to test the

antifungal effects of Botong (Barritonia, asiatica) seeds against powdery mildew using

eggplants. Hence, the recognizing the government’s agenda towards sustainable

agricultural farming and food production.

13
Statement of the Problem

This study was conducted to determine the effectiveness of Barringtonia asiatica

seed extract as an antifungal agents against Powdery Mildew in Eggplant (Solanum

melongena).

Specifically, answers to the following questions were sought:

1. How effective is the Barringtonia asiatica seed extract in disinfecting the eggplant

from Powdery Mildew in terms of:

A. Area of infection

B. Number of days to eliminate Powdery Mildew in eggplant (Solanum

melongena)

2. What is the most effective concentration of Barringtonia asiatica seed extract

needed to disinfect the eggplant (Solanum melongena)?

3. Is there any significant difference among the three treatments in terms of area of

infection and number of days to eliminate powdery mildews?

Hypotheses

Hypothesis Alternative

There is a significant difference between the effects of different concentrations of

Barringtonia asiatica seed extract as an antifungal agent against White Powdery Mildew

in eggplants (Solanum melongena).

14
Null Hypothesis

There is no significant difference between the effects of different concentrations

of Barringtonia asiatica seed extract as an antifungal agent against White Powdery

Mildew in eggplants (Solanum melongena).

Significance of the Study

Findings of this study will benefit our farmers in combatting pests and diseases in

crop production. This will help them increase their crop yield without depleting the soil

quality for future use. Eventually, there will be an increase in the agricultural profit and

better maintained physical health conditions due to least intake of harmful vapors from

synthetic fungicides.

Results of this study will provide innovative farming techniques to some of our

local government agencies such as the Department of Agriculture and the Department

of Science and Technology. Data provided could serve as baseline information for

future researches and related investigations.

Finally, our environment and the organisms living in it, including us humans, have

at least device its way to prevent our home from total deterioration from synthetic

chemicals. This kind of study promotes conservation and preservation of resources

without compromising the needs of the future generations.

15
 MATERIALS AND METHODS

A. Research Design

The Single Group Design was utilized in this study wherein three (3) treatments

were used and replicated four (4) times.

T1 R1 T1 R2 T1 R3 T1 R4

T2 R2 T2 R1 T2 R3 T2 R4

T3 R3 T3 R2 T3 R1 T3 R4

Figure 1: Experimental Layout Design

Wherein:

Treatment 1 (T1) = 50% Botong (Barringtonia Assiatica) and 50% Water

Treatment 2 (T2) = 75% Botong (Barringtonia Assiatica) and 25% Water

Treatment 3 (T3) = 100% Botong (Barringtonia Assiatica

16
B. Materials

This study used the following materials:

15 pcs of Botong Sterilized white cloth

Sterilized Surgical Gloves 500ml Beaker
Eggplants- 16pcs. Knife
Spray Bottle Distilled Water
Mortar and pestle Grinder

C. Methods and Procedure

1. Collection of Materials

1.1 Preparation of Laboratory Apparatus

All the laboratory equipment/apparatus were washed in free flowing water. After

the equipment were washed, they were placed in a clean disinfected tray with cover.

1.2 Collection of Botong (Barringtonia assiatica)

Botong (Barringtonia assiatica) fruit was collected from Brgy. Pambuhan,

Mercedes Camarines Norte by the researchers. The seedlings was randomly selected

by the Department of Agriculture with the help of the qualified scientist.

1.3 Planting and Caring of the Eggplant

The area of plantation was selected by the Department of Agriculture with the

help of the qualified scientist, the area will selected based from the temperature (21-30

C).

17
 The seedling was randomly selected. After the selection 12 seedlings was

planted into 16x18 size of seed bags and was placed in the Department of Agriculture to

avoid contamination of other microorganism.

Daily watering of the eggplant was applied and conducted to sustain the moisture

of soil.

1.4 Collection of Powdery Mildew

The Powdery Mildew was collected from the Department of Agriculture with the

help of the qualified scientist.

Powdery Mildew was inoculated in the leaves of each plant by putting the leaves

of powdery mildew infected eggplant at the top of uninfected eggplant.

The area of infection of powdery mildew was monitor and measure by the

researchers every 5 days within 15 days.

2. Extraction of the Seed Extract

The seed was grounded and pounded using blender and mortar and pestle. The

extract was squeezed out using the sterilized cloth and was stored in refrigerator to

avoid spoilage.

3. Preparation of Treatments

- Treatment 1 (50% Botong Seed Extract, 50% Water) fifty (50) ml of Botong seed

extracted stored in a tightly sealed sterilized bottle. After that botong seed

18
 extract was mixed in 50 ml of water, creating a 100 ml solution that is 50%

water and 50% botong seed extract

- Treatment 2 (75% Botong Seed Extract, 25% Water) seventy-five (75) ml of

Botong seed extracted stored in a tightly sealed sterilized bottle. After that

botong seed extract was mixed in 25 ml of water, creating a 100 ml solution

that is 25% water and 75% botong seed extract

- Treatment 3 (100% Botong Seed Extract) one-hundread (100) ml of Botong

seed extracted stored in a tightly sealed sterilized bottle.Water was no longer

mixed in this treatment to create a solution that is 100% botong seed extract

4. Application of Treatments to Uncontrolled Area

After 12 days of fungi infection, treatment was applied every 2 days directly to the

infected parts of the plants and was monitor daily by the researchers. The fungi

decrease in the area of infection was monitor by the researchers daily.

5. Testing

The antifungal activity of Botong (Barringtonia assiatica) seed extract was tested

against powdery mildew by applying the Botong seed extract in the inoculated pathogen

at an interval of three up to 15th day time interval. This was done three times with

different concentration of Botong (Barringtonia assiatica). To determine which

concentration is the most effective. The researchers will observe and compute the

percent inhibition, disease incidence and the statistical analysis.

19
 Flow Chart

Collection of Planting of the

Materials Eggplant

Extraction of Seed Collection and

Extract Culturing of White
Powdery Mildew

Application of Testing
Treatments to
Uncontrolled Area

Statistical Analysis
of Data

Figure 5: Flow Chart of Experimentation

20
D. Statistical Analysis

The data obtained was analyzed using techniques of One-Way Analysis of

Variance (ANOVA), to test the effectiveness of Botong Seed Extract as antifungal agent

against Powdery Mildew and if there any significant difference between the different

treatments against Powdery Mildew in eggplants.

The following formula were used:

" Lx2
Tss = L.X2 -
N

1
Ssb = r ∑(sum of each column) 2 - CLX)2

Ssw = Tss – Ssb

Dft = N -1

Dfb = no. of column - 1

Mssb = Ssb
Dfb

Mssw= Ssw
Dfw

F = Mssb
Mssw

21
 RESULTS

This study aimed to determine the effectiveness of Botong (Barringtonia asiatica)

seed extract as an anti-fungal agent against the Powdery Mildew in eggplant (Solanum

melongena). Significant difference among the treatments prepared were likewise

measured to identify the most effective mixture in inhibiting fungal activity. Data

gathered were arranged and presented using tables to systematically answer the

posted objectives.

1. Effects of Botong (Barringtonia asiatica) Seed Extract as an Antifungal

Agents Against Powdery Mildew in Eggplant

A. Area of Infection

Table 1

Area of Infection of Fungi (in millimeter) After 12 Days of Infecting the Eggplant

TREATMENTS REPLICATES TOTAL MEAN

R1 R2 R3 R4
T1 75mm 75mm 75mm 75mm 225mm 75mm
T2 75mm 75mm 75mm 75mm 225mm 75mm
T3 75mm 75mm 75mm 75mm 225mm 75mm

Table 1 shows the area of infection of powdery mildew after 12 days of infecting

the eggplant. After the inoculation, an average area of 75 mm was identified as the

observation site for the antifungal effect of the Botong seed extract per eggplant. The
22
smaller the area becomes after the application of the extract, the more effective is its

anti-fungal property against the White Powdery Mildew.

Table 2

Area of Infection of Fungi (in millimeter) After 2 Days of Application of Treatment

TREATMENTS REPLICATES TOTAL MEAN

R1 R2 R3 R4
T1 25mm 5mm 20mm 15mm 65mm 21.66mm
T2 10mm 20mm 15mm 15mm 60mm 20mm
T3 20mm 5mm 0mm 30mm 55mm 18.33mm

Table 2 shows the area of infection of powdery mildew after 2 days upon

application of treatments. It was revealed that Treatment 3, which is composed of 100%

Botong seed extract, is the most effective concentration since it has the lowest

computed mean of 18.33 mm.

TABLE 3

Summary of Area of Infection Before and After the Application of Treatments

Area of Infection before Area of Infection After

Treatments Difference
Application of Treatment Application of Treatment
T1 225mm 65mm 160mm
T2 225mm 60mm 165mm
T3 225mm 55mm 170mm
Total 765mm 180mm 585mm

Table 3 shows the comparative effects of Botong seed extract to White Powdery

Mildew before and after the application of treatments. Treatment 3 reveals the biggest

23
difference in the decrease of area which is 170mm, indicating the most effective among

the treatments used. However, data further imply that despite the slight differences in

the area of infection, all treatments were proven effective in disinfecting white powdery

mildew since the affected area are reduced from its original size.

B. Number of Days Eliminating White Powdery Mildew in Eggplant

Table 4

Number of Days to Disinfect the Eggplant After Application of Treatments

Treatments Replicates Total Mean

R1 R2 R3 R4
T1 8 5 3 3 19 4.75
T2 3 6 3 6 18 4.5
T3 6 4 2 3 15 3.75

Table 4 shows the antifungal effect of Botong seed extract against powdery

mildew in terms of the number of days of disinfection. Data above revealed that

Treatment 3 gained an average days of 3.75 followed by Treatment 2 and 1

respectively. The data showed that there is a slight difference in the effectiveness of the

three treatments as to the days of disinfecting the eggplant using the extract.

24
 2. Most Effective Concentration of Barringtonia asiatica Seed Extract Needed

to Disinfect the Eggplant (Solanum melongena)

Table 5

Comparison on the Effectiveness of the Different Treatments Used in

Disinfecting White Powdery Mildew on Eggplants.

Area of Infection Number of Days of

Treatments
Disinfection

T1 21.66mm 4.75
T2 20mm 4.5
T3 18.33mm 3.75

Table 5 shows the comparison of the data based on the two indicators set to

determine the most effective level of concentration used in the study. From the above

table, Treatment 3 revealed to be the most effective mixture among the three treatments

since it has the least size of the area of infection and least number of days of

disinfection. Consequently, this was followed by Treatment 2 and 3.

25
3. Significant Difference Among the Three Treatments in Terms of Area of

Infection and Number of Days to Eliminate Powdery Mildews

Table 6

SS df MS F

Between 1, 138.487 2 8.316702 15.2

Within 16.633404 2 569.239 F- tab. 1% =

8.02

Total 1155.111404 3 F– tab 5% =

4.26

ANOVA Table for the Significant Difference Based on the Area of Infection

Table 5 shows the data computed from the Analysis of Variance. At 1% level of

significance, the computed F-value is 15.2. This is greater than the tabular value of

8.02 which indicates that there is a significant difference among the different levels of

concentrations of Botong seed extract.

26
 TABLE 7

ANOVA Table for the Significant Difference Based on the Number of Days

SS df MS F

Between 30.75 2 15.375 3.780564946

Within 0.9038745 2 0.45193725 F- tab. 1% =

8.02

Total 1155.111404 3 F– tab 5% =

4.26

of Eliminating White Powdery Mildew

ANOVA computation revealed that at 1% level of significance, the computed F-

value is 3.78. This is lower than the tabular value of 8.02 indicating that there is no

significant difference among the three treatments used in terms of the number of days

of infection.

27
 DISCUSSION

The present study focused on the antifungal property of a local plant specie

found in Mercedes, Camarines Norte called Botong (Barringtonia asiatica) against the

White Powdery Mildew in eggplant (Solanum melongena). Effectiveness is measured

according to the area of disinfection and the number of days to disinfect the fungi from

the eggplant.

Based from the results presented, it was revealed that Treatment 3, composed of

100% Botong seed extract, most effectively eradicated white powdery mildew on the

leaves of the eggplants. It was further revealed that there is a significant difference in

the effects of the different treatments used. The same level of treatment exhibited

shortest number of days as to its antifungal activity.

The excellent antifungal property of many plants including Botong, is attributed to

the chemical compounds present in its extract. Based from the chemical analysis

conducted by the Department of Science and Technology Botong seed extract contains

alkaloids, triterpenes, glycosides and a high concentration of saponins and tannins.

Alkaloid is a chemical substance that originated from plants and is composed of

carbon, hydrogen and oxygen. They occur in many genera of plants. Alkaloids can be

found in almost all parts of these plants, including the leaves, roots, seeds, and bark.

Each plant part usually contains several chemically related alkaloids.

(http://science.howstuffworks.com/alkaloid-info.htm)

The antifungal activities of many phenol compounds isolated from Holm Oak

Quercus ilex leaves, belonging to the classes of flavonoids, proanthocyanidins, and

phenol acids, have been examined against fourteen fungal species (Karioti et al., 2011).

28
Two coumarins, scopoletin and isoscopoletin, two simple phenol acids, protocatechuic

acid and isovanillic acid and one flavonoid, eriodictyol separated from the aerial parts of

Centaurea spruneri, showed fungistatic activity at 0.259–2.38 μmol/ml and fungicidal at

0.69–2.6 μmol/ml against all fungi tested.

Similarly, according to Morteza-Semnani K et.al, the methanolic extract and total

alkaloids of the aerial parts of Glaucium oxylobum exhibited good activity against

Microsporum gypseum, Microsporum canis, Trichophyton mentagrophytes and

Epidermophyton floccosum. Four alkaloids, dicentrine, glaucine, protopine, and alpha-

allocryptopine, were identified as the compounds responsible for the antifungal activity

of this plant. (http://www.ncbi.nlm.nih.gov/pubmed/12837370)

The toxic anti-bacterial or anti-fungal activities of plant alkaloids have been

shown in a number of studies. Recently Joosten, Lotte et.al reported findings of similar

studies. Accordingly, quinolizidine alkaloid extracts (inhibitory concentrations of 62.2–

500 μg/ml) from the aerial parts of Lupinus angustifolius and Genista vuralii showed

activity against several different bacterial species. Anti-fungal effects by alkaloids also

have been found for several plant associated fungi by bioassay experiments.

Further, several quinolizidine alkaloids were mentioned that inhibited the

germination of conidia Erysiphe graminis. Results showed that two furoquinoline

alkaloids showed activity against the phytopathogenic fungus Cladosporium

cumcumerinum. The inhibitory concentrations of the two alkaloids, dictamnine and

haplopine, was 25 μg/ml and originated from root bark of Dictamnus dasycarpus.

Another study showed that the isoquinoline alkaloids, corynoline and

acetylcorynoline, inhibited the fungal growth of Cladosporium herbarum at a

29
concentration of 3 μg/ml. Similar report revealed that steroidal alkaloids from the

rhizomes and roots of Veratrum taliense inhibited the growth of the phytopathogenic

fungi, Phytophthora capisis and Rhizoctonia cerealis in concentrations of 80–200 μg/ml.

(Joosten, Lotte et.al 2010)

Saponins, on the other hand, are secondary plant metabolites that occur in a

wide range of plant species. They are stored in plant cells as inactive precursors but are

readily converted into biologically active antibiotics by plant enzymes in response to

pathogen attack. These compounds can also be regarded as ‘preformed’, since the

plant enzymes that activate them are already present in healthy plant tissues. (Figen

Mert-Turk, 2005)

The production of low-molecular-weight antimicrobial molecules within plants is

one component of defense against pathogens. Among them, preformed antimicrobial

compounds (phytoanticipins) are the first biochemical barriers against pathogens.

Saponins (a group of phytoanticipins) are present constitutively in plants and play

important roles in plant defense.

A variety of biological roles have been postulated for different saponins, involving

allelopathic activity, defense against insects and pathogens. The mechanism of

antifungal action of saponins is not well understood but it is believed that they complex

with sterols in the cell membrane, leading to pore formation and consequent loss of

membrane integrity. Saponins that have been studied in detail in relation to their

potential role in plant defense against attack by phytopathogenic fungi are triterpenoid

avenacins in oat roots and the steroidal glycoalkaloids a-tomatine in the leaves of

tomato. (Figen Mert-Turk, 2005)

30
 Related study was done using three saponins, named minutoside A (1),

minutoside B (2), minutoside C (3), and two known sapogenins, alliogenin and

neoagigenin,were isolated from the bulbs of Allium minutiflorum Regel. The isolated

compounds were evaluated for their antimicrobial activity. All the novel saponins

showed a significant antifungal activity depending on their concentration and with the

following rank: minutoside B > minutoside C minutoside A. (Barile, Elisa et.al 2006 )

Moreover, Saponin rich-extracts (SREs) from Balanites aegyptiaca fruit

mesocarp (BE), Quillja saponaria bark (QE), and Yucca schidigera (YE) have been

tested against common phytopathogenic fungi (Pythium ultimum, Fusarium oxysporum,

Alternaria solani,Colletotrichum coccodes, and Verticillium dahliae). The inhibitory effect

of these extracts was measured in vitro and the concentrations that will reduce the

colony diameter of fungus to 50% of the control (DRC50 values) were determined.

Dose-dependent fungal and/or saponin-specific results were obtained. The highest

concentration of BE (4%) showed high (81.1%) and moderate (34.7%) growth

inhibitions against P. ultimum and A. solani, respectively, whereas weak growth

inhibition or weak growth stimulation occurred against F. oxysporum, C. coccodes, and

V. dahliae. The highest concentration (4%) of QE showed moderate growth inhibition

(35.9–59.1%) of all fungi except C. coccodes. YE showed moderate to very high (54.1–

100%) growth inhibition of all fungi tested. The results of this study suggest that

selected SREs can play an important role in controlling these fungi, especially under

organic management.

Tannins are defined as phenolic compounds of high molecular weight ranging

from 500 to more than 3000 which they found in plants leaves, bark, wood and bound to

31
proteins that form insoluble or soluble tannin-protein complexes. It is one of the most

important secondary metabolites. They have positive effects on animals by anti-

microbial, anthelmintic, protein bypassed effects in ruminants.(Hassanpou, 2011)

Other secondary metabolites, including terpenes, phenolics and nitrogen (N) and

sulphur (S) containing compounds, defend plants against a variety of herbivores and

pathogenic microorganisms as well as various kinds of abiotic stresses. This substance

protect plants against herbivory, pathogenic microbes and various abiotic stresses as

well as specific plant responses to pathogen attack, the genetic control of host-

pathogen interactions (Mazid, 2011).

Secondary metabolites are relevant to agriculture. The very defensive

compounds that increase the reproductive fitness of plants by warding off fungi,

bacteria, and herbivores may also make them undesirable as food for humans. (Ördög

Vince, 2011)

Simple triterpenes are components of surface waxes and specialized

membranes and may potentially act as signaling molecules, whereas complex

glycosylated triterpenes (saponins) provide protection against pathogens and pests.

Related evidences mentioned above confirmed the combinations of suitable

substances present in plant extract, such as alkaloid, triterpenes, tannins, and saponins,

resulted in even greater increase in the antifungal potential of Botong (Barringtonia

assiatica) seed extract against White Powdery Mildew (WPM) in eggplant. These further

verify why Treatment 3 or the 100% botong seed extract demonstrated significant

antifungal effect against WPM as to the area of disinfection.

32
 Consequently, if the number of days is to be considered for the eradication of

WPM, results showed that there is no significant difference. This implies that all

treatments are effective antifungal agents regardless of the length of time of

disinfection.

However, no statistical account as to the percentage of anti-fungi substances

were identified from the applied treatments since the study delimited its scope to the

indicators set by the researchers only.

33
 CONCLUSION

The effectiveness of the anti-fungal property of Botong (Barringtonia, assiatica)

seed extract was measured in terms of the size of the area of disinfection 2 days after

the treatment and the number of days it eradicated the White Powdery Mildew (WPM)

on eggplants leaves. Observation area infected with fungus was set at an average of

75mm per eggplant leaf.

Different levels of concentrations were used as treatments namely: T1, Botong

(Barringtonia Assiatica) and 50% Water; T2, 75% Botong (Barringtonia Assiatica) and

25% Water; and T3, 100% Botong (Barringtonia Assiatica).

Comparative results showed that T3 significantly decreased the area of infection

from 75 mm to 18.33 mm; T2 with a mean decrease of 20mm and T1, 21.66mm

respectively. Analysis of Variance revealed significant difference among the effects of

the three treatments.

At 1% level of significance, the computed F-test value is 15.2, greater than the

tabular value of 8.02. Since the computed F-test value is greater than the tabular value

of 8.02, null hypothesis is rejected. Thus, there is a significant difference in the effect of

the Botong seed extract against white powdery mildew on eggplants.

This suggests that if a farmer would use Botong seed extract to eradicate white

powdery mildew on eggplants, T3 or 100% extract, being the most effective level of

concentration, must be used to effectively treat fungal infestation.

Similar result was revealed as to the number of days the Botong seed extract

eradicated the white powdery mildew using the different treatments. Length of days

were as follows: T1, 4.75 days, T2, 4.5 days and T3, 3.75 days. Treatment 3 has the

34
shortest number of days to eradicate White Powdery Mildew, signifying the most

effective among the three.

Analysis of Variance revealed that the computed value of 3.78 is lower than the

tabular value of 8.02 at 1% level of significance indicating no significant difference. This

is manifested by the slight difference on the number of days the Botong seed extract

disinfected the infested eggplant. Thus, all treatments are equally effective based on the

number of days it eradicated white powdery mildew on eggplant.

If a farmer would consolidate all the findings of this study, it would be best for him

to choose T3 or the 100% extract of Botong seed. Although all treatments are similarly

effective as to the number of days it can disinfect white powdery mildew, however, it is

best to consider that T3 was the most effective level of concentration among the three

as exhibited in the size of area of infection.

35
 RECOMMENDATIONS

Based from the results and findings of the research, it was therefore

recommended that the result of the study be communicated among the scientific

community for further in-depth investigations particularly on the utilization of more

improved extraction technique. In this manner, the quality and quantity of the antifungal

extract might be better improved for massive production and consumption.

It is further recommended that more similar researches be made to promote

sustainable farming techniques particularly in the Philippines.

36
 REFERENCES

A. BOOKS

1. Franck E. Dayan, Franck E., Charles L. Cantrell, Stephen O. Duke. Natural

Products in Crop Protection. Natural Products Utilization Research Unit,
Agricultural Research Service, United States Department of Agriculture,
University, MS 38677, USA

B. ARTICLE

1. Tirado, Reyes & David, Bedoya. Agrochemical use in the Philippines and its
consequences to the environment. Greenpeace Southeast Asia 24 KJ Street,
East Kamias Quezon City, Philippines, 1102.February 2008

2. Wightwick, Walters; Roberts, Allinson; Graeme, Reichman; Suzanne and

Neal Menzies Environmental Risks of Fungicides Used in Horticultural
Production Systems, Fungicides, Odile Carisse (2010). (Ed.),ISBN: 978-953-
307-266-1

3. Pretty, J. N., J. I. L. Morison, and R. E. Hine. 2003. Reducing Food Poverty

by Increasing Agricultural Sustainability in Developing Countries. Agriculture,
Ecosystems & Environment 95:217-234.

4. Mazid M, Khan TA, Mohammad F. Role of Secondary Metabolites in Defense

Mechanisms of Plants. Biology and Medicine, 3 (2) Special Issue: 232-249,
2011. eISSN: 09748369, www.biolmedonline.com

5. Ördög Vince, Molnár Zoltán Debreceni Egyetem, Nyugat-Magyarországi

Egyetem, Pannon Egyetem. Plant Physiology. (2011)
http://www.tankonyvtar.hu/en/tartalom/tamop425/0010_1A_Book_angol_01_n
ovenyelettan/ch03s05.html

6. Albert P. Aquino, Jamaica Angelica P. Deriquito, and Meliza A. Festejo.

Organic Agriculture Act Towards a Greener Philippines. 2013

C. JOURNALS

1. Karioti, A.; Soković, M.; Ćirić, A.; Koukoulitsa, C.; Bilia, R.A. & Skaltsa, H.
(2011). Antimicrobial Properties of Quercus ilex L. Proanthocyanidin Dimers
and Simple Phenolics: Evaluation of Their Synergistic Activity with
Conventional Antimicrobials and Prediction of Their Pharmacokinetic Profile.

37
 Journal of Agricultural and Food Chemistry, Vol.59, No. 12, pp. 6412-6422,
ISSN 0021-8561

2. Figen Mert-Turk . Saponins Versus Plant Fungal Pathogens. Journal of Cell

and Molecular Biology 5:13-17, Halic University. Printed in Turkey.2006.

3. Hassanpour, et.al Plants and Secondary Metabolites (Tannins): A Review

International Journal of Forest, Soil and Erosion (IJFSE).. Vol.1. No.1. 2011

D. PUBLISHED THESIS

1. Stefani, Angelo Jr., D’Arc Felício, Joanna & de Andréa, Mara M.Comparative
Assessment of the Effect of Synthetic and Natural Fungicides on Soil
Respiration. Published: 7 March 2012

2. Veljić, M.; Tarbuk, M.; Petar D.M.; Ćirić, A.; Soković, M. & Marin, M.
(Antimicrobial Activity of Methanol Extracts of Some Genuine Mosses from
Serbia. Pharmaceutical Biology, Vol. 46, No. 12, pp. 871-875, ISSN 1388-
0209 (2008)

3. Duke, O.S.; Dayan, E.F.; Romagni, G.J. & Rimando, M.A. (2000). Natural
products as sources of herbicides: current status and future trends. Weed
Research, Vol. 40, pp. 99-111, ISSN1365-3180

4. Ramesha Thimmappa, Katrin Geisler,Thomas Louveau,Paul O'Maille, and

Anne Osbourn. Triterpene Biosynthesis in Plants. Annual Review of Plant
Biology.Vol. 65: 225-257 (Volume publication date April 2014).First published
online as a Review in Advance on January 29, 2014.DOI: 10.1146/annurev-
arplant-050312-120229

38
 ACKNOWLEDGEMENT

The Researchers would like to express their deepest gratitude to the following

persons who made this study a success:

Above all, our Almighty God, our Father in heaven, for all the provision and

guidance throughout the realization of this research.

To Mr. Isagani F. Musa and Mischelle Hidalgo our Research Advisers, for

technical and intellectual supports; for pursuing and inspiring us to keep on keeping on.

To Mrs. Angeline M. Ross, our Class Adviser, for understanding and

considerations, allowing us to cope with our missed lessons during the conduct of this

study.

To Mr. Harry Felomino, our Adult Scientist, for sharing his expertise, knowledge

and technical supports needed in our research.

And lastly, to our beloved families, for all the support they have given us- morally,

spiritually, and financially; for understanding and for believing in us. Thank you for all

your prayers and patience.

To God be the glory.

The Researchers

39
 APPENDICES
A. Photographs of Procedure and
Experimentations

B. Computation of the F- test of Tables

2, 5, and 7

40
 APPENDIX A

Preparation of Materials Needed

Grinder Knife Mortar and Pestle

Beaker Sterile Gloves Graduated Cylinder

Sterile Cloth Spray Bottle

41
 Gathering of Botong

Picking botong fruit from tree

Finding Botong Tree

Colected botong fruit

42
Collection and Culturing of Powdery Mildew

Collected Powdery Mildew

Inoculating Powdery Mildew

43
 Extraction of the Seed Extract

Collected Botong fruit Pounding Botong Seed Botong Seed ready for

extraction

Washing Botong fruit Grinding Botong Seed

Extracting the seed extract

Cutting Botong fruit Slicing botong seed

Botong fruit spited in to two Extracting botong seed

44
 Application of Treatments to Uncontrolled Area and Testing

Eggplant leaves infected

with powdery mildew
Treatment number 2 Treatment number 3
(75%botong seed extract, (100%botong seed
25% water) extract)

Measuring Area of
Infection
Arranging set-up

Treatment number 1
(50%botong seed extract,
50% water)

Tracing the Area of Application of Treatments

Infection

45
Statistical Analysis
Table 2

TREATMENT REPLICATES TOTAL MEAN

R1 R2 R3 R4
T1 25mm 5mm 20mm 15mm 65mm 21.66mm

T2 10mm 20mm 15mm 15mm 60mm 20mm

T3 20mm 5mm 0mm 30mm 55mm 18.33mm

Y1 = 𝟐𝟓+𝟓+𝟐𝟎+𝟏𝟓
𝟒

=21.66

Y2 = 𝟏𝟎+𝟐𝟎+𝟏𝟓+𝟏𝟓
𝟒

=20
𝟐𝟎+𝟓+𝟎+𝟑𝟎
Y3 = 𝟒

= 18.33
𝟐𝟏.𝟔𝟔+𝟐𝟎+𝟏𝟖.𝟑𝟑
Y= 𝟑

Sb = 3 (21.66 – 19.996)2 + 3 (20 – 19.996)2 + 3 (18.33 – 19.996)2

= 16.633404

Fb = 3-1

=2
𝟏𝟔.𝟔𝟑𝟑𝟒𝟎𝟒
Msb= 𝟐

46
 =8.316702

SW = (20-21.66) 2+(5-21.66) 2+(20-21.66) 2+(15-21.66) 2+(10-20) 2+(20-20) 2+(15-20) 2+ +(15-20)

2
+(20-18.33) 2+(5-18.33) 2+(0-18.33) 2+(30-18.33) 2

=1,138.478

Fw = 𝟑(𝟒 − 𝟏)

=9
𝟏𝟏𝟑𝟖.𝟒𝟕𝟖
Msw= 𝟗

𝟏𝟐𝟔.𝟒𝟗
F =𝟖.𝟑𝟏𝟔𝟕𝟎𝟐

. =15.2

Table 4

Number of Days to disinfect the eggplant after Application of Treatment

Treatment Replicate Total Mean

R1 R2 R3 R4

T1 8 5 3 3 19 4.75

T2 3 6 3 6 18 4.5
T3 6 4 2 3 15 3.75

Days to Disinfect
𝟖+𝟓+𝟑+𝟑
Y1 = 𝟒

=4.75
𝟑+𝟔+𝟑+𝟔
Y2 = 𝟒

=4.5
𝟔+𝟒+𝟐+𝟑
Y3 = 𝟒

=3.75

47
 𝟒.𝟕𝟓+𝟒.𝟓+𝟑.𝟕𝟓
Y= 𝟑

=4.3333

Sb = 3 (4.75 - 4.3333)2 + 3 (4.5 – 4.3333)2 + 3 (3.75 – 4.3333)2

= 𝟏. 𝟖𝟎𝟕𝟗𝟗

Fb = 4-1

=2
𝟏.𝟖𝟎𝟕𝟗𝟗
Msb = 𝟐

=0.9038745

Sw= (8-4.75) 2+(5-4.75) 2+(3-4.75) 2+(3-4.75) 2+(3-4.5) 2+(6-4.5) 2+(3-4.5) 2+ +(6-4.5) 2+(6-3.75)
2
+(4-3.75) 2+(2-3.75) 2+(3-3.75) 2

=30.75

Fw = 3(4-1)

=9
𝟑𝟎.𝟕𝟓
Msw= 𝟗

𝟑.𝟒𝟏𝟔
F=
𝟎.𝟗𝟎𝟑𝟖𝟕𝟒𝟓

. =3.780564946

48
49