Bioefficacy of Bitter Vine (Mikania micrantha), Tuba-tuba (Jatropha curcas), and Paragis

(Eleusine indica) Plant Extracts against Fusarium oxysporum f.sp. cubense

Tagum City National High School

Research Proposal

Group Category

Proponents:

Frences Ann B. Villamor

Gyle C. Boctoto

Marian Elloisa P. Gulay

Mr. Henry F. Haranay

Research Adviser

April 2019
 Bioefficacy of Bitter Vine (Mikania micrantha), Tuba-tuba (Jatropha curcas), and Paragis
(Eleusine indica) Leaf Extracts against Fusarium oxysporum f.sp. cubense

Rationale

Bananas are monocotyledonous plants in the genus Musa belonging to the family
Musaceae (Abdullahi et al.,2018). Philippines is one of the world’s top producers of bananas.
Banana industry in our province is very important because it composes 70% of our economy
(Notarte, 2018). Fungi inflict losses up to 90% of agricultural crop. Bananas suffer from various
types of fungi diseases, such as Mycosphaerella leaf spots, Black and Yellow Sigatoka, Eumusae
leaf spot, Mycosphaerella speckle, and lastly the Panama disease ( Abdulahhi et al., 2018) Panama
disease (or Fusarium wilt of banana) is a plant disease that infects banana caused by the fungus
Fusarium oxysporum f.sp. cubense (Foc). This fungus is a soil-borne infection that kills banana. It
enters the plant through the roots. As it goes upward, it stops up the vascular tissues and obstructs
the flow of water and supplements. The first visible banana fusarium wilt symptoms are stunted
growth, leaf distortion and yellowing, and wilt along the edges of mature, lower leaves (Dyer,
2018). As for now, the farmers are continuing their effort to have this kind of eradication which is
burning. add other methods of treating the disease (common and innovative practices). state what
you want to happen to panama disease.

Mikania micrantha (Bitter vine) came from the family of Asteraceae is known as mile-a-
minute weed because of its fast-growing characteristics. It has been present in south china since
the 1980s and had caused adverse impact on agricultural production in the established area because
it steals the photosynthesis of other plants. Phytochemical tests of the crude extract, partitionates
and fractions of M. micrantha had detected the presence of tannins, polyphenols, alkaloids,
saponins and triterpenoids (Matawali et al., 2016). Also, in the study of Zhuang Shi-hong and
others, it also has four compounds which was isolated from ethanol extracts of M. micrantha, these
are stigmasterol, deoxymikanolide, dihydromikanolide and Beta-sitosterol which has antifungal
properties. explain what will it do to the fungus. pangita pud literature/studies about the percent
composition of these substances in the plant.
 Jatropha curcas (Tuba-tuba) is a physic nut in the spurge family, Euphorbiaceae. Leaf
extracts of J. curcas had demonstrated insecticidal effects against crop pests such as Helicoverpa
armigera and Sitophilus zeamais. Its leaf extracts yield alkaloids, flavonoids, saponins, tannins,
phenolic compounds, steroids, terpenoids (Igbinosa et al., 2009). Leaves contain apigenin, vitexin,
isovitexin used for malaria, rheumatic and muscular pains. According to Yin and others, they found
that 9-hexadecenoic acid, a component present in J. curcas leaf extracts, has antifungal properties.
explain what will it do to the fungus. pangita pud literature/studies about the percent composition
of these substances in the plant.

Eleusine indica (Paragis) is a tufted annual grass belonging to the family poaceae formally
known as Gramineae family. It is an invasive annual weed found all over the warmer areas of the
world. E. indica is used in herbal medicine in different continents. It is used as antidiarrhea (Lans,
2007), diuretic and anthelmintic (Gunjar et al, 2012, Gruyal et al, 2014), and management of
fertility (Ashidi et al, 2013). It has also been shown to have antidiabetic and antimalarial activity
(Okokon et al, 2015) as well antioxidant and anti-inflammatory activity (Sagnia et al, 2014). The
results of the phytochemical test revealed the presence of alkaloids, flavonoids, cardiac glycosides,
tannins and acidic compounds. (Alaekwe et al., 2015). The compounds isolated from the
chloroform and methanol extracts of E. indica were found to be derivatives of Hexadecanoic acid,
phosphatidylethanolamine and ethanediyl ester. The compounds isolated are 1 – [[[(2-
aminoethoxy)hydroxyphosphinyl]oxy]methyl]1-,2-ethandiylester and Hexadecanoic acid for the
chloroform and methanol extracts respectively.These extracted compounds are known to be anti
cancerous, antifungal , and anti – convulsant and are used in treating various viral and fungal
related diseases. This confirmed the traditional uses of E. indica. Explain why you have chosen
these plants to study against the disease. pangita pud literature/studies about the percent
composition of these substances in the plant.

Research Questions:

1. Can the extract of Mikania Micrantha, Jatropha curca, and Eleusine indica influence the growth of
Fusarium oxysporum f.sp. cubense?

What is the measure of the zone of inhibition produced by the treatments of plant extracts, such as:

A. Mikania
B. Jatropha
 C. Eleusina

What is the measure of the zone of growth produced by the combination of extracts such as:

A. mikania and jatropha

B. mikania and eleusina
C. jatropha and eleusina, and
D. mikania, jatropha, and eleusina?

2. What particular plant extract has the highest capability in inhibiting the growth of Fusarium oxysporum
f.sp. cubense?

3. What is the minimum concentration level of each extract needed to suppress the growth of Fusarium
oxysporum f.sp. cubense?

D. Mikania
E. Jatropha
F. Eleusina

what is the minimum concentration level of each combined extract needed to suppress the growth of
fusarium:

A. mikania and jatropha

B. mikania and eleusina
C. jatropha and eleusina, and
D. mikania, jatropha, and eleusina?

4. Which has the highest capability of inhibiting the growth of Fusarium oxysporum f.sp. cubense? Is it the
pure extract, the combination of two different extracts or the combination of the three extracts?

what the most effective treatment in inhibiting the growth of fusarium in terms of the zone of growth?

Hypothesis

If Mikania micrantha, Jatropha curcas, and Eleusine indica extracts contain chemicals that can
suppress the growth of Fusarium oxysporum f.sp. cubense, then it will create a zone of growth. show
the relationship between variables then predict (e.g. the zone of growth will stay the same if the
mikania plant extracts was treated to fusarium) , kung pila kabook inyong extracts mao pud ang
kadaghanon sa hypothesis… isa-isahon gyud kada extract.. then ang final hypothesis mao ni:
there is an optimum zone of growth observed in fusarium if the combined extracts of mikania,
jatropha, and eleusina was used

Objectives/ Expected Outcomes

The researchers want to suppress fusarium wilt in banana plants that is caused by
Fusarium oxysporum f.sp. cubense by utilizing the plant extracts of Mikania micrantha, Jatropha
curcas, and Eleusine indica as an antifungal agent. we expect…. (that the combination of
extracts would produce better results than that of a single plant extract).

Materials and Methodology

A. Preparation of Media

The medium to be used is Potato Dextrose Agar (PDA). Measure 500g of potato, 45g of dextrose,
45g of agar and 2L of distilled water. Wash 500 g potato, peel off the skin and slice them into
small pieces. Boil the potatoes in 2 liters distilled water for an hour in an open vessel or pressure
cooker. Filter through eight cheesecloth layers. Discard the substantial portion; then add 45g of
dextrose and agar to the liquid part of the potato, Mix it thoroughly and return to heat until the agar
is fully dissolved (around 40-50 min). Withdraw the media from heat and dispense it in sterilized
Erlenmeyer flasks. Cover each container with a piece of loosely crimped aluminum foil, so the foil
will not fall off but does not make a tight seal. Place the flasks in an autoclave and operate it
following the manufacturer's instructions to sterilize the PDA at 121°C (250 degrees F) at a
pressure of 103,421 Pascal (15 PSI) for 15 minutes. Shut off the autoclave and let it return to
atmospheric pressure, then remove the flasks and let them cool to handling temperature. Fill the
sterilized Petri dishes halfway with PDA solution. include how many petri plates with agar will be
produced. (e.g. there will be 30 petri plates with pda that will be used in this experiment).

B. Isolation and accession of Fungi

Fusarium oxysporum f.sp. cubense will be isolated from the pseudostem of infected banana plant.
Samples of infected banana plant will be obtained in Mankilam, San Miguel, Kapalong, Magdum,
and Tagum City. include safety procedure in this section (e.g. with personal protective equipment,
we will be isolating the fungus from an infected banana plant; delikado baya ang fusarium, basin
mukuyanap). state where will you do this experiment (asa mo mag-isolate), dili pwede sa field…
I guess pwede ni mahitabo sa PAGRO… just check on them.
C. Culturing of Fungi

explain what is the purpose of this step. Cut the discoloured vascular strands from inner banana
pseudostem into small pieces approximately 0.5 cm. and then rinsed 2 – 3 times with distilled
water after soaked in 1% Sodium hypochlorite for 2 – 3 min. Place the tissue sections in Water
Agar (WA).When mycelium grown from the tissue sections, transfer it to Potato Dextrose Agar
(PDA) and incubate at 25º C for 7 – 10 days, until getting pure culture.

D. Collection of plant samples

The leaves of tuba-tuba (Jatropha curcas), the whole plant of bitter vine (Mikania micrantha) and
paragis (Eleusine indica) will be collected from Mangga, Visayan Village, Tagum City and will
be assessed by the Provincial Agricultural Office, Mankilam, Tagum City for authenticity.

E. Preparation of Plant extracts

Prepare the leaves of tuba-tuba, bitter vine, and and paragis by weighing 350g each. Each plant’s
leaves will be It will be blended and will be macerated with 500 ml 95% Ethanol in room
temperature for three days and be stirred occasionally. After that this procedure, the extract will
be filtered filter the extract using Whatman No. 1 filter paper. Then, it will be concentrated by
evaporation at 40°C in a rotary evaporator. asa buhaton ang rotary evaporation? The whole plants
of bitter vine and paragis will undergo the same process.

F. Experimentation

This experimentation will be laid out with Complete Randomized Block Design (CRBD) with nine
treatments, each with three replicants in five trials. The expected outcome of each trial is that the
experimental variables will create a zone of growth. after the extraction, the treatments will now
commence. the following setups will be followed in testing the plant extracts….

Testing will be done through poisoned food technique. Each extract will be incorporated
into the prepared medium. then, the fungus will be transferred to the petri plates.

NOTE: INCLUDE DIAGRAMS kung asa nga SPOTS sa PETRI PLATES ibutang ang (mga)
fungus, simple drawing lang ninyo.

example:
Prepare the following treatment

setup extract

1 5 mL of mikania (100% extract)

2 5 mL of jatropha (100% extract)

3 5 mL of eleusina (100% extract)

4 2.5 mL of mikania and 2.5 mL of jatropha

(100% extract)

5 etc… (100% extract)

# positive control (fungicide) (% solution)

# negative control (no treatment)

the treatments will be replicated 5 times to determine the consistency of the results. the experiment
will be done at ___________ which will be assisted by (QS Name / Designated Supervisor Name).
the procedures in this experimental plan are adapted from (Name kung asa gikan ang procedure)
and was validated by our qualified scientist (Name of QS).

The positive control variables will be treated with commercial fungicide specifically Chlorotalonil
(state concentration) and the negative control variable will be no treatment.

To obtain the data, we have to observe the growth of Fusarium oxysporum inside the petri dish if
there is a zone of growth. Zone of growth will be collected every 24 hours for 5 days after
inoculation. qualitative data will also be obtained using photo-documentation and actual
observations.

Treatment 1- Pure bitter vine plant extract (100% concentration)

Measure 9ml of the bitter vine plant extract

Treatment 2- Pure tuba-tuba leaf extract (100% concentration)

Measure 9ml of the tuba-tuba leaf extract

Treatment 3- Pure paragis plant extract (100% concentration)

Measure 9ml of the paragis plant extract

Treatment 4- Combination of bitter vine and tuba tuba extracts (50% concentration each)

Measure 4.5mL each for bitter vine and tuba-tuba extracts

Treatment 5- Combination of tuba tuba and paragis extracts (50% concentration each)

Measure 4.5mL each for tuba-tuba and paragis extracts

Treatment 6- Combination of bitter vine and paragis (50% concentration each)

Measure 4.5mL each for bitter vine and paragis extracts

Treatment 7- Combination of bitter vine, tuba-tuba, and paragis extracts w(33.3% concentration
each)

Measure 3mL each for bitter vine, tuba-tuba and paragis leaf extracts

Treatment 8- Chlorothalonil

This is a commercial fungicide

Treatment 9- Distilled water

Measure 9mL distilled water

Trial 1 2 3 4 5 Mean
Treatment
T1
(100% pure bitter vine extract)

T2
(100% pure tuba-tuba extract)

T3
(100% pure paragis extract)

T4
(50% concentration of each bitter
vine and tuba- tuba extract)
T5
(50% concentration of each tuba-
tuba and paragis extract)
T6
(50% concentration of each bitter
vine and paragis extract)
T7
(33.3% concentration of each bitter
vine, tuba- tuba, and paragis
extract)

T8
(Commercial fungicide)

T9
(Distilled water)

Experimental Design isagol ni nga section sa EXPERIMENTATION section sa taas

This experimentation will be laid out with Complete Randomized Block Design (CRBD)
with nine treatments, each with three replicants in five trials. The expected outcome of each trial
is that the experimental variables will create a zone of growth.

Testing will be done through poisoned food technique. Each treatment will be incorporated
into the prepared medium. Mix well and transfer the medium into three petri dishes (for the three
replicants). The bacteria will be transferred as well to the said plates.
 Data Analysis

There will be experimental and control variable. The experimental variable will be treated with
M.micrantha, J.curcas, and E.indica extracts at a specific concentration. The positive control
variables will be treated with commercial fungicide specifically Chlorotalonil and the negative
control variable will be distilled water.

To obtain the data, we have to observe the activity of fungi Fusarium oxysporum inside the petri
dish when treated with bitter vine and tuba-tuba leaf extract to see if there is a zone of growth.

Zone of growth will be collected after 24, 48, and 72 hours after inoculation.

quantitative data that will be collected in this study are (1) zone of growth of fusarium, (2) daily
mean zone of growth for each treatment, (3) grand mean zone of growth for each treatment. then
the data will undergo statistical analysis for comparison of treatments. specifically, analysis of
variance (ANOVA) will be utilized to determine which treatment is more effective than the other
treatments. This study will be using ANOVA testing for data analysis.

Risk Assessment and Safety

According to General Chemical Guidelines, the following safety practices is a must for minimizing
risk when working with hazardous chemicals. The student researchers must wear safety gears and
protective devices and have a background about the do’s and don’ts in relation to the study.

The student researchers must be adhered by qualified scientists or designated laboratory personnel
to ensure that no harm or causalities might occur during the experimentation. Proper care of the
equipment that will be used is a must. include: the experiment will not start, unless there is the
presence of a qualified scientist or designated supervisor.

F. oxysporum f.sp. cubense (Foc) has a biosafety level of 1. BSL 1 (Biosafety classification is
based on U.S Public Health Service Guidelines). explain further what does a BSL-1 mean and its
proper protocol when dealing with this kind of biosafety.

Ethanol is a colorless, volatile and highly flammable liquid that has a slight odor. Because of its
incredible versatility, ethanol mixes very well with other solvents and water, as well as chlorides
and hydrocarbons. Being this versatile, ethanol is used for a great many things – but it can also be
quite dangerous.

Exposure to ethanol can be in vapor form (breathing it in), skin/body contact or ingestion. All are
serious and need to be managed appropriately to ensure more damage is not incurred while trying
to attend to the exposure: Inhalation – if you are exposed to ethanol vapors, move to a well-
ventilated area to access fresh air. Contact emergency medical personnel for further assistance.
Skin contact – should ethanol come into contact with your skin, gently wash the area with warm
water and soap. If the skin is still irritated, seek medical assistance for further treatment. Contact
with eyes – if ethanol splashes into your eyes, find a flush station and flush eyes for at least 15
minutes. Contact emergency medical personnel. Ingestion – lay down and contact emergency
medical personnel immediately. Do not induce vomiting as it can create more damage. Do not
drink anything else.

Bibliography

Kubura, Abdullahi Khadijat, Adebola Mathew Omoniyi, and Ajayi Hammed Olayiwola.
"Antifungal efficacy of three plant extracts in the suppression of panama disease in banana plants."
(2018).
Australia, ACIAR. “The Science of Stopping Panama Disease.” YouTube, YouTube, 18 Feb.
2018,
www.youtube.com/watch?v=GAZGa1v7u8A&fbclid=IwAR2KBVxrhiU1xymQq0YguxeE00mL
hjrq1eC-oQgHPJ_OVHXOGHcCLvhLDo8.
Dyer, Mary H. “Fusarium Wilt Of Banana: Managing Of Fusarium Wilt In Bananas.” Gardening
Know How, www.gardeningknowhow.com/edible/fruits/banana/fusarium-wilt-in-
bananas.htm?fbclid=IwAR1GgYvR_kk5Bt_yAYT2dsaBZA4GikWsR3_HiDads2EkPfDHc_5zH
7I8NZA.
PCAARRD, DOST. “Fusarium-Resistant Banana.” YouTube, YouTube, 5 Feb. 2018,
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SevN18loIzUpDNcXtuGJdiT_KUKxK4.
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en.wikipedia.org/wiki/Jatropha_curcas.
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medical benefits." Journal of medicinal plants research 6.14 (2012): 2691-2699.
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pharmacology 3.2 (2009): 058-062.
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ELECUSINE INDICA." Acta Scientiae et Intellectus ISSN 2410 (2015): 9738.
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agritech.tnau.ac.in/farm_enterprises/Farm enterprises_ Mushroom_Culture media.html.
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Fusarium oxysporum f. sp. cubense causing banana wilt." International Journal of Agricultural
Technology 12.7.2 (2016): 2181-2185.
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TIMETABLE OF ACTIVITIES
 DATE TIME LOCATION DESCRIPTION
April 11, 2019 3:00 PM - 9:00 PM At Home. Conversation Online communication
via messenger with the groupmates.
Giving ideas regarding
robotics.
April 12, 2019 9:00 AM - 9:00 PM At Home. Conversation Online communication
via messenger with the groupmates.
Giving ideas regarding
life science
April 13, 2019 1:00 PM - 5:00 PM Villamor’s Residence Gathering Ideas regarding
Staphylococcus aureus &
Planning.
April 15, 2019 1:00 PM - 5:00 PM City Library Studying about S. aureus
& Searching about
MagnaportheGrisea
April 16, 2019 1:00 PM - 5:00 PM Villamor’s Residence Looking for the potential
extract against S. aureus
April 17, 2019 1:00 PM - 4:00 PM Villamor’s Residence We decided to change
study and look for the
potential extract against
MagnaportheGrisea and
XanthomonasOryzae

April 19, 2019 3:00 PM - 5:00 PM Villamor’s Residence We decided to change the
study. We studied about
Tungro Virus and
searched for the potential
extract against to it.
April 20, 2019 10:00 AM - 4:00 PM Villamor’s Residence We searched its related
literature and consulted
our research teacher about
our study. And again, we
changed our study.

April 22, 2019 10:00 AM - 5:00 PM Villamor’s Residence We decided to go back to

our previous study which
is XanthomonasOryzae.
We started to make our
Research Proposal.
April 23, 2019 2:00 PM - 4:00 PM PAGRO Consulting about the
methodology or
procedure regarding our
study and we decided to
change our study again.
April 24, 2019 10:00 AM – 4:00 PM Villamor’s Residence We started to make our
research proposal again.
 April 25, 2019 10:00 AM – 4:00 PM Villamor’s Residence Continuing in making
research proposal.
April 26,2019 8:00 AM – 4:00 PM PAGRO Looking for a qualified
scientist to consult our
research proposal.
May 1 - May 3 8:30 AM – 4:00 PM At Home. Conversation Vacation/Planning for our
via messenger study
May 6 - May 10 8:30 AM – 4:00 PM At Home. Conversation Vacation/Planning for our
via messenger study
May 13 - May 17 8:30 AM – 4:00 PM UIC Searching for a laboratory
Tagum Doctors Hospital that can provide our
TCNHS Science needed
Laboratory equipment/Gathering
materials for our
experiment
May 20 - May 24 8:30 AM – 4:00 PM UIC Searching for a laboratory
Tagum Doctors Hospital that can provide our
TCNHS Science needed
Laboratory equipment/Gathering
materials for our
experiment
May 27 - May 31 8:30 AM – 4:00 PM UIC Searching for a laboratory
Tagum Doctors Hospital that can provide our
TCNHS Science needed
Laboratory equipment/Gathering
materials for our
experiment
June 3 - June 7 8:30 AM – 4:00 PM Laboratory Conducting the
experiment
June 10 - June 14 8:30 AM – 4:00 PM Laboratory Conducting the
experiment
June 17 - June 21 8:30 AM – 4:00 PM Laboratory Conducting the
experiment
June 24 - June 28 8:30 AM – 4:00 PM Laboratory Conducting the
experiment
July 1 - July 5 8:30 AM – 4:00 PM Laboratory Conducting the
experiment
July 8 - July 12 8:30 AM – 4:00 PM Laboratory Conducting the
experiment
July 15 - July 19 8:30 AM – 4:00 PM Villamor’s Residence Writing the research
paper
July 22 - July 27 8:30 AM – 4:00 PM Villamor’s Residence Writing the research
paper
July 29 - August 2 8:30 AM – 4:00 PM Villamor’s Residence Writing the research
paper
August 5 - August 9 8:30 AM – 4:00 PM Villamor’s Residence Writing the research
paper
August 12 - August 16 8:30 AM – 4:00 PM Villamor’s Residence Writing the research
paper
August 19 - August 23 8:30 AM – 4:00 PM Villamor’s Residence Writing the research
paper
August 26 - August 30 8:30 AM – 4:00 PM Villamor’s Residence Writing the research
paper