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.

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.

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.

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.

Research Questions:

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

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

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

4. Will the mixture of two different extract affect the growth of Fusarium oxysporum f.sp. cubense?

5. Related to the 4th question, which mixture has the highest capability in inhibiting the growth of
Fusarium oxysporum f.sp. cubense?
6. Will the mixture of the three extracts affect the growth of Fusarium oxysporum f.sp. cubense?

7. 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?

Hypotheses

If a zone of inhibition can be created by the extracts of Mikania micrantha, Jatropha

curcas, and Eleusine indica, then it is efficient for the suppression of growth of Fusarium
oxysporum.

If cultured Fusarium oxysporum is applied with the extracts of Mikania micrantha,

Jatropha curcas, and Eleusine indica then it can make a zone of inhibition.

Objectives/ Expected Outcomes

The researchers want to treat/suppress or prevent the fusarium wilt in banana plants that
is caused by Fusarium oxysporum by utilizing the plant extracts of Mikania micrantha, Jatropha
curcas, and Eleusine indica as an antifungal agent.

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.
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.

C. Culturing of Fungi

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), and 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.

E. Preparation of Plant extracts

Prepare the leaves of tuba-tuba,and the whole plant of bitter vine and paragis weighing 350g
each, It will be blended and will be macerated with 1500 ml 95% Ethanol in room temperature
for three days and stirred occasionally. After that, filter the extract using Whatman No. 1 filter
paper. Then, it will be concentrated by evaporation 40°C in a rotary evaporator.

F. Experimentation

Prepare the following treatment

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

Measure 9ml of the bitter vine leaf extract

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

Measure 9ml of the tuba-tuba leaf extract

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

Measure 9ml of the paragis leaf extract

Treatment 4- Mixture of treatments 1 and 2 (50% concentration)

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

Treatment 5- Mixture of treatments 2 and 3 (50% concentration)

Measure 4.5mL each for tuba-tuba and paragis leaf extracts

Treatment 6- Mixture of treatments 1 and 3 (50% concentration)

Measure 4.5mL each for bitter vine and paragis leaf extracts

Treatment 7- Mixture of treatments 1, 2, and 3 (33.3% concentration)

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

Experimental Design

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)

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.

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.

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)

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,
www.youtube.com/watch?v=R2f1_qSHUlw&fbclid=IwAR2PzHy14yAbcO59f5_zaoWRM9rbh
SevN18loIzUpDNcXtuGJdiT_KUKxK4.
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“Jatropha Curcas.” Wikipedia, Wikimedia Foundation, 10 July 2019,
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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guajava, and Andrographis paniculata." The Scientific World Journal 2014 (2014).
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indica." Int J Pure App Biosci 3.1 (2015): 257-64.
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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.
McIntosh, Philip. “How to Make Potato Dextrose Agar.” Hunker,
www.hunker.com/13406994/how-to-make-potato-dextrose-agar.
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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

that can provide our
Tagum Doctors Hospital needed
TCNHS Science equipment/Gathering
materials for our
Laboratory
experiment

May 20 - May 24 8:30 AM – 4:00 PM UIC Searching for a laboratory

that can provide our
Tagum Doctors Hospital needed
TCNHS Science equipment/Gathering
Laboratory materials for our
experiment

May 27 - May 31 8:30 AM – 4:00 PM UIC Searching for a laboratory

that can provide our
Tagum Doctors Hospital needed
 TCNHS Science equipment/Gathering
Laboratory 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