(1) PROJECT PROFILE

Project Title: An In Vitro Investigation: Antifungal Activity of Calamansi (Citrus microcarpa) Peels Essential Oil
Against Rice Blast (Magnaporthe oryzae)
Names of Project Proponents: Althea D. Cordero Region: IV-A CALABARZON
Division: Cavite
School: Tanza National Comprehensive High School (TNCHS) Grade Level: Grade 12
Project Duration (number of months): 6 months
Email: adc402320150107@depedcavite.com.ph Contact number: 0947-234-7163

(2) CATEGORY OF RESEARCH (4) THEME

Physical Science Food Safety
✔ Life Science Water Conservation
Robotics and Intelligent Mathematics Renewable Energy
Mathematics and Computational Sciences Cyber Security
Traffic/Road Congestion
Health
Disaster Mitigation
(3) ✔ Agriculture and Environment
✔ Individual Others (please specify)
Team

(5) EXECUTIVE SUMMARY

Just this February 2022, rice blast (Magnaporthe grisea) has affected at least 602 hectares of rice farms in three

provinces of the Cagayan Valley region. The blast infection has already destroyed rice crops in 22 communities in Isabela,

Quirino, and Cagayan (Villamore 2022). Rice blast is caused by the fungus Magnaporthe oryzae, which also is among

the most common diseases affecting rice crops globally. Its effects include either neck or panicle blast, which destroys

the rice grain during the seed laying stage, or leaf blast, which weakens or kills seedlings (Hatanaka 2012, 1). On the

other hand, calamansi (Citrus microcarpa), a native citrus popular in the Philippines, has been detected to have D-

limonene which is known for its promising nature in the essential oil of its peels. Although the calamansi peels, which
account for the majority of the fruit weight, are considered waste in the country (Palma et al. 2019, 108-110). This study

therefore aims to evaluate the essential oil from C. microcarpa (calamansi) peels' potential antifungal activity against the

rice blast fungus, M. oryzae. The fruit of calamansi will be bought at the public market of Tanza, Cavite and the rice blast

fungi (M. oryzae) will be bought from University of the Philippines Los Baños, Laguna. The extraction of essential oil

using steam distillation and the identification of the constituents that compose C. microcarpa peels essential oil using Gas

chromatography–mass spectrometry (GC-MS) will be done in the Department of Science and Technology (DOST). To

have wider reference for results interpretation, two antifungal assays will be done namely Poison Food (PF) technique

and Volatile Activity (VA) assay. The researcher will then use one-way analysis of variance (ANOVA) test to identify

the presence of significant difference between tests. SPSS software will be used to examine all statistical analyses. The

researcher expects positive results for this project including the presence of important constituents in C. microcarpa peels

EO, comparable percentage inhibition of mycelial growth of the treatment applied with C. microcarpa EO to that of the

positive control’s result, and presence of significant difference between tests. This research can then aid the agriculture

industry in terms of solutions to plant-pathogen problems.

This study is about the evaluation of the essential oil from C. microcarpa (calamansi) peels' potential antifungal

activity against the rice blast fungus, M. oryzae. It is estimated to be done within 6 months, starting from the formulation

of the topic up till the final defense. An 8,000 to 10,000 peso investment would be sufficient to fund this project.

(6) INTRODUCTION

Rice plays a very important role in the ASEAN countries as a primary staple food. The ASEAN countries consume

up to 22% of the rice worldwide (Cumagun, Lopez, & Tharreau, 2015, 81). Rice (Oryza sativa) is one of the most in

demand cereal crops all throughout the planet, and is the main food source for more than half of the world’s population,
68% of this is from Asian population (Durgeshlal et. al, 2019, 48). As the demand in rice increases day by day because

of the global population overgrowth, it is estimated that by the year 2050, total agricultural production needs to increase

by 60% to meet the global food demands (Tsaneva et. al 2019). However, crucial observation should be done due to the

insect pests and diseases that cause negative effects to the crop production (Cumagun, 2015, 160). Just this February

2022, rice blast (Magnaporthe grisea) has affected at least 602 hectares of rice farms in three provinces of the Cagayan

Valley region. The blast infection has already destroyed rice crops in 22 communities in Isabela, Quirino, and Cagayan

(Villamore 2022).

Fungi are one of the most common types of microbes responsible for plant disease. Over the last few decades,

outbreaks of these pathogens have cost the agriculture business millions of dollars around the world. Because of their

effectiveness, synthetic fungicides were commonly utilized to solve the problem. However, despite their effectiveness,

these compounds can harm the plant, non-targeted creatures, and the environment (Li 2015).

Rice blast is caused by the fungus Magnaporthe oryzae, which also is among the most common diseases affecting

rice crops globally. Its effects include either neck or panicle blast, which destroys the rice grain during the seed laying

stage, or leaf blast, which weakens or kills seedlings (Hatanaka 2012, 1). The action of the fungi starts when fungal spores

with specific adhesive ejected from the tip of each spore attach to leaves. Then the spores will damage the leaf cuticle

after germination to permit invasion (Cha et. al 2011).

Despite the numerous strategies established to combat the fungus, the fungus continues to pose a significant danger

to global rice supply. Because of the frequent administrations, the fungus developed resistance, resulting in limited

efficacy of standard fungicides against M. oryzae (Hatanaka et. al 2012). Therefore, new fungicides are in the interest of

researchers. The new fungicide requires properties like antimicrobial and plant origin. It is already proven that plant
extracts can control plant diseases like those caused by fungi. Calamansi, for example, is reported to have antifungal

activity.

This study is about the evaluation of the essential oil from C. microcarpa (calamansi) peels' potential antifungal

activity against the rice blast fungus, M. oryzae. The fruit of calamansi will be bought at the public market of Tanza,

Cavite and the rice blast fungi (M. oryzae) will be bought from University of the Philippines Los Baños, Laguna. The

extraction of essential oil using steam distillation and the identification of the constituents that compose C. microcarpa

peels essential oil using Gas chromatography–mass spectrometry (GC-MS) will be done in the Department of Science

and Technology (DOST).

(6.1) RATIONALE/SIGNIFICANCE

Rice (Oryza sativa) is grown on about 147 million hectares around the world, mostly in densely populated areas.

Its prices are a sensitive political matter in the Philippines because of its importance in the economy and the people's diet.

As a result, the Rice Tariffication Law, which was passed in March 2019, has sparked criticism. To finally comply with

the World Trade Organization's (WTO) principles and rules, the policy reform eliminated the quantitative import limits

and replaced them with ad valorem tariffs. This policy reform has ignited a raging controversy that has regularly spilled

into the mainstream media. The main argument is that reducing paddy prices would be disastrous for small rice farmers,

who are already impoverished and vulnerable to market and other shocks such as the risk for the national food security

due to the increase of import dependence (Balié et al. 2021, 289-303).

Moreover, environmental phenomena such as rising global temperatures, diminishing acreage, depleting water

supplies, and other abiotic and biotic stresses are putting strain on rice production, putting food supply security at risk
too. And among these biotic stresses that affect rice productivity, the most important are the diseases. One of which is the

rice blast. Rice blast has extensive geographical distribution and destructiveness under favorable conditions causing it to

become a major rice disease produced by the fungus Magnaporthe oryzae (Carrillo et al., 2021).

Rice blast disease is a major threat to rice production globally. This disease is caused by the hemibiotroph

filamentous fungus Magnaporthe oryzae (anamorph Pyricularia oryzae), which causes the loss of enough rice to feed 60

million people per year. M. oryzae has a unique biology that allows it to secrete a wide range of small-secreted proteins

in order to minimize or circumvent host pathogen surveillance. Unlike other plant diseases, M. oryzae has a

hemibiotrophic life cycle, in which the fungus first suppresses the plant immune system before causing plant cell death.

Due to the fungus's capacity to quickly adapt and mutate in order to evolve resistance to many rice varieties, neither

traditional breeding nor chemical treatments have been successful in managing this disease. Without a question, rice blast

disease continues to pose a threat to world rice production and food security (Fernandez and Orth 2017, 2-15). Therefore,

the researcher aims to investigate new and innovative solutions to this problem by attempting to use biochemical extract

or specifically, essential oil from Citrus microcarpa (calamansi) peels against the rice blast fungus, M. oryzae.

This could help in reducing and lessening plant diseases caused by the fungi. This study will be helpful for students

who are investigating the same field and this might be a helpful reference. The farmers can also benefit from this study

as this can help in providing organic fungicides to them to avoid great destruction of yields and low production of crops

caused by the rice blast fungus, M. oryzae. Lastly, this study can also be beneficial for the community by helping the

farmers for the great yield loss of rice to lessen their expenses.
(6.2) SCIENTIFIC BASIS/THEORETICAL FRAMEWORK/MATHEMATICAL THEORY INVOLVED

Plant disease control methods differ depending on the host plant, the pathogen, the intensity of their interaction,

and other environmental conditions. Chemical agents, particularly fungicides such as Benzimidazoles, Cadmium

compounds, Carbamic acid derivatives, and Copper compounds, have become an essential aspect of crop production in

many regions of the world, resulting in higher yield and income. However, the overuse of these fungicides caused a slew

of environmental and ecological issues. For example, methyl bromide was eventually reported to be a Class 1 stratospheric

ozone depletion. Many synthetic fungicides have been discovered to have unexpected impacts in species other than their

intended targets. A vast number of fungicides have also been shown to have negative effects on domestic animals, wildlife,

birds, honey bees, fish, and aquatic invertebrates, which is a serious problem because there is no particular treatment for

fungicide poisoning in domesticated animals or humans. As a result of the aforementioned challenges, research into novel

antifungal medications that are inexpensive, have no non-target adverse effects, and, most significantly, are eco-friendly

and green is rising. In line with this, aside from giving flavor and fragrance, plant essential oils readily offer themselves

as one of the greatest solutions in this subject (Kumar et al. 2020, 38-39).

Steam distillation of aromatic plants, particularly those used as scents and flavorings, can yield essential oils.

Around 3000 EOs have been isolated from diverse plant species, but only around 300 are commercially viable and used

in the fragrance, medicinal, culinary, and agricultural industries. Superior and inferior bioactive components make up

EOs, which are principally responsible for their biological activities. Essential oils are made up of 20-60 different

chemical components, with major components present in relatively high quantities (20–70%) and minor components

present in minimal amounts. Terpenes (e.g., pinene, limonene, myrcene, terpinene, p-cymene), aromatic phenols, and

terpenoids have been discovered to play important roles in the composition of numerous essential oils. GC-MS (Gas

chromatography–mass spectrometry) is used to calculate equilibrium estimations and EO values (Sil et al. 2020, 1885).
 Additionally, essential oils and their derivatives are also used to control multiple plant pathogenic microorganisms.

Essential oils from plants such as basil, fennel, citrus, lemongrass, rosemary, oregano, and thyme have shown strong

antifungal activity against a wide range of flora pathogens. Numerous researches have investigated and proven the

antifungal properties of essential oils. Most literature publications mention that, aside from fungicidal and fungistatic

effects, its methods of action vary depending on the instance. The potential of EOs to disrupt cell wall and cell formation,

cytoplasmic stability, and the impact of cellular cell damage and macromolecule escape all contribute to their cytotoxic

character in their surroundings. Thus, combining botanicals and natural items with essential oils can undoubtedly be of

aid in achieving a proper balance between green and pesticide chemistry (Sil et al. 2020, 1886-1888).

D-limonene, a common monoterpene molecule found in plants and abundant in the pericarp of citrus fruits, is a

promising natural substance that is now being studied for cancer prevention. Commercially, 90–95 percent D-limonene

may be produced from lemon pericarps and orange essential oils. However, limonene can also be found in a variety of

other citrus fruits (Palma et al. 2019, 108). It also exhibits fungicidal and insecticidal properties, suggesting that it could

be used to make natural pesticides. This was proven by the efficacy of free D-limonene and D-limonene-loaded

nanoemulsions against four agricultural diseases, including Pyricularia oryzae, after tests that showed positive and

promising results (Feng et al. 2020, 2-7).

Calamansi (Citrus microcarpa) is a native citrus that grows throughout Southeast Asia, but is only popular and

common in the Philippines. All year-round, the fruit is available and is widely used for a variety of purposes. Although

the calamansi pericarps, which account for the majority of the fruit weight, are considered waste in the country. Among

the other citrus fruits, D-limonene may also be detected in the essential oil of calamansi peels, with a yield of 92.67

percent of the total essential oil, which is less than the 94.88 percent found in orange pericarps by Aissou et al. (2017). It

is, however, higher than the fraction of 84.73 percent observed in lemon essential oil by AL-Jabri and Hossain (2018). A
plentiful and low-cost supply of raw material from calamansi pericarp waste, as well as a simple and low-cost extraction

method, promote the economic feasibility of this resource. And even though there are numerous promising studies on D-

limonene and other citrus essential oils, little is known about Citrus microcarpa essential oil, particularly its

pharmacological activity and potential uses (Palma et al. 2019, 108-110). Therefore, with the relevant information

mentioned, the researcher aims to study the potential of calamansi peels essential oil as an antifungal agent against the

rice blast fungus, Magnaporthe oryzae.

(6.3) OBJECTIVES

General: This research aims to investigate the Antifungal Activity of Calamansi (Citrus microcarpa) Peels Essential Oil

Against Rice Blast (Magnaporthe oryzae).

Specific:

● To determine the yield of C. microcarpa (calamansi) peels essential oil from steam distillation.

● To identify the constituents that compose C. microcarpa (calamansi) peels essential oil.

● To determine the amount of each constituent present in C. microcarpa (calamansi) peels essential oil in

percentage.

● To discover the effects of C. microcarpa (calamansi) peel essential oils on M. oryzae.

● To identify which concentration of the C. microcarpa (calamansi) peels essential oil will show the greatest

antifungal activity against M. oryzae. Respectively:

a. 50 µg/mL

b. 100 µg/mL

c. 150 µg/mL
 ● To determine the significant difference in the resulting antifungal activity between the three concentrations (50

µg/mL, 100 µg/mL, 150 µg/mL) of C. microcarpa (calamansi) peels essential oil against M. oryzae.

● To determine the significant difference in the resulting antifungal activity between the three treatments (C.

microcarpa (calamansi) peels essential oil, positive control, negative control) against M. oryzae.

(7) REVIEW OF LITERATURE

Conceptual Literature

Citrus microcarpa

Citrus microcarpa tree has branches near to the ground, a height of 3 to 5 meters high, dark green leaves, bears

white fragrant flowers and round fruits. A calamansi fruit contains yellow to orange pulp which is very juicy and green

seeds ranging from 1 to 5 in number, though some fruits contain no seeds (Acero and Morte 2017, 29). The calamansi

fruits are widely used in the Philippines as condiments, for its juice, and for its medicinal uses. (Acero and Morte 2017).

Calamansi peel is reported to have medicinal properties such as antimicrobial agent that both kills bacteria and fungi.

Phytochemicals such as monoterpenes, sesquiterpene hydrocarbons, and limonene are present in its peels and those

constituents act as fungicide (Cheong et al. 2012).

Magnaporthe oryzae

Magnaporthe oryzae is the fungus that causes rice to have rice blast which is one of the major diseases that affect

rice crops worldwide. The damage it causes results in either neck or panicle blast, which harms the rice grain during the

seed setting stage or leaf blast, which debilitates or kills seedlings (Hatanaka et al. 2012). The action of the fungi starts
when fungal spores with specific adhesive ejected from the tip of each spore attach to leaves. Then the spores will damage

the leaf cuticle after germination to permit invasion (Cha et al. 2011).

Fungi

Fungus is an organism which collects food by getting nutrients from an outside source. Different types of fungi

can be observed today varying in their sources of food such as in soil, wood, decaying organic matter, or living plants and

other organisms. Fungi can also be distinguished from its size ranging from single-celled organisms that are invisible to

the naked eye to a number of the largest multicellular ones. A hundred thousand of plant diseases are caused by fungi

including up to 70% of the most important ones, resulting in an economic loss of billions of dollars each year. Vast disease

to seeds, seedlings, mature plants, and aging plants can be caused by these plant pathogens, resulting in the lower growth

and reproduction of crops. Aside from that, forest trees and wooden structures can also be attacked by fungi (Ammirati

and Seidl 2009).

Essential Oils

Essential oils are liquids that are isolated from plants when introduced to solvents. It is a concentrated hydrophobic

liquid containing volatile. Scientists gave interest to essential oils obtained from many plants. It also became very popular

due to its bioactive components acting as antimicrobial agents. The phenolic compounds that can be found in essential

oils could be used to prevent growth of many spoilage and pathogenic microorganisms in foods. Usually, the effectiveness

of essential oils could be achieved in higher concentrations by direct addition, and so there comes another method of

treatment called vapor contact (Malo et al. 2013).

Steam Distillation

Steam distillation is the most popular method used to extract and isolate essential oils from plants for use in natural

products. This happens when the steam vaporizes the plant material volatile compounds, which eventually go through a

condensation and collection process. Steam distillation is proven economical, fast process for collecting essential oils,

and highly efficient. It is also already widely used in the country primarily for the collection of essential oils from

lemongrass (Bugayong et al. 2019).

Related Studies

Antifungal Activity of Three Different Ethanolic Extract against Isolates from Diseased Rice Plant (Durgeshlal C., Sahroj

Khan M., Prabhat S. A., and Aaditya Prasad Y. 2019)

Rice blast and sheath blight are two of the most serious fungal diseases affecting rice farmers today. These diseases

have a significant impact on productivity and have become a problem in terms of consumption. The researchers of this

study aimed to determine the antifungal activity of three different plant ethanolic leaf extract available in the Philippines

against the rice diseases. There were varying concentrations of different plant extracts for the sheath blight, and a hundred

percent concentration was used for the rice blast. Food Poisoned Technique Assay was done to assess the antifungal

activity of the extracts made, three replicates were made. Based on the results gathered, the plant D. metal, J. carus

showed the highest antifungal activity at 100% concentration against the isolated pathogen causing sheath blight.

Whereas, J. carus and R. tuberosa gained the highest antifungal property against rice blast. In comparison to mancozeb,

the three plant extracts demonstrated a high percentage of mycelial inhibition. As a result, extracts from these three plants

have the active potential to inhibit fungus growth and can be used as a bio fungicide to control rice blast and sheath blight

infection. Because these bio fungicides are derived from plants, their negative impact on the environment and other
organisms will be mitigated. They may also aid the government's goal of determining how to limit the use of chemical

fungicides. In vivo trials are recommended to demonstrate the effectiveness of plant health management in the field.

Efficacy of plant extracts against the rice blast fungus Magnaporthe oryzae. (Lemtukei D., Kihara J., Thi Q. N., and Ueno

M. 2016)

Thi and his colleagues studied the fungicidal activity of Houttuynia cordata and Artemisia indica extracts against

Magnaporthe oryzae in 2016. M. oryzae were suspended in an H.cordata extract, as well as A.indica. Separately, it was

transferred onto glass slides. M. appressorium formation and spore germination were studied using this method. oryzae

were significantly inhibited by the two extracts when compared to the control, distilled water. Because of the positive

results, antifungal substances in both extracts can be suggested. To summarize, H. cordata, as well as A. indica extracts

have the potential to be effective fungicides for plant diseases such as rice blast disease.

Chemical composition and cytotoxicity of Philippine calamansi essential oil (Palma E., Cruz P., Cruz D., Bugayong A.,

and Castillo A. 2018)

In a research conducted by Bugayong and her co researchers last 2018, the volatile composition, ƅ-limonene

content, and cytotoxicity of calamansi essential oil were determined. The essential oils were obtained by steam distillation

and characterized using Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy and Gas Chromatography

Analysis. MTT assay is used to observe the cytotoxicity of CM EO using human mammary tumor cell line MCF-7 and

Chinese hamster ovary non-tumor cell line AA8. The characterization of CM EO showed that ƅ-limonene is the major
constituent in the essential oil. While the cytotoxicity revealed that the IC50 of CM EO on MCF-7 is 7.98 ± 1.77 and

AA8 has 16.15 ± 8.35 µg/mL. The results state that the cytotoxicity in MCF-7 is higher than in AA8 by the CM EO and

it can be suggested that the ƅ-limonene is responsible for the effects and perhaps, the terpenes also. In conclusion,

Calamansi essential oil has a good potential to be an organic low-cost chemopreventive and chemotherapeutic agent.

Antifungal activity of plant extracts with potential to control plant pathogens in pineapple (Costa H. B., Fernandes P. B.,

Meira D. D., Sales M. C., and Ventura J. A. 2016)

The researchers used plate-hole method in vitro for the screening of the antifungal potential of the extracts. The

study is observed in qualitative manner wherein 29% were effective against C. paradoxa and 46% for the F. guttiforme

of among the 49 mother tinctures. The natural extracts of mother tincture of four plants, resin of one plant and crude

extracts of another plant are effective against the fungi, F. guttiforme. These treatments showed similar results as for the

commercial fungicide. The results concluded that mother tinctures can effectively control phytopathogens. This study

provides developments of bioactive natural products with the added benefit of being environmentally safe.

Antifungal activities of medicinal plants extracts of Ivorian pharmacopoeia (Mathieu K. A., Marcel A. G., Djè D. B.,

Sitapha O., Adama C., and Joseph D. A. 2014)

 For the preparation of the plant extracts, homogenization process was done and each of it was subjected to

Sabouraud agar by the method, agar slanted double dilution. Ketoconazole was used as measures for anti-fungal trials.

One thousand cells of Candida albicans were implanted on the priorly prepared medium culture. The anti-fungal

parameters values (minimal fungicidal concentrations [MFC] and IC50) were evaluated for the determination of the

antifungal activity. All extracts exhibited antifungal activities that varied in effectivity from plant species to another based

on the results. Eight of them had a satisfactory anti-candida activity while extracts from Terminalia species were the most

potent. One of them was Terminalia superba extracts that possessed the strongest activities. The aqueous and

hydroethanolic extracts of T. superba were more active twice and four times compared with ketoconazole. While the

aqueous extract of Guarea cedrata showed the worst antifungal activity. As for the conclusion, all of the medicinal plant

extracts exhibited antifungal activities and the most active was T. superba.

Green-odour compounds have antifungal activity against the rice blast fungus Magnaporthe oryzae (Hatanaka A.,

Motoyama T., Osada H., Sariah M., and Tajul M. I. 2012)

A study by Tajul and his colleagues reported the antifungal activity of Green-odour compounds against

Magnaporthe oryzae. To observe the biological activity of the compounds, trans-2-hexenal, cis-3-hexenol, n-hexanal,

and cis-3-hexenal against M. oryzae, these compounds were applied onto rice plants in laboratory condition. The two

compounds, trans-2-hexenal and cis-3-hexenal, revealed effective disease suppression, followed by n-hexanal, and cis-

3-hexenol with weak effect. Compounds, trans-2-hexenal and cis-3-hexenal exhibited a growth suppression on M. oryzae.

These two compounds actually inhibited appressorium formation at lower concentration than the growth suppression.

Concluding, results showed that the two compounds are the most potent compounds in inhibiting the growth and

appressorium formation of M. oryzae and so these compounds could be used as fungicides against rice blast.
Development and optimization of antifungal packaging for sliced pan loaf based on garlic as active agent and bread aroma

as aroma corrector. (Carballo G., Catalá R., Galet V., Gavara R., Mozos R., and Muñoz P. 2019)

Mozos and his colleagues aimed to develop antimicrobial films containing garlic extract to be applied as active

packaging for preservative-free sliced pan loaf, with the goal of extending its shelf-life in a study in 2019. First, they have

prepared the chemicals and solutions that they will use. Penicillium expansum, fungi were isolated from natural sliced

bread, and was grown on potato dextrose agar (PDA) in polystyrene. Petri dishes for 7 days at 28°C. They did film

preparations and garlic extract and bread aroma inhibition assay. They used ANOVA test for statistical analysis. The

antifungal effect of garlic extract and bread aroma in vapor phase P. expansum was determined by the disc diffusion

method. 0.1 microliter of garlic extract resulted in growth inhibition of P. expansum, whilst 0.25 microliter proved lethal

for this mold.

In vitro Evaluation of Insecticidal and Antifungal potencies of fruit peel extracts of pomegranate (Punica granatum)

(Chaieb I., Hamouda A. B., Laarif A., and Remadi M. D. 2015)

Hamouda and colleagues tested the insecticidal and antifungal activities of various Punica granatum fruit peel

extracts in vitro against two pests (Myzus persicae and Phthorimaea operculella) and four fungi (Botrytis cinerea,

Fusarium sambucinum, Penicillium digitatum and Aspergillus niger). Adults treated with aqueous, ethanol, and methanol

had mortality rates of 29 percent, 53 percent, and 55 percent, respectively. The researchers also observed similar effects

on Phthorimaea operculella larval penetration into potato tubers and the number of eggs laid by the potato tuber moth. In
terms of antifungal activity, the results showed that the effectiveness of all extracts was only observed in the mycelial

growth of Penicillium digitatum and Fusarium sambucinum when compared to the control. For the fungus Botrytis

cinerea and Aspergillus niger, there was no significant difference between the treatment and the control. Concluding, the

protection of plants from diseases and pests may be managed by these promising botanical extracts.

Antifungal Activity of Essential Oil of Eucalyptus camaldulensis Dehnh. against Selected Fusarium spp. (Gakuubi M.,

Maina A., and Wagacha J. 2017)

The researchers of this study targets to evaluate the antifungal activity of Eucalyptus camaldulensis Dehnh.

essential oil against five different Fusarium spp. That is commonly seen on maize. The essential oil of the E.

camaldulensis underwent steam distillation extraction in a modified Clevenger-type apparatus. The chemical

compositions acquired were characterized by gas chromatography mass spectrometry. Poisoned food technique was used

to determine the percentage inhibition of mycelial growth, minimum inhibitory concentration, and minimum fungicidal

concentration of the EO on the test pathogens. The antifungal activity of the varying concentrations of the essentials oil

was evaluated using disc diffusion method. The most abundant compounds identified in the EO were 1,8-cineole (16.2%),

α-pinene (15.6%), α-phellandrene (10.0%), and p-cymene (8.1%). At a concentration of EO, all test pathogens showed

full suppression of mycelial growth of 7-8 μL/mL after it was incubated for 5 days. On the test fungi, the minimum

inhibitory and fungicidal concentrations of the EO were in the range of 7-8 μL/mL and 8–10 μL/mL, respectively. These

findings support E. camaldulensis.'s fungicidal properties. The essential oils of E. camaldulensis and their potential

applications in the management of economically valuable Fusarium spp. as well as potential possibilities to synthetic

fungicides.
Antifungal activity of Ocimum canum Essential oil against Toxigenic Fungi isolated from Peanut Seeds in post-harvest

in Benin (Adjou S., Kouton S., Dahouenon-Ahoussi E., Sohounhloue C.K., and Soumanou Mohamed M. 2012)

The purpose of this research is to determine the inhibition of Aspergillus flavus and Aspergillus parasiticus

isolated from peanut.Their aflatoxin production was stimulated by essential oils extracted from fresh Ocimum canum

leaves. Minimal obstructiveness. The oil's minimal fungicidal concentration (MFC) and minimal (MIC) were determined.

The acquired EO through hydrodistillation was analyzed using gas chromatography-mass spectrometry analysis and was

able to identify 30 components. The results showed that O. canum essential oil contains pronounced anti-fungal activity

against the growth of the Aspergillus flavus and A. parasiticus. The results obtained by the disk diffusion method showed

75% of inhibition of A. parasiticus growth for the essential oil when compared with control (Nystatin). The results of

anti-aflatoxin organic assay, given in table-6, showed that EO of O. canum has important aflatoxin inhibition potential on

the toxigenic strain Aspergillus parasiticus at 1.5µl/mL, aflatoxin production by A. parasiticus was inhibited.

Essential oil of Rosmarinus officinalis L. from West Highlands of Algeria: Chemical characterization and in vitro

antifungal activity against Fusarium oxysporum f. sp. albedinis (Boukhobza Z., Boulenouar N., Cheriti A., and Kadri Z.

2021)

Boukhobza and colleagues conducted a study regarding the antifungal activity of Rosmarinus officinalis,

considering that there are only few investigations of its essential oil (EO) chemically and biologically. In this aspect, the
chemical composition derived from the leaves and stem of R. officinalis were assessed through GC/MS analysis. Their

antifungal activity against Fusarium oxysporum f. sp. albedinis were also evaluated. The results of the GC/MS analysis

revealed that monoterpenes (oxygenated 84.9%, 52.4%) and hydrocarbons (12.2%, 8.4 %) were the dominating classes

of chemicals in both the leaves and the stem. The predominant component was 1,8-cineole (leaves: 54.4%, stem: 29.7%),

classifying these EOs as 1,8-cineole chemotype. The antifungal activity of EOs in vitro was assessed using micro

atmosphere and direct contact techniques. After a 7-day incubation utilizing the direct contact approach, the best

inhibitory action against Foa was identified by estimated growth reduction (RGR= 0.398; RGR=0.383) with EOs from

the leaves and stem, correspondingly.

(8) METHODOLOGY

Procurement and preparation of plant materials

In order to accumulate Citrus microcarpa peels, the researcher will buy 3.5 kilograms of calamansi fruits in the

town market of Tanza, Cavite. The researcher will thoroughly pick and purchase newly harvested calamansi fruits to

assure their freshness. Since at least 3 kilograms of fruit peels will be needed for the preparation of essential oils, the

calamansi juices will then be squeezed out, as it is not necessary for the present study. To reduce waste, the juice will be

stored and refrigerated for other purposes. The peels will be carefully rinsed with warm water (40 °C) prior to treatment

to remove dirt. Afterward, the researcher will store the 3 kilograms of calamansi peels in a clean container before

surrendering it to DOST-CED Technical Services.

Extraction of Calamansi Peels Essential Oil

The stored fresh peels of C. microcarpa will be submitted to the Department of Science and Technology (DOST)-

Chemicals and Energy Department located in Taguig, Metro Manila for essential oil extraction. Following the standard
procedures of the institution, the peels will be oven dried for 15 minutes at 95 °C. After drying, a portion of 150 grams of

dried calamansi peels will be subjected to steam distillation at a temperature of 100 °C for 1 hour. This process will be

repeated three times to get a sufficient amount of essential oil for antifungal tests and chemical analysis. Gas

chromatography-Mass spectrometry (GC-MS) analysis will also be undertaken to determine the major constituents and

overall composition of the essential oil (Adjou et al. 2012, 20-21).

Once the C. microcarpa EO is received by the researcher, it will be stored in air-tight sealed glass vials covered

with aluminum foil at 4 °C until further use.

Procurement of Rice Blast Fungal Strain

The researcher will obtain the rice blast, Magnaporthe oryzae, fungal strain from the University of the Philippines-

Los Baños, Laguna (UPLB). It will be maintained on Potato Dextrose Agar (PDA) Medium and stored at 4°C until

further utilization.

Preparation of Positive Control

The researcher will prepare a 500 ppm concentration of Mancozeb by dissolving 625 mg of Mancozeb WP 80%

in 1000 mL of sterile distilled water, as described by Durgeshlal et al. (2019, 52). The mixture will then be gently stirred

until homogenized. The researcher will cover the container with aluminum foil and keep it until it is needed.

Antifungal Assay

The fungicidal activity of C. microcarpa EO against M. oryzae will be determined by employing the poisoned

food (PF) technique provided by Gakuubi, Maina, and Wagacha (2017, 2-3), as well as the volatile activity (VA) assay

described by Znini et al. (2013) with slight modification.

 I. Poisoned Food (PF) Technique

In the PF technique, the researcher will prepare specific initial concentrations (50 µg/mL, 100 µg/mL, 150 µg/mL)

by adding an appropriate amount of C. microcarpa EO containing 20 μg/mL of Tween-80 to cooled sterile molten PDA

(45 °C), followed by manual rotation in a sterile Erlenmeyer flask to disperse the oil in the medium. After the

emulsification, 20 mL of the medium will be transferred onto 90mm x 15mm sterile petri dishes with care given to avoid

air bubble trapping. For around 1 hour, the medium will be allowed to solidify at room temperature. Once they are

completely solidified, each petri plate will be aseptically inoculated at the center with 6 mm mycelial discs taken from

the perimeter of actively proliferating regions of 7-day-old M. oryzae colonies with the help of a sterile cork borer. The

same procedure will be performed with positive (Mancozeb 80%) and negative (Normal Saline Solution) controls. Each

treatment will have three replicates, and the plates will be incubated at 28 °C for 7 days. Each day, the fungal colony

diameter will be measured and recorded using a Vernier caliper.

II. Volatile Activity (VA) Assay

In the VA assay, the petri plates measuring 90 mm x 15 mm will be filled with 20 mL of sterile PDA media and

allowed to solidify. Following this, a 6 mm mycelial disc cut from the perimeter of a 7-day-old M. oryzae mycelium

culture will be seeded into each petri dish. The petri dishes will be inverted and 8 mm sterile filter paper discs will be

impregnated with the concentrations (50 µg/mL, 100 µg/mL, 150 µg/mL distilled water with 5% Tween 20) of C.

microcarpa EO, which will be attached to the inverted lid (1 disc per lid). For the incubation period, the researcher will

wrap the petri dishes with parafilm along the rim to observe the release of volatile components; these will be inverted and

incubated for 7 days at 25 ± 2 °C. In this technique, three replicates will also be conducted for each treatment and control.

Moreover, the diameter of the mycelial growth will be measured using a Vernier caliper along two perpendicular lines

passing through the center of the dish.

Calculation of Percentage Inhibition of Mycelial Growth

For both antifungal assays, the percentage inhibition of the mycelial growth of the test fungi by the essential oil

will be calculated using the formula stated in the research of Boukhobza et al. (2021, 46):

𝑑𝑐 − 𝑑𝑡
Inhibition of mycelial growth (%) = 𝑥 100
𝑑𝑐

Where dc represents the mean diameter of the fungal colony in control, while dt is the mean diameter of the fungal

colony in treatment.

Transferring of Experiments and Distinction of Effects

The minimum inhibitory concentration (MIC) will be defined as the lowest EO concentration at which no growth

occurs, i.e. no change in mycelial disc diameter. The researcher will examine the minimum fungicidal concentrations

(MFCs) of C. microcarpa EO on M. oryzae as follows to determine whether the EO has a biocidal effect on the test fungi

(Gakuubi, Maina, and Wagacha 2017, 3). To distinguish between the essential oil's fungistatic and fungicidal effects on

the target organism, the inhibited fungal discs of the oil-treated plates will be reinoculated into freshly made PDA petri

plates, and their growth revival will be examined after 72 hours at 28 °C incubation. The minimum fungicidal

concentration will be defined as the lowest concentration of oil at which no growth appears on the plates following

subculturing. If growth resumes, it is considered fungistatic; otherwise, it is regarded as fungicidal (Znini 2013, 1767).

Data Analysis

In order to evaluate whether there will be any significant differences among the percentage inhibition of mycelial

growth of C. microcarpa EO and the controls (Mancozeb and NSS) against the rice blast causing pathogen, M. oryzae,
the researcher will use the one-way analysis of variance (ANOVA) test. In accordance with the data analysis of Durgeshlal

et al. (2019, 52), least significant difference (LSD) (p < 0.05) will be employed for a post hoc test to determine which of

the treatments will be substantially different against M. oryzae. SPSS software will be used to examine all statistical

analyses.

Hypotheses

H0: There will be no yield of C. microcarpa (calamansi) peels essential oil from steam distillation.

H0: There will be no constituent identified from C. microcarpa (calamansi) peels essential oil.

H0: The amount of each constituent present in C. microcarpa (calamansi) peels essential oil in percentage will be

0%.

H0: C. microcarpa (calamansi) peels essential oil will have no effect on M. oryzae.

H0: No concentration (50 µg/mL, 100 µg/mL, 150 µg/mL) of C. microcarpa (calamansi) peels essential oil will

show the greatest antifungal activity against M. oryzae.

H0: There will be no significant difference in the resulting antifungal activity between the three concentrations (50

µg/mL, 100 µg/mL, 150 µg/mL) of C. microcarpa (calamansi) peels essential oil against M. oryzae.

H0: There will be no significant difference in the resulting antifungal activity between the three treatments (C.

microcarpa (calamansi) peels essential oil, positive control, negative control) against M. oryzae.

Risk and Safety

In order to ensure the safety of the researcher, they will require supervision from a plant pathologist when handling

and disposing of the fungi and chemicals. They will also be supervised when utilizing laboratory equipment. Furthermore,

personal protective equipment (PPE) such as lab gowns, latex gloves, and masks will be worn at all times. To guarantee

that there will be no trace of potential infections, hand washing before and after exiting the laboratory will be strictly
enforced. Pre and post-use sterilizing of laboratory equipment will also be performed. Additionally, the researcher shall

thoroughly sterilize and disinfect the experimentation site.

(9) EXPECTED OUTPUTS AND POTENTIAL IMPACTS

In this research, it is expected to have a high yield of the essential oil from steam distillation, it is also expected to

get the needed phytochemical constituents from the essential oil gathered. The researcher will be expecting high efficiency

of C. microcarpa peels essential oil as an antifungal agent against M. oryzae. The varying concentrations are expected to

have different effects on the inhibition zone. It is also expected for the C. microcarpa treatment to be comparable to that

of the positive control. If the findings are proven positive, this research will aid in the provision of organic fungicides to

farmers in order to prevent the rice blast (Magnaporthe oryzae) from destroying yields and causing low crop production.

This could serve as a model for future researcher to understand better problems of the agricultural field, and be able to

develop new researches that are important for the environment. And lastly, the community could profit from it for it is

expected to increase the efficiency and quality of rice being consumed by millions of people.
(10) WORK PLAN AND TARGET DELIVERABLES

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