Antifungal Activity of Corn (Zea mays) Cob Extract

against F. oxysporum (Fusarium oxysporum)

Paula Louise P. Atienza

Szophia Valeiree H. Ceniza

Mia Isabela R. Cuevas

Jer Marvielle B. Lasac

Sabelle Angelica B. Marzan

Jonas Clyde M. Oli

Kathleen Anne Salazar

Maria Juliana R. Vergara

Sta. Rosa Science and Technology High School

 Abstract

The main goal of this study was to utilize the antifungal activity of corn (Zea mays) cob to inhibit

the growth of Fusarium oxysporum, a fungi known to be resistant to most available drugs. The

researchers obtained the extract using the maceration method to release the necessary

phytochemicals for inhibition. Three different concentrations for the extract were employed for

antifungal testing which are: twenty five percent, fifty percent and seventy five percent. There

were three trials for each concentration in correspondence to the Randomized Complete Block

Design. The zones of inhibition for all concentrations were equivalent to zero. Using the

Analysis of Variance and Pearson-R Correlation, at the conventional level of significance, the

computed f and r-values are smaller than their respective tabular values. Based on the

abovementioned result, the decision to accept all null hypotheses were formulated. It was

concluded that the various concentrations of the corn cob extract have no significant effect on the

size of inhibition. Furthermore, there is no significant relationship between the extract and the

fungi. Numerous researches have reported the presence of phytochemicals in corn cobs, however,

this study proved that it is not sufficient to inhibit the growth of Fusarium oxysporum. In

accordance to the results of this study, the researchers highly recommend finding other plant

waste materials with potential antifungal properties, applying the extract to other fungi and

discovering other methods to stimulate the antifungal activity of corn cobs. It also proposes the

utilization of the extract for other areas of concern such as antibacterial and anticoagulation

fields.

Antifungal Activity of Corn (Zea mays) Cob Extract against F. oxysporum (Fusarium

oxysporum)
 The Philippines is an agricultural country with a land area of 30 million hectares, 47

percent of which is used for farming. Contrary to the massive production of crops, several

problems are besetting the farming industry such as the spread of pathogenic microbes. Crop

diseases have an effect on the income of farmers and the health of consumers. Several

researches have been conducted in an attempt to provide low-cost and efficient solutions to crop

blights, scabs and wilts.

Corn (Zea mays)

Corn is the second most important crop in the Philippines that contributes significantly to

the country’s economic development. Corn wastes are considered one of the top agricultural

wastes in the Philippines. The combustion of these wastes pose serious risks on the environment

and the health of farmers. The cob is the part of the corn ear on which the kernels grow. This

part will become the waste product once the kernels have been consumed. Phytochemical

studies revealed that corn cob contains liquid crystalline oils, flavonoids, phenolic compounds

and this anthocyanins which result in the anti-inflammatory, antibacterial and antioxidant

properties of the cob. Carvacrol, thymol and menthol are phenols used chiefly as fungicides,

moisturizers and disinfectants to treat fungal infections such as eczema and rashes

(botanicalonline.com).

Total Phenolic Contents of Corn By- Products

According to Dong et. al (2014), three of the most commonly known by- products of corn

(Zea mays) are used in different herbal medicine due to its rich chemical deposits within it and

was proven to be safe. Various studies show that the three are rich in phenolic compounds,

flavonoids, kerotinoids and different antioxidants and other antibacterial properties. Stigma
maydis is proven to have the richest contents among these parts. It contains exceptional

bioactivities which are due to hydroxyl groups found in these phenolic compounds. Corncobs

have almost the same properties with the former but the study also showed that anthocyanins

were present which is a type of flavonoid. On the other hand, corn cobs and corn husks are easier

to obtain than stigma maydis because of a higher yield in production and most of these are

thrown out.

Maize grain is the third most consumed cereal, surpassed only by rice and wheat. It is

ranked as one of the cereals with the highest content of phenolic compounds. In maize grain, the

phenolic acids: ferulic acid and p‐coumaric acid predominate, which are highly abundant in their

bound forms. However, other phenolics such as anthocyanins, flavonoids, and flavanols have

been identified in colored maize grains. Flavonoids possess important biological activities such

as antioxidant, antimutagenic, and microbicidal. The flavonoids reported to be present in maize

are anthocyanins, flavonols, and proanthocyanins (Salinas-Moreno et al., 2016).

Fusarium oxysporum

Fusarium oxysporum is a soil-borne pathogenic fungus comprising of over 120 special

forms, each specific to a unique plant host. F. oxysporum causes fusarium wilt, a vascular

wilting disease characterized by root rot, seed blight, rotten fruits and discolored leaves followed

by the impairment of the whole plant (Campbell et al, 2014). According to Gutierrez (2016),

approximately 15,000 hectares of land have been affected by the Fusarium wilt that is considered

as one of the first disease that affects perennial and annual plants to have spread globally. Dry

weather and low moisture accelerates the development of F. oxysporum. Attempts of complete

eradication have only produced insignificant results since mycelia growth displayed high

adaptation ability in different environmental conditions. The most effective solution is soil
sterilization, a method that requires a lot of equipment and is of long duration. Alternatively,

some farmers use commercial fungicides with limited effects on the fungus and negative effects

on the plant (Bush, 2014).

According to Puig (2014), Fusarium wilt is a global threat to all banana growing areas

around the world. The massive losses of bananas, specifically ‘Cavendish’, in Davao region’s

banana industry is caused by this disease. Because of this, a huge area of lands in Davao del

Norte and ComVal were abandoned. One of the causal organisms is Fusarium oxysporum f. sp.

cubense Tropical Race 4 (Foc TR4). This affects all banana cultivars growing in the region.

Fusarium wilt in the region remains to be a problem in some Asian countries, Australia, South

and Central America and Africa, notwithstanding disease control.

Fusarium wilt in Cavendish bananas around Davao on the southern Philippine island of

Mindanao was caused by Foc VCG 01213 that was found both on the Mindanao and Luzon

islands, and was isolated mainly from the Silk cultivar Latundan. VCG 0122 was related with the

Cavendish cultivar Grande Naine in the Davao del Norte area. Three different VCGs were

isolated from Grande Naine, namely VCGs 0122, 01213 and 01216. (Mostart et al, 2017)

Numerous crops have been known to be host plants for Fusarium species causing soil-

borne diseases. Though extensive studies have been performed, little is known about the fungus

stimulating or inhibiting properties. In the present study, the effect of flavonoid compounds on

the germination and fungal growth of soil-borne pathogen Fusarium oxysporum was observed.

Myricetin and luteolin exhibited a low stimulating activity on microconidia germination of F.

oxysporum, whereas the other flavonoids were inactive at different concentrations. Individual

flavonoid concentrations resulted in a small increase of fungal growth. However, the lowest

flavonoid concentrations showed an inhibiting effect on fungal growth. This suggests that low
flavonoid concentrations exhibit antimicrobial properties against F. oxysporum (Steinkellner &

Mammerler, 2016).

The Philippines has and continues to undergo an economic crisis when it comes to the

agricultural sector. The growth and quality of the crops can be affected by a lot of factors. One

of these is the susceptibility of the plant to different pathogenic microbes. Fusarium oxysporum

is a fungus that enters the host through the roots and grows on the xylem, blocking the vascular

and preventing the transport of water and nutrients. Species in the Solenaceae family such as

bananas, eggplants, pepper, potatoes and watermelons are the most susceptible to fusarium wilt,

commonly known as Panama disease. This fungus can be dormant for 30 years before resuming

virulence and is resistant to most antimycotic medications due to its rapid mycelia growth and

ability to survive in the absence of effective biological, chemical and physical control measures

(Ploetz, 2015).

Fungal-pathogen inflicted diseases have been a major challenge to agriculture, health as

well as national economy. The resistance of Fusarium oxysporum to a wide variety of chemicals

has stimulated the search of new alternatives for control measures. The use of plant extracts are

one of the most promising, effective, safer and eco-friendly method to eradicate the pathogen

from ripe tomato. Out of 9 extracts, it was found that almost all extracts at 25% concentration

were effective in reducing the mycelium growth of Fusarium oxysporum f. sp. lycopersici. The

highest inhibition (87%) of mycelium growth of this pathogen was observed when treated after

the plant extract of Calotropis proceraon at 25% concentration and lowest inhibition (27%) of

this pathogen was observed when treated after the plant extract of Momordi cacharantia on at

5% concentration (Mahmud, Nasrin & Podder, 2018)

 Fusarium oxysporum is an ascomycete facultative fungus recently known as a serious

emerging opportunistic pathogen of human and other animals. Antifungal susceptibility test

revealed the concentration dependent growth inhibition of lawsone against F. oxysporum with

minimum inhibitory concentration (MIC) at 100 μg/mL. Ultra-structural analysis indicates the

prominent damage on cell wall of the mycelium after lawsone treatment, and imples that it could

increase the membrane permeability and disintegration of cells leading to cellular death.

Lawsone can induce the reactive oxygen species (ROS) level in the filaments of F. Oxysporum

which activates the autophagy related pathways due to the oxidative stress. Results showed the

loss of viability and germination capacity of F. oxysporum spores by lawsone in concentration

dependent manner. F. oxysporum infected zebrafish has recovered after lawsone therapy as a

topical treatment suggesting that lawsone is a potential natural antifusariosis agent (Dananjaya,

et al., 2017).

Extraction of Phenolic Compounds

The extraction process is a key component to efficiently obtain a great amount of

chemical constituents in organic materials. Phenolics are secondary metabolites that are found in

several fruits, grains and different parts of plants. It is considered to have a wide range of

biological activities, such as antioxidant, antibacterial, antifungal and provides cardio-

protection. The solvent to be used can greatly affect the amount and type of phenolic compounds

to be extracted. Ethanol and methanol with different concentrations are commonly used

whenever phenolics, flavonoids, kerotenoids and tannins are extracted on plants. The study used

100ml of water, 50% ethanol, 80% ethanol, 50% methanol, 80% methanol and ethyl acetate to

test each in 2g- sample powders of three by- products of corn (corncobs, cornhusk and stigma

maydis) in for a day. It was then extracted via ultrasonic cleanerat 40°C thrice, and then
concentrated through a rotary evaporator. After different tests it was proven that 80% ethanol can

be used to effectively extract more from the by- products of corn (Dong et. al, 2014).

According to Saikia and Mahanta (2013), it was reported that the phenolic content and

flavonoid content of cooked vegetables are different from the uncooked form as it could have

effect like destruction, release, and transformation of the phytochemicals in the vegetable.

Application of heat could change the structural integrity and cellular matrix of vegetables when

cooked. This means it can have either positive and negative effects on the total flavonoid content

of the vegetable. In the study, it was shown that boiling the vegetable lead to a decrease in the

total flavonoid content.

The goal of extraction is to separate soluble plant metabolites, leaving behind insoluble

cellular marc (residue). The initial crude extracts commonly contain a complex mixture of

alkaloids, glycosides, phenolics, terpenoids and flavonoids. Maceration is a widely-used method

of extraction used in medicinal plant research. It involves soaking powdered plant materials in a

stoppered container with a solvent at room temperature for a minimum period of 3 days. This

process intends to soften and break the plant’s cell wall in order to release soluble

phytochemicals. The mixture is then pressed or strained by filtration. Conventionally, the choice

of solvent will determine the type of compound extracted from the samples (Azwanida, 2015)

In this study “Effectiveness of Maceration Periods with Different Extraction Solvents on

in-vitro Antimicrobial Activity from Fruit of Momordica charantia L.”, research has shown that

“longer maceration time often leads to chemical changes, particularly oxidation of phenolic

compounds such as flavonoids. It was the reason why there were no antifungal activity observed

for hexane that was macerated at 12 and 24 hours. But the presence of other phytochemicals that
conferred the activity might be the reason why mild antifungal activity was observed at 48 hours

maceration time.” (Yang et al, 2014)

Over the past few decades, antifungals have decreased in effectivity due to the emergence

of drug-resistant fungi and toxic reactions. F. oxysporum is resistant to most available antifungal

drugs. This study aims to utilize the antifungal activity of uncooked corn cobs to eliminate

Fusarium oxysporum. It will yield a significant difference between the sizes of inhibition and

concentration of the corn cob extracts. The researchers also predict that there will be a

significant relationship between the amount of corn cob extract and its antifungal activity against

F. oxysporum. The extract will be applied to a culture of the aforementioned fungi instead of the

infected plant to limit the effect of extraneous variables. This study provides a good chance of

reducing the cost of sterilization and the use of fungicides with little impact on the infected plant.

Problem Statement

The researchers hypothesize that:

1) There is a significant difference between the sizes of inhibition and the concentration of the

corn cob extract at 25%, 50% and 75% and;

2) There is a significant relationship between the corn cob extract and the fungi, Fusarium

oxysporum.

Scope and Delimitation

The research study focus on extracting the uncooked corn cobs to test its antifungal

activity. It also focus on the potential of the extract against the fungi, Fusarium oxysporum. It

covers the use of laboratory and laboratory equipment such as florence flask and beaker.
 Nevertheless, it does not seek to include the testing of the corn cob extract to an actual

plant affected by the said disease. It is also limited that the corn cob extract can completely

alleviate the fungi.

Significance of the Study

The study will benefit the farmers as the number of crops to be affected by Fusarium wilt

will be lessened. This will benefit them as a component to create a natural antifungal agent rather

than commercial antifungals. It will also benefit the community since the number of corn cob

wastes will decrease and be reused to agriculture in the locality. Future Researchers will also

benefit as they may come up with a more comprehensive study about the effect of the corn cob

extract against F. oxysporum.

This study will prove a safer and more effective alternative component to create

fungicides using corn cobs, lessening environmental wastes and producing an accessible natural

remedy for Fusarium wilt.

Method

Research Design

The appropriate experimental design for the study is the Randomize Complete Block

Design (RCBD). This design will be used to determine the cause of change by using successive

treatments. There will be randomization process to designate samples for the experimental group

of the study. RCBD implies that there will be repetition of the experiment and the treatments

used.

Measures
 The study will use the Kirby-Bauer method, a common technique to measure the zone of

inhibition. This is the appropriate technique to measure the antifungal activity of corn cob extract

against F. oxysporum. The results will be measured by examining the area in which the fungi is

unable to grow because of the extract that will be applied. The areas will be measured in

millimeters and will undergo statistical treatment to see changes in significance of the different

concentrations.

Procedure

One thousand grams of corn cobs will be collected in the city of Santa Rosa in

Laguna. The corn cobs will be subjected to air-dry for an average of 3 days. After air-drying the

cobs were manually crushed using mortar and pestle. The researchers used manual crushing

instead of using millers to monitor the solubility of the cob and limit the contact of the sample

with contaminated materials. Approximately 1000 grams of pulverized corn cobs were then

soaked in 95% ethanol. The mixture followed a 1:3 ratio which implies that 250 grams of corn

cobs will be soaked in 750 ml of ethanol. This process was done under the following conditions:

at room temperature, using tightly-sealed containers at a duration of two (2) days. The initial

crude extract was then subjected to filtration using regent containers, funnels and filter papers.

Water bath was the final step for maceration which includes the exposure of the extract to

steam which allowed evaporation and condensation to take place. This was done to remove any

trace of ethanol in the extract. A stoichiometric amount of 25 ml of corn cob extract was placed

in an amber bottle and delivered to the laboratory in National Sciences Research Institute of the

University of the Philippines Diliman College in Quezon City for antifungal testing.

Microbial suspension was prepared from a 7-day old culture of F. oxysporum. 0.1%

peptone water was used as a suspending medium.

 For each trial, Pre-poured Potato Dextrose Agar (PDA) plates were inoculated with the

microbial suspension by swabbing the agar surface. The cotton swab on an applicator stick was

dipped into the microbial suspension, rotated a few times, and pressed firmly on the inside wall

of the tube to remove excess inoculum. The swab was streaked over the whole agar surface. This

process was repeated two more times, rotating the plate 60° each time to ensure even

distribution. Three (3) equidistant wells were made on the agar plate using a cork borer. Two

hundred (200) μl of the sample in different concentrations was placed in each well.

The PDA plates were incubated at room temperature for 3 days. The clearing zones of

each plates were measured in millimeters.

Results

Table 1 shows the summary table of significant difference between the sizes of inhibition

and different concentrations of corn cob extract at 25%, 50%, and 75%.

In 25% concentration, the sizes of inhibition of trials 1, 2, and 3 are all 0.0. In 50%

concentration, the sizes of inhibition of trials 1, 2, and 3 are all 0.0. In 75% concentration, the

sizes of inhibition of trials 1, 2, and 3 are also 0.0.

The following data was calculated using Analysis of Variance. Based on the data shown

in the table below, the obtained F-value obtained was 0. The table showed the analysis of

variance of the gathered data at α=0.05 level of significance. The null hypothesis is accepted

because F value is less than the F-critical (0<5.1433).

This implied that there is no significant difference between the sizes of inhibition and

different concentrations of Corn cob extract at 25%, 50%, and 75%.

 Table 1
Significant difference between the sizes of inhibition and the concentration of the corn cob
extract at 25%, 50% and 75%.

Zone of inhibition (mm)

Concentrations
Trial 1 Trial Trial 3
2

25% 0 0 0

50% 0 0 0

75% 0 0 0

Sources SS df MS

Between Groups 0 2 0

Within Groups 0 6 0

Total 0 8

F value Fcrit

Decision: Accept the H o

0 5.1433

Table 2 shows the summary table of significant relationship between the corn cob extract

and the fungi, Fusarium oxysporum.The concentrations of the corn cob extract are 25%, 50%,

and 75% with three samples of each. In 25% concentration, the sizes of inhibition are all 0.0. In

50% concentration, the sizes of inhibition are all 0.0. In 75% concentration, the sizes of

inhibition o are also 0.0.

 The following data was calculated using Pearson-R Correlation. Based on the data shown

in the table below, the obtained Rcomputed was 0. The table showed that the pearson-r correlation of

the gathered data at α=0.05 level of significance is equivalent to 0.632. The null hypothesis is

accepted because Rcomputed value is less than the Rtabular.

. This implied that the different concentrations of Corn cob extract and the sizes of

inhibition in each trial does not have any correlation with each other.

Table 2
Significant relationship between the corn cob extract and the fungi, Fusarium oxysporum.

Concentrations Zone of inhibition (mm)

1 0.25 0.0

2 0.25 0.0

3 0.25 0.0

4 0.50 0.0

5 0.50 0.0

6 0.50 0.0

7 0.75 0.0

8 0.75 0.0

9 0.75 0.0

Decision: Accept rcomputed 0

:

H o
 Discussion

This study utilized the antifungal activity of corn cobs to inhibit the growth of Fusarium

oxysporum. Antifungal tests have determined that the zones of inhibition for all concentrations of

the extract were equivalent to 0. All null hypotheses were accepted with respect to the statistical

treatments. This suggests that the corn cob extract has no significant effect on the growth of the

fungi. F. oxysporum is a drug-resistant microorganism that produces enzymes which are

necessary to degrade lignocellulosic biomass to sugars. It also has a recognized ability to survive

without any pathogenic activity which implies that any attempt of inhibition might encounter

some difficulties. Contrary to previous studies regarding the pharmacological activity of the corn

plant, the phenolic acids and flavonoids present in corn cobs were not sufficient to inhibit the

growth of the fungi. According to a study by Mahmud, Nasrin and Podder (2018), the

recommended concentration for inhibition is 25 percent while Anasontzis and Christakopoulos

identified that F. oxysporum ferments monosaccharides to ethanol. Based on the abovementioned

studies, the researchers were able to conclude that three factors have contributed to hindrances in

the inhibition of the fungi: the method of extraction, amount of the extract and the tolerance of F.

oxysporum to ethanol.

Limitations of the Study

The study only seeks to inhibit Fusarium oxysporum using corn cob extract. It does not

seek to include other parts of the corn plant for the extract. The researchers only used maceration

as a method of extraction in the said study. It focused on the potential of the corn cob extract

against Fusarium oxysporum that is tested on the laboratory not on the plant infected by

Fusarium wilt.
Implication of Findings to Theory and Practice

In this study, the inhibition of F. oxysporum using corn cob extract was not observed,

primarily due to the effect of extraneous variables and other factors concerning the methods and

samples. During the experimentation and analyzation processes, the researchers have identified

the significant contributions of the study. Specifically, it is the first research to have used corn

cob as an antifungal agent against Fusarium oxysporum. And it is also proposes the feasibility of

using the cob extract as an alternative, cheap component for natural fungicides directed to other

microorganisms besides F. oxysporum.

Similarly, the results of this study indicate that the fungi is widely resistant to almost, if

not all, drugs or organic materials with pharmacological properties. Studying other plant waste

materials, finding other ways to stimulate the antifungal activity of corn cob such as using

another mode of extraction and applying it to other microorganisms is highly recommended.

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Curriculum Vitae