RUNNINGHEAD: Antifungal Activity of Wild Sunflower against Fusarium oxysporum 1

The Antifungal Activity of Wild Sunflower (Tithonia diversifolia) Extract

against Fusarium oxysporum

Victor Jr. B. Botigan

10- Juniper

Pines City National High School

Daryl C. Magalgalit
Research Adviser

July 31, 2017

Date

2017-2018
School Year
Antifungal Activity of Wild Sunflower against Fusarium oxysporum 2

Chapter 1

Background of the Study

Two years ago the Strawberry Farm in Benguet experienced Fusaruium wilt. The

strawberries’ leaves were dicolored and gone dry. The damage did not affect the normal

production of strawberries in Benguet. Farmers were alarmed because their income was affected

(GMA News, 2015).

Several areas in Benguet, Ifugao, and Nueva Vizcaya reported an alarming damage of

sweet potatoes due to Fusarium wilt. The damage affected the people who rely on sweet potato

as their staple food (Alipit, 2015).

“Fusarium wilt is the number one cause of vascular wilt characterized by the interveinal

yellowing of the leaves followed by complete yellowing of the lower, older leaves. Infected vine

stem ruptures with brown to purple discoloration. This disease is caused by the fungi Fusarium

oxysporum and was first reported in Kayapa, Nueva Vizcaya in 2011 with 30 to 85 percent

disease incidence in affected villages (Backian et al., 2014). This disease fairly spread through

the movement of infected planting materials. Sweet potato farmers in Kayapa, Nueva Vizcaya

describe this disease as “nappit” that slowly spreads until the whole plant dies. Accordingly,

sweet potato roots harvested from infected plants taste bitter with foul odor and colored black.”

(BSU RD4, 2017).

Obafemi et al., (2006) found out that, ethyl acetate and methanol extracts of TIthonia

diversifolia (wild sunflower) inhibits the growth of Candida Albicans. These events inspired the

researcher to conduct a study on the antifungal activity of wild sunflower against Fusarium

oxysporum. The farmers and consumers may benefit from the results of the study.
Antifungal Activity of Wild Sunflower on Fusarium oxysporum 3

Statement of the Problem

The study aims to determine the antifungal activity of wild sunflower extract on

Fusarium oxysporum.

Specifically, the study aims to answer the following questions:

1. What are the antifungal components of wild sunflower extract?

2. What concentration (25%, 50%, 75%, 100%) of wild sunflower extract best inhibits the

growth of Fusarium oxysporum?

3. Is there a significant difference between the mean inhibition zones of the bromuconazole

and the extract that best inhibits the growth of Fusarium oxysporum?

Hypotheses

1. There are antifungal components of wild sunflower extract.

2. The concentration of wild sunflower extract that best inhibits the growth of Fusarium

oxysporum is 75% extract by 25% distilled water.

3. There is a significant difference between the mean inhibition zones of the bromuconazole

and the extract that best inhibits the growth of Fusarium oxysporum.

Scope and Delimitation

The study aims to determine the Antifungal Activity of Wild sunflower extract against

Fusarium oxysporum. The extract that will be utilized for the study is the ethanolic extract of

Wild sunflower (Tithonia diversifolia). The study does not cover but encourage the use of other

varieties of sunflower. The study will be conducted at the D.O.S.T. Laboratory of Pines City

National High School from July 20, 2017 to September 4, 2017.

Antifungal Activity of Wild Sunflower against Fusarium oxysporum 4

Significance of the Study

The results of the study is important because the findings may lead into the development

of natural products that may inhibit the growth of Fusarium oxysporum on plants and soil. The

consumers nowadays are aware of the effects of synthetic fungicides to the human health and the

environment. They avoid vegetables and fruits that were applied with synthetic fungicides,

affecting the income of the farmers and traders. Wild sunflower may also be a source of income

for cultivators because they may trade the wild sunflower to the Agriculture sector that may use

it for further studies and the development of natural products against fusarium.

Review of Related Literature

“Fusarium wilt caused by the soil borne fungus, Fusarium oxysporum Schlectend.: Fr. f.

sp. lycopersici (Sacc.) W.C. Snyder and H.N. Hansen, is one of the most devastating diseases of

tomato. It affects greenhouse and field grown tomatoes in warm vegetable production areas. The

disease is characterized by yellowed leaves and wilted plants with minimal or absent crop yield.

There may be a 30 to 40% yield loss due to the disease and this may go up to 80% under

favorable weather conditions. The pathogen invades the root epidermis and extends into the

vascular tissue. It colonizes the xylem vessels producing mycelium and conidia. The

characteristic wilt symptoms appear as a result of severe water stress, mainly due to vessel

clogging (Nirmaladevi et al., 2016).”

“Fusarium oxysporum is a ubiquitous root-infecting fungal pathogen that causes wilt

disease on several plant species including Arabidopsis thaliana. F. oxysporum is considered a

hemibiotrophic pathogen because it begins its infection cycle as a biotroph but later changes to a

necrotroph. In the biotrophic phase, F. oxysporum establishes infection via the roots and travels
Antifungal Activity of Wild Sunflower on Fusarium oxysporum 5

towards the vasculature. Upon perception of fungal elicitors, plants mount a basal defence

response (PTI) characterised by an oxidative burst, cell wall callose deposition and

transcriptional changes which is designed to inhibit microbial colonisation (Lyons et al., 2015).”

Fungi are resistant to antimicrobial agents however the methanol and chloroform extract

of Wild sunflower (Tithonia diversifolia) was able to inhibit the growth of a Candida Albicans in

vitro. The phytochemical constituents that are present in the extracts were tannins and saponins

(Ogundare, 2007).

“Leaf extracts from Ageratum conyzoides, Spilanthes filicaulis and Tithonia diversifolia

(Asteraceae) were evaluated for their antifungal activities against three leafspot fungi such as

Cochliobolus lunatus, Fusarium lateritium and Fusarium solani. In this study, ethanol was used

as an extraction solvent and bioactivity screening was done by poisoned food technique. All the

extract concentrations ranging from 8 - 120 mg/ml in Potato Dextrose Agar (PDA) medium were

significantly (P<0.05) toxic to the test fungi in vitro with their inhibition potentials being

concentration and species dependent (Ilondu et al., 2014).”

According to the study of Obafemi et al., (2006) the Hexane, Ethyl Acetate and Methanol

extract of Tithonia diversifolia manifested strong antifungal activity on Candida Albicans with a

recorded inhibition zone of 25 mm for Hexane and Ethyl Acetate extract and 22mm for

Methanol extract respectively.

In vitro efficacy of three organic solvent (petroleum ether, chloroform and methanol)

extracts of Tithonia diversifolia leaves were tested against nine plant pathogenic fungal species.

The results revealed that each fungal species may show differential growth responses to different

solvent extracts of the same plant species. The PE extract could inhibit the growth of nine fungal
Antifungal Activity of Wild Sunflower against Fusarium oxysporum 6

species while the CH and ME extracts inhibited the growth of 3 and 5 fungal species,

respectively. Colony growth of A. alternata could not be inhibited by any of the extracts. The

highest inhibitory effects were observed with Petroleum Ether extract against D. oryzae, A.

solani and A. flavus (Linthoingambi and Mutum 2013).

Methodology

Materials and Methods

Collection and preparation of wild sunflower extract.

The sunflower leaves will be gathered and washed using tap water and will be air dried

for 3 days. The leaves will be weighed before soaking in 70% ethanol and macerating for 3 days.

Filter papers will be used for filtration. The liquid obtained will be subjected to water bath to

obtain sunflower extract. 3 different concentrations of the extract will be made: 15%, 30%

Phytochemical Analysis.

“Crude extract samples from the four collection sites: UP Drive, Military Cut-off near the

Baguio General Hospital, area inside the Girl Scout of the Philippines Headquarters

(GSP), residential area in Barangay Tomay, Benguet were submitted to the Natural Science

Research Unit (NSRU) of St Louis University, Baguio City, Philippines and were qualitatively

tested for the presence of physiologically active plant constituents like alkaloids, steroids,

anthraquinones, flavonoids, saponins, tannins and polyphenols and cyanogenic glycosides

(Gutierrez et al. 2015).”

Antifungal Activity of Wild Sunflower on Fusarium oxysporum 7

Table 1: Phytochemical Compounds in leaves of T. diversifolia

Phytochemical Compounds Remarks

Alkaloids +ve
Anthraquinones +ve
Flavonoids +ve
Polyphenols +ve
Saponin +ve
Cyanogenic glycocides +ve
Steroids +ve
+ve
Key: +ve = positive

Preparation of medium.

The medium that will be used in Antifungal assay is Potato Dextrose Agar (PDA). The

composition of Potato Dextrose is 4 gm/L of potato extract and 20 gm/L of dextrose. For

solidification 20 gm/L of Mueller Hinton agar will be added. These constituents will be mixed,

autoclaved and will be poured into Petri plates for solidification. These plates will be used for

antifungal assay (Neela, Ismat, and Shamsi, 2014).

Isolation of F. oxysporum.

The fungus will be isolated from infected sweet potato leaves. The leaves will be rinsed

thoroughly in tap water. The leaves will be disinfected using ten percent (10%) bleach. The

leaves will be rinsed three times in sterile-distilled water, will be dried on sterile filter paper and

will be plated onto Potato dextrose agar (PDA) medium amended with streptomycin sulphate

(300 mg/l). Fungal isolates will be incubated one week at 24°C.

The zone of inhibition of the different concentrations of wild sunflower extract.

Plant extracts will be tested for their efficacy against the fungi by using Well diffusion

method. The agar will be inoculated by spreading 200µL of inoculum using a sterile glass rod.
Antifungal Activity of Wild Sunflower against Fusarium oxysporum 8

One well will be drilled aseptically into the agar using a sterile 6mm. cork borer. 30µL of the

treatments will be introduced into the well. The set ups will be incubated for 7 days at

tempearature of 23 degrees Celcius. The inhibition zone of the treatments will be measured using

a foot rule in mm after 7 days of incubation. The Distilled water will be the negative control and

the bromuconazole will be the positive control, respectively.

Statistical Analysis

Table 1. The antifungal activity of the different concentration of Tithonia diversifolia

extract

Inhibition zone
Treatments
T1 T2 T3 Mean

1. Distilled water

2. 25% Extract :75 SDW

3. 50%Extract :50% SDW

4. 75%Extract :25% SDW

5. 100% Extract

6. Bromucanazole

KEY: SDW=Distilled Water; T=Trial

Mean= S/N

where: S=the sum of the inhibition zone of the treatment, N=the number of replicates
Antifungal Activity of Wild Sunflower on Fusarium oxysporum 9

Table 2. T-Test between bromucanozole and and the extract.

Treatment n Mean Standrad Deviatoin

bromucanazole 3

Concentration 3

Total 6

KEY: n=number of treatments