Chapter 1

INTRODUCTION

Background of the Study

Black mold is a disease affecting the vegetables and fruits which

decreases the global productivity. Black mold caused in onion can occasionally

be seen in the field at harvest; it is a post-harvest disease and can cause

extensive losses in storage under tropical conditions (Tyson and Fullerton, 2004).

Bartholomew (2009) stated that this disease on plants is associated with a

plant pathogen called Aspergillus niger. Aspergilli are ubiquitous in nature. They

are geographically widely distributed, and have been observed in a broad range

of habitats. Aspergillus spores are in the air we breathe almost everywhere we

go. It is those who have severe fungal allergies and other immune deficiencies

that could become very sick intake of A. niger spores.

In addition, Kier (2013) asserted that the Aspergillus niger contains toxins

that can make people with weak immune systems become very sick and can

sometimes result in death. These toxins can be inhaled by humans, most

commonly people who work around plants or peat, and can cause a lung disease

called Aspergillosis, which has infected over 300,000 people worldwide.

On the other hand, E. hirta L. has been proven to have wide

pharmacological activities like antifungal, and antibacterial. Khan et al., (2011)

researched about the antifungal activity of E. hirta L. (methanolic extract) on A.

flavus. Bhaskara et al., (2010) have reported antifungal of E.hirta L. where they

worked on the leaves. Yazdani et al., (2012) have screened plants from Malaysia

against A. flavus where they used the shoots of E. hirta L. Lagnika et al., (2012)

have studied the antifungal activity. Ethanolic extract of E.hirta L. showed an

antifungal activity against A. niger using paper disc diffusion method.

In some related studies, researchers mostly analyzed the antifungal

activity of the whole plant of Euphorbia hirta. Therefore, we investigated

specifically the leaf part of the E. hirta to provide wider knowledge of its

antifungal activity. In this work, the antifungal activity of ethanolic extract of E.

hirta L. would increase the productivity of fruits and vegetables for it has the

ability to fight against the Black mold associated with a fungi, A. niger. The

findings of the study would benefit the society because it can also treat severe

fungal allergies and other immune deficiencies due to the intake of A. niger

spores (Bartholomew, 2009) and especially to our farmers to lessen the rejected

crops due to this plant pathogen. This study aims to provide knowledge about the

possibility antifungal activity of Euphorbia hirta which could help farmers create a

product that can avoid black molds in fruits and vegetables especially to that in

demand market products. There is a need to explore this plant for its potency as

well as its security.

Statement of the Problem

This study aimed to determine the feasibility of ethanolic extract of Tawa

tawa (Eurphorbia hirta Linn.) in inhibiting the growth against Aspergillus niger a

plant pathogen, specifically it sought to answer the following questions:

1. What are the phytochemicals present in Euphorbia hirta?

2. What is the level of inhibition of the growth of Aspergillus niger as an

effect of ethanolic extract of E. hirta?

3. Is there a significant difference between the inhibition activity of

ethanolic extract of Euphorbia hirta and Agrozeb (positive control) in

inhibiting the growth of Aspergillus niger?

4. Is there a significant difference between the inhibition activity of

ethanolic extract of Euphorbia hirta and Aqueous Euphorbia hirta extract

in inhibiting the growth of Aspergillus niger?

Hypotheses

This section of the study showed the possible results and answers

towards the inhibition of ethanolic extract of Tawa tawa (Eurphorbia hirta Linn.)

against Aspergillus niger.

1. There is no significant difference between the inhibition activity of

ethanolic extract of Euphorbia hirta and Agrozeb (positive control) in

inhibiting the growth of Aspergillus niger.

2. There is no significant difference between the inhibition activity of

ethanolic extract of Euphorbia hirta and Aqueous Euphorbia hirta extract

in inhibiting the growth of Aspergillus niger.

Scientific Literature

This section of the study presented various literatures and studies which

gave relevance to the investigation and will provide the readers a clear picture of

what was being emphasized in the study.

Euphorbia hirta

According to the description from Flora of (Zhonghuabencao, 1999) , E.

hirta is a small annual, branched herb prostrate to ascending with branches

reaching 70 cm in height, reddish or purplish in color, with abundant latex and is

covered with short hairs. Its leaves are opposite, distichous, and simple; its

obvious stipules are linear. The leaf blades of E. hirta are lanceolate-oblong, long

elliptic, or ovate-lanceolate; its base is very unequal; one side is cuneate, the

other side is round; the apex is almost acute, and its margins are finely toothed,

often with a purple blotch near the midvein.

The inflorescence of E. hirta has a terminal or axillary cluster of flowers,

called a ‘cyathium’, with several cyathia densely clustered into a cyme. The

flowers of E. hirta are unisexual; the male flowers are sessile, the bracteoles are

linear, fringed, the perianth is absent, and possesses one stamen, whereas the

female flowers have short pedicel, the perianth is rimmed, the ovary is superior,

covered with short hairs, three-celled, possesses three styles, minute, and the

apex is two-fid. The fruit of E. hirta is exerted, acutely three-lobed, base truncate,

covered in short hairs, and three-seeded. The seeds are oblong, four-sided

prismatic, slightly wrinkled, pinkish brown, and caruncle absent. Flowering

duration of individual plant is usually throughout the year. E. hirta often grows in
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cultivated areas in lowland, paddy fields, gardens, roadsides, and waste places.

They prefer dry condition, from sea-level up to 2000 m altitude.

Antibacterial/Antifungal Activity of E. hirta

Jackson et al. (2009) evaluated the antimicrobial activity of nystatin and

the methanol extract of the leaves of E. hirta against Candida albicans using the

checkerboard method. The results showed that some combinations of the extract

with nystatin could be synergistic in activity for some ratio combinations and

similar for some others. The antimicrobial activity of ethanol extracts of the aerial

parts of E. hirta was then investigated. A remarkable antimicrobial effect has

been revealed against Escherichia coli (enteropathogen), Proteus vulgaris,

Pseudomonas aeruginosa, and Staphylococcus aureus. The diameters of

inhibition zones were 21, 19, 23, and 19 Salmonella typhi, Klebsiella pneumonia,

P. aeruginosa, and fungus species Aspergillus niger, Aspergillus fumigatus,

Aspergillus flavus, and Rhizopus oryzae. Antimicrobial activity was attributed to

tannins, flavonoids, alkaloids, glycosides, proteins, sterols, and saponins.

Moreover, leaves collected from August to December showed more significant

antimicrobial activities (Suresh et al., 2008).

Ethanol as Solvent

There are many techniques to recover antioxidants from plants, such as

Soxhlet extraction, maceration, supercritical fluid extraction, subcritical water

extraction, and ultrasound-assisted extraction. However, extraction yield not only

depend on the extraction method but also on the solvent used for extraction. The
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presence of various antioxidant compounds with different chemical

characteristics and polarities may or may not be soluble in a particular solvent

(Turkmen et. al., 2006). Polar solvents are frequently used for recovering

polyphenols from plant matrices. The most suitable solvents are aqueous

mixtures containing ethanol, methanol, acetone, and ethyl acetate. Ethanol has

been known as a good solvent for polyphenol extraction and is safe for human

consumption. Methanol has been generally found to be more efficient in

extraction of lower molecular weight polyphenols, whereas aqueous acetone is

good for extraction of higher molecular weight flavanols (Dai and Mumper, 2010).

The maximum total phenolic content was obtained from barley flour by extraction

using a mixture of ethanol and acetone (Bonoli et. al., 2004). Antioxidant phenols

in barley (Hordeum vulgare L.) flour: comparative spectrophotometric study

among extraction methods of free and bound phenolic compounds

Aspergillus niger

Aspergillus niger contains several toxins, some harmless and others

harmful to certain people. The main toxins it contains are malformin C, and

ochratoxin A. niger can be as beneficial as it is harmful, though. Through

fermentation, it can produce useful enzymes that can be used in the production

of corn syrup, Beano, wine and cider (Kumar 2010).

Fortunately, most people can handle the inhalation of a moderate amount

of A. niger spores. Aspergillus spores are in the air we breathe almost

everywhere we go. There has actually been a case where a 70 year old man had
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to have his foot amputated because there was a “painful black ‘gangrenous

appearing’ mass on his foot. “Tissue samples showed not only branching

hyphae, but dark pigmented fungal fruiting heads with double sterigmata in which

Aspergillus niger was identified”. This may be a case of a sickly man that was

just unfortunate enough to come into contact with A. niger, but the so-called

“harmless” fungi can effect healthy people in rare cases. Otomycosis, an ear

infection that can be very painful, can be caused by Aspergillus niger. Allergic

reactions can be severe when an individual that is very allergic to fungi. “When

inhaled, A. niger can cause hypersensitivity reactions such as asthma and

allergic alveolitis” (EPA.gov). This is uncommon, but nearly fatal if the wrong

person became infected.

A. niger can also affect plants, such as onions and tomatoes. Small

animals, such as mice and chicks were fed moldy soybeans with A. niger on

them, and the subjects subsequently died after digestion. Onions are a common

plant that A. niger likes to inhabit, causing spoilage and can then result in

economic loss in farming communities. Mangoes, grapes and tomatoes are also

victims of the pathogen, as well as 34 other genera of plants. Lastly, A. niger can

cause the rotting of wood and other hard surfaces. Wood surfaces have been

found softening or deteriorating because of the contamination of A. niger. There

have also been reports of A. niger effecting very random substrates such as

polyvinyl acetate, polyester-type polyurethanes, and even English style

crumpets.
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Aspergillus niger does not discriminate when it comes to what it wants to

contaminate. However, this very durable fungus is nothing to be worried about,

unless you have a weak immune system or have a sensitive allergy to fungi, or if

you are a mouse. There are also remedies that can be taken if one contracts

Otomycosis or another common fungal infection. There are many other more

harmful fungi that are in the same family (A. fumigatus) to be worried about, so

be sure you do not inhale too many A. niger spores, and don’t eat heavily molded

fruits that are covered in a black substance, and you should be fine.

Agrozeb as Positive Control

Common Name: Mancozeb

Crops: Banana, mango, grape, papaya, potato, tomato, cantaloupe,

cucumber, squash, melon, watermelon, celery, cabbage, cauliflower, broccoli,

lettuce, onion, eggplant, pepper, rice, corn, peanut, beans, peas, mongo,

soybean, cotton, ornamentals

Target Pests: Sigatoka, Anthracnose, Scab, Black Leaf Streak, Fruit rot,

Early/Late Blight, Downy Mildew, Leaf Spot, Purple Blotch, Botrytis Leaf Blight,

Sheath Blight, Rust.

Ahir and Maharshi (2008) reported that pre-harvest application of

carbendazim + mancozeb at 0.2 per cent concentration found most effective

against black mould rot of onion caused by A. niger. Maharshi et al. (2009) found

out that carbendazim (1000 ppm) gave complete control of blue mould rot (
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Penicillium italicum) of kinnow mandarin followed by carbendazim 12 % +

mancozeb 63 % (1000 ppm) in both pre- and post- inoculation treatments.

According to (Patel, 2009) incorporation of carbendazim (12%) +

mancozeb (63%) at 500 to 1000 µg/ml concentrations gave complete mycelial

growth inhibition of A. niger. Verma and Tikoo (2003) reported pre inoculation

treatment of carbendazim (0.05 %) found most effective against P. digitatum

infecting mandarin orange than post-inoculation treatment. Pre harvest sprays of

saaf (0.1 %), benomyl (0.1 %), carbendazim (0.1 %) and mancozeb (0.2 %) gave

100, 100, 87.4 and 73.38 per cent disease reduction over control, respectively at

15 days of storage against P. digitatum on onion in storage conditions (Raju and

Naik, 2006).

Agrozeb was used in the study as a positive control because of its

feasibility to fight various fungi. It has also the ability to control disease in

different fruits and vegetables caused by the infection of Aspergillus niger.