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CHAPTER I

THE PROBLEM AND ITS SETTING

Introduction

Tomato or the Lycopersicum esculentum is one of the most widely and

economically significant vegetables crops. This plant is a member of nightshade

family or solanaceace. This fruit is believed to be originally domesticated in Europe

during the 19th century and in Incas of South America. It is a hairy, maybe a bushy or

vinelike, range in height from 3 to 6 feet (0.9-1.8 meters) and a strong smelling

spreading plant. It takes 45 to 50 days for the fruits to mature. The size, shape, and

color of the fruits vary according to the variety. Some tomatoes are small and round-

like pears are plums, spherical as cherries and other nutrients. Specifically, it

contains the nutrients called lycopene anthocyanin that improve the skins ability to

protect against harmful UV rays, carotene and prevent prostate cancer (pfaf.org).

Commonly tomato is attacked by the different diseases and pests in all stages

of its development from seeds emergence. The tomato soft rot (Erwinia carotovora)

is considered as one of the most severe postharvest diseases that causes

deterioration in the tomato fruit. Usually the bacteria enter through swollen, water

logged lenticels (pores) on tomato specifically on the stem end and wounds. The

bacteria are carried on seed pieces, borne in water and soil and sometimes can be

carried by the insect bodies and equipment. Slimy or watery substance oozes from

break in the blister until it becomes black and foul smelling appears.


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On the other hand, garlic (Allium sativum) has a potential to cure disease and

fight against bacteria and fungi. This plant is pungent, spicy flavor that mellows and

sweeten considerably when cooking. When the garlic is being crushed it yields a

substance called allicin, this is a powerful substance that is present in this ancient

plant. Allicinhas been found to be the compound that is responsible for the hot

sensation of the raw garlic. The chemicals open thermo TRP (Transient Receptor

Potential a channels that are responsible for the burning sense of heat in foods)

(Gibson et al. 2005).

This study will provide an idea and answers to the questions that the

researcher wants to answer. Specifically the researcher wants to know the efficacy

of garlic extract against tomato soft rot.

Statement of the Problem

This study will be undertaken to find out if the garlic has a potential in curing

the tomato soft rot.

Specifically the researcher wants to provide answers to the following questions:

1. Does garlic (Allium sativum) clove extract exhibit potential curing of tomato

soft rot?

2. At what concentration of garlic (Allium sativum) clove extract most effective?


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Scope and Delimitation

This study will be delimited on the efficacy of garlic or Allium sativumagainst

tomato Soft rot or the Erwinia carotovora. Specifically this study will focus on the

concentration of garlic extract which could be effective against the former stated

disease. The researcher will use the filter paper as a test disc in the different

treatments. The researcher will use four treatments and will be replicated three

times.

This study will be conducted in the Mindanao State University under the

laboratory condition within the second semester of the school year 2011-2012;

moreover this study aims to find out if the garlic extract truly affects the growth of the

Erwinia carotovora. The researcher also wanted to know what concentration level of

garlic extract is most effective in suppressing the growth of Erwinia carotovora.

Significance of the Study

The main objective of the researcher is to provide ideas that could be very

important to the following:

The Agricultural Sectors, this study will provide an idea that aside from

using those synthetic pesticides they will be given an option and an alternative form

of treatment in treating the tomato soft rot. In addition with, this study will also help

the agricultural sectors in terms of money, aside from spending too much amount

just to buy synthetic chemicals in order to ensure the quality of their harvested

products they will be provided an option in the form of garlic extract that is easy to do

or can be prepared in the home and does not cost too much.


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The Business Sectors, this study will give an idea that garlic extract is

effective in treating disease in tomato specifically the tomato soft rot. They can

improve the study by doing several studies that are related to the findings of this

study. They can commercialize the garlic extract and can help to the farmers by

selling the products in a cheaper price comparing to those synthetic products. Also

they can easily spread the idea of using garlic extract as an alternative form of

treating the tomato soft rot diseases.

The Researcher, it will help in adding knowledge not only on the researchers

part but also to others. The findings of this study will help the researcher realize that

the diversity of the worlds flora also embodied with diversity ofcapacity and ability.

On the other hand, this study will help the researcher in dessiminating knowledge

and information to others that from the very common plant which is garlic, it has an

enzyme that can stop the disease that normally attack the tomato fruits.

The Other Researcher, this research will serve as a template to the other

study that the other researcher will conduct. The other researcher will give

suggestions on what related studies are necessary to be conducted.

Moreover, because the garlic is widely provided throughout the country this

study is being provided by its advantages because of the abundance of the plants

that are needed in this study.


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CHAPTER II

REVIEW OF RELATED LITERATURE AND RELATED STUDY

This chapter presents the review of related literature and studies that provide

important insights, ideas and facts that will help in the development of this study.

RELATED LITERATURE

Bioassay

Bioassay (Biological Assay) is a procedure for the determination of the

concentration of a particular constituent of a mixture. A Bioassay is a measurement

of the effects of a substance on living organisms.

Biological Assay is method of measuring the effects of a biologically active

substance using an intermediate in vivo or in vitro tissue or cell model under

controlled conditions. It includes virulence studies in animal fetuses in utero, mouse

convulsion bioassay of insulin, quantitation of tumor-initiator systems in mouse skin,

calculation of potentiating effects of a hormonal factor in an isolated strip of

contracting stomach muscle, etc

(http://www.bionewsonline.com/m/what_is_bioassay.htm)

A bioassay is a single step within a microarray experiment. There are 3 types

of bioassays. A physical bioassay corresponds to wet-lab microarray experimental

step. Measured bioassays correspond to a situation after feature extraction has

been performed. A derived bioassay corresponds to data processing experimental

steps.
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A physical bioassay is created by applying some amount of some biomaterial to a

microarray. Bioassay treatment events (e.g., wash, apply blocking agent etc.)

transform physical bioassays into new physical bioassays. A particular type of

bioassay treatment is image acquisition

(http://www.mged.org/Workgroups/MAGE/bioassay.html)

On the other hand, bioassays are procedures that can determine the

concentration of purity or biological activity of a substance such as vitamin,

hormone, and plant growth factor. While measuring the effect on an organism, tissue

cells, enzymes or the receptor is preparing to be compared to a standard

preparation. Bioassays may be qualitative or quantitative. Qualitative bioassays are

used for assessing the physical effects of a substance that may not be quantified,

such as abnormal development or deformity. An example of a qualitative bioassay

includes Arnold Adolph Berthold's famous experiment on castrated chickens. This

analysis found that by removing the testes of a chicken, it would not develop into a

rooster because the endocrine signals necessary for this process were not available.

Quantitative bioassays involve estimation of the concentration or potency of a

substance by measurement of the biological response that it produces. Quantitative

bioassays are typically analyzed using the methods of biostatistics

(file:///E:/Bioassay.htm, 2011).

Erwinia species

Erwinia is a genus of Enterobacteriaceae bacteria containing mostly plant

pathogenic species which was named for the first phytobacteriologist, Erwin Smith. It
http://www.mged.org/Workgroups/MAGE/bioassay.htmlhttp://en.wiktionary.org/wiki/qualitativehttp://en.wiktionary.org/wiki/quantitativehttp://en.wikipedia.org/wiki/Deformityhttp://en.wikipedia.org/wiki/Arnold_Adolph_Bertholdhttp://en.wikipedia.org/wiki/Biostatisticshttp://en.wikipedia.org/wiki/Enterobacteriaceaehttp://en.wikipedia.org/wiki/Planthttp://en.wikipedia.org/wiki/Pathogenhttp://en.wikipedia.org/wiki/Pathogenhttp://en.wikipedia.org/wiki/Planthttp://en.wikipedia.org/wiki/Enterobacteriaceaehttp://en.wikipedia.org/wiki/Biostatisticshttp://en.wikipedia.org/wiki/Arnold_Adolph_Bertholdhttp://en.wikipedia.org/wiki/Deformityhttp://en.wiktionary.org/wiki/quantitativehttp://en.wiktionary.org/wiki/qualitativehttp://www.mged.org/Workgroups/MAGE/bioassay.html


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is a gram negative bacterium related toE. coli,Shigella,SalmonellaandYersinia. It

is primarily a rod-shaped bacterium. A well-known member of this genus is the

speciesE. amylovora, which causes fireblight on apple, pear, and other rosaceous

crops. Erwinia carotovora (also known as Pectobacterium carotovorum) is another

species, which causes diseases in many plants (wikimedia foundation, inc, 2010).

Erwinia carotovora belongs to Kingdom Bacteria, Phylum Proteobacteria

Class Gamma Proteobacteria, OrderEnterobacteriales, FamilyEnterobacteriaceae,

and Genus Erwinia(Winslow et al.1990).

It is stated that the Erwinia species are primary plant pathogens; some cause

plant soft rot diseases. These species is believed to be the producer of the enzymes

that hydrolize the pectin between individual plant cells. This causes the plant cells to

separate from each other. Erwinia is gram-negative bacteria that are colorless after

the alcohol wash, the addition of safranin (LPS), lipoproteins and phosphocialized

functions when counterstained with a red dye after which they appear pink (Tortora,

G.; Funke, B and Case, C. 2005).

These bacteria are able to cause disease in almost any plant tissue it

invades. It is a very economically important pathogen in terms of postharvest losses,

and a common cause of decay in stored fruits and vegetables. Decay caused by E.

carotovora is often referred to as bacterial soft rot (BSR). Most plants or plant parts

can resist invasion by the bacteria, unless some type of wound is present. High

humidity and temperatures around 30C favor development of decay. Mutants can

be produced which are less virulent. Virulence factors include: pectinases,
http://en.wikipedia.org/wiki/Escherichia_colihttp://en.wikipedia.org/wiki/Escherichia_colihttp://en.wikipedia.org/wiki/Escherichia_colihttp://en.wikipedia.org/wiki/Shigellahttp://en.wikipedia.org/wiki/Shigellahttp://en.wikipedia.org/wiki/Shigellahttp://en.wikipedia.org/wiki/Salmonellahttp://en.wikipedia.org/wiki/Salmonellahttp://en.wikipedia.org/wiki/Salmonellahttp://en.wikipedia.org/wiki/Yersiniahttp://en.wikipedia.org/wiki/Yersiniahttp://en.wikipedia.org/wiki/Yersiniahttp://en.wikipedia.org/wiki/Erwinia_amylovorahttp://en.wikipedia.org/wiki/Erwinia_amylovorahttp://en.wikipedia.org/wiki/Erwinia_amylovorahttp://en.wikipedia.org/wiki/Fireblighthttp://www.wikimediafoundation.org/http://en.wikipedia.org/wiki/Bacteriumhttp://en.wikipedia.org/wiki/Proteobacteriahttp://en.wikipedia.org/wiki/Proteobacteriahttp://en.wikipedia.org/wiki/Enterobacterialeshttp://en.wikipedia.org/wiki/Enterobacterialeshttp://en.wikipedia.org/wiki/Enterobacteriaceaehttp://en.wikipedia.org/wiki/Enterobacteriaceaehttp://en.wikipedia.org/wiki/Enterobacteriaceaehttp://en.wikipedia.org/wiki/Postharvest_losseshttp://en.wikipedia.org/wiki/Postharvest_losseshttp://en.wikipedia.org/wiki/Enterobacteriaceaehttp://en.wikipedia.org/wiki/Enterobacterialeshttp://en.wikipedia.org/wiki/Proteobacteriahttp://en.wikipedia.org/wiki/Bacteriumhttp://www.wikimediafoundation.org/http://en.wikipedia.org/wiki/Fireblighthttp://en.wikipedia.org/wiki/Erwinia_amylovorahttp://en.wikipedia.org/wiki/Yersiniahttp://en.wikipedia.org/wiki/Salmonellahttp://en.wikipedia.org/wiki/Shigellahttp://en.wikipedia.org/wiki/Escherichia_coli


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cellulases, (which degrade plant cell walls), and also proteases, lipases, xylanases

and nucleases (along with the normal virulence factors for pathogens Fe

acquisition, LPS integrity, multiple global regulatory systems) (wikimedia foundation,

Inc, 2010).

Garlic (Allium sativum) cloves

Garlic (allium sativum) is classified under the Kingdom Plantae, clade

Angiospermae and Monocots, order Asparagales, family Amaryllidaceae, subfamily

Allioideae, genus Alliumand species sativum. Its close relatives include the onion,

shallot, leek, chive, and rakkyo(Dalmacio, 2008).

There are over 600 subvarieties of garlic, a member of the Lily family and a

close relative to shallots, onions, leeks and chives. But garlic falls into one of two

main categories: hardneck and softneck.

Hardneck garlic (Allium sativum ophioscorodon) produces fewer cloves than

softneck, and is better suited to northern, cooler climates. Each plant produces a

garlic scape, a central shoot with a flower atop it. Hardneck garlic is usually grown

by small farmers, and many claim that it has more robust flavor than softneck

varieties.Although the precise taxonomy of garlic is somewhat muddy, hardneck

varieties break down into five main types: Porcelain, Purple Stripe, Marbled Purple

Stripe, Glazed Purple Stripe and Rocambole, and three varieties of weakly bolting

hardnecks that often produce softnecks: Creole, Asiatic and Turban (Steiner, 1998).
http://www.wikimediafoundation.org/http://www.wikimediafoundation.org/http://en.wikipedia.org/wiki/Shallothttp://en.wikipedia.org/wiki/Leekhttp://en.wikipedia.org/wiki/Chivehttp://en.wikipedia.org/wiki/Rakkyohttp://en.wikipedia.org/wiki/Rakkyohttp://en.wikipedia.org/wiki/Rakkyohttp://en.wikipedia.org/wiki/Chivehttp://en.wikipedia.org/wiki/Leekhttp://en.wikipedia.org/wiki/Shallothttp://www.wikimediafoundation.org/http://www.wikimediafoundation.org/


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Softneck garlic (Allium sativum sativum) is grown in warmer climates. Most

garlic in the supermarket is the softneck variety. Softneck garlic does not produce

garlic scapes, and it produces more cloves than hardneck garlic. Softneck garlic can

be broken down into two varietal groups: Artichoke and Silverskin (New York Times,

2011).

Dating back over 6,000 years, garlic is native to central Asia, and has long

been a staple in the Mediterranean region, as well as a frequent seasoning in Asia,

Africa, and Europe. It was known to Ancient Egyptians, and has been used

throughout its history for both culinary and medicinal purposes. In 1858, Louis

Pasteur observed garlic's antibacterial activity, and it was used as an antiseptic to

prevent gangrene during World War I and World War II. More recently, it has been

found from a clinical trial that a mouthwash containing 2.5% fresh garlic shows good

antimicrobial activity, although the majority of the participants reported an

unpleasant taste and halitosis (Walker, 1994).

The juice from the cloves is used as an insect repellant. It has a very strong

smell and applied to any stings in order to ease the pain. An extract of the plant can

be used as a fungicide. Garlic is used in treating blight and the garlic clover is being

spread among those fruits that are being stored in order for the fruit to delay from

rotting (Marteneou, 2001).

When crushed, Allium sativum yields allicin, an antibiotic and antifungal

compound (phytoncide). It has been claimed that it can be used as a home remedy

to help speed recovery from strep throat or other minor ailments because of its
http://en.wikipedia.org/wiki/Ancient_Egyptianshttp://en.wikipedia.org/wiki/Culinaryhttp://en.wikipedia.org/wiki/Medicinalhttp://en.wikipedia.org/wiki/Louis_Pasteurhttp://en.wikipedia.org/wiki/Louis_Pasteurhttp://en.wikipedia.org/wiki/Antiseptichttp://en.wikipedia.org/wiki/Gangrenehttp://en.wikipedia.org/wiki/Halitosishttp://en.wikipedia.org/wiki/Allicinhttp://en.wikipedia.org/wiki/Antibiotichttp://en.wikipedia.org/wiki/Phytoncidehttp://en.wikipedia.org/wiki/Home_remedyhttp://en.wikipedia.org/wiki/Strep_throathttp://en.wikipedia.org/wiki/Strep_throathttp://en.wikipedia.org/wiki/Home_remedyhttp://en.wikipedia.org/wiki/Phytoncidehttp://en.wikipedia.org/wiki/Antibiotichttp://en.wikipedia.org/wiki/Allicinhttp://en.wikipedia.org/wiki/Halitosishttp://en.wikipedia.org/wiki/Gangrenehttp://en.wikipedia.org/wiki/Antiseptichttp://en.wikipedia.org/wiki/Louis_Pasteurhttp://en.wikipedia.org/wiki/Louis_Pasteurhttp://en.wikipedia.org/wiki/Medicinalhttp://en.wikipedia.org/wiki/Culinaryhttp://en.wikipedia.org/wiki/Ancient_Egyptians


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antibiotic properties. It also contains the sulfur-containing compounds alliin, ajoene,

diallylsulfide, dithiin, S-allylcysteine, and enzymes, B vitamins, proteins, minerals,

saponins, flavonoids, and Maillard reaction products, which are not sulfur-containing

compounds. Furthermore, a phytoalexin (allixin) was found, a nonsulfur compound

with a -pyrone skeleton structure with antioxidant effects, antimicrobial effects,

antitumor promoting effects, inhibition of aflatoxin B2 DNA binding, and neurotrophic

effects (Lin,1998). Prostaglandins A2 and Fla were isolated from a homogenized

garlic extract. High-molecular weight fructans and agglutinins (homo- and

heterodimeric mannose-binding lectins) were also isolated from garlic.

Allicinis the major odor principle that is produced by the enzymatic action of

alliinase on alliin; it is decomposed by heat and alkali but is unaffected by dilute

acids in solution. Composition of garlic products depends on product form.

Thiosulfinates (e.g., allicin) were found to be released only from garlic cloves and

garlic powder products; vinyl dithiins and ajoenes were found only in products

containing garlic macerated in vegetable oil; diallyl, methyl allyl, and dimethyl sulfide

series components were exclusive to products containing the oil of steam distilled

garlic (Funke,2005).

Allicin is a substance, an organic substance that usually found in the garlic.

Allicin in its pure form exhibits i.antibacterial activity against a wide range of gram-

negative and gram-positive bacteria, including multi drug-resistant enterotoxicogenic

strains of Echerichia coli; ii) also exhibits antifungal activity, specifically against

candida albicans; iii) also, anti-parasitic activity, including some major human

intestinal protozoan parasites such as Entamoeba histolytica and Giardia lambia; iv)
http://en.wikipedia.org/wiki/Sulfurhttp://en.wikipedia.org/wiki/Alliinhttp://en.wikipedia.org/wiki/Ajoenehttp://en.wikipedia.org/wiki/Allylcysteinehttp://en.wikipedia.org/wiki/Enzymehttp://en.wikipedia.org/wiki/Vitamin_Bhttp://en.wikipedia.org/wiki/Proteinhttp://en.wikipedia.org/wiki/Mineralhttp://en.wikipedia.org/wiki/Saponinhttp://en.wikipedia.org/wiki/Flavonoidhttp://en.wikipedia.org/wiki/Maillard_reactionhttp://en.wikipedia.org/wiki/Phytoalexinhttp://en.wikipedia.org/wiki/Allixinhttp://en.wikipedia.org/wiki/Pyronehttp://en.wikipedia.org/wiki/Pyronehttp://en.wikipedia.org/wiki/Allixinhttp://en.wikipedia.org/wiki/Phytoalexinhttp://en.wikipedia.org/wiki/Maillard_reactionhttp://en.wikipedia.org/wiki/Flavonoidhttp://en.wikipedia.org/wiki/Saponinhttp://en.wikipedia.org/wiki/Mineralhttp://en.wikipedia.org/wiki/Proteinhttp://en.wikipedia.org/wiki/Vitamin_Bhttp://en.wikipedia.org/wiki/Enzymehttp://en.wikipedia.org/wiki/Allylcysteinehttp://en.wikipedia.org/wiki/Ajoenehttp://en.wikipedia.org/wiki/Alliinhttp://en.wikipedia.org/wiki/Sulfur


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and also it exhibits anti-viral activity. The main antimicrobial effect of allicin is due to

its chemical reaction with thiol groups of various enzymes, e.g. alcohol

dehydrogenase, thioredoxin reductase, and RNA polymerase, which can affect

essential metabolism of cysteine proteinase activity involved in the virulence of E.

histolytica. (Ankri and Mirelman, 2001).

Tomato Plant (Lycopersicum esculentum)

Tomato (Lycospersicum esculentum) is classified under the Kingdom Plantae,

Phylum Angiospermae, Class Eudicots, Order Solanales, Family Solanaceae, and

under the Genus Solanumand Species lycopersicum.

Tomato or the Lycopersicum esculentumas stated in the introduction of this

study, this plant is a member of nightshade family. This plant is a bushy plant and it

bears a fruit when unripe, its color green and turns red or orange when it is riped.

This plant is used in many ways; ingredients in several dishes, sauces, juices,

catsups, seasoning and medicines. On the other hand, the stem of tomato can be

used in paper manufacture and others. In the time of harvesting tomato is varying

according to its purpose for which you grow tomatoes. You can harvest them either

mature green or pink and red ripe (Ibsen,1999).

The tomato soft rot is brought about by the action of enzymes (pectolytic and

cellulitic) on the peptic substances and cellulose of the cell walls. As a result of these

enzymes the host tissues disintegrate and their protoplast die. The factors which

predispose the fruits and vegetables to soft rot under stored conditions are humidity,

temperature, presence of bruises or lesions or attack by other patogens. It can be


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carried on insect bodies and equipments. These bacteria retain its life and can grow

between the temperatures of 32F to 90F of which optimal growth between 70 to

80F. Bacterial soft rot occurs on a wide range of crops and one of the most severe

post harvest diseases in some commodities (Ibsen and Gary, 2000).

This disease first appears as small, tannic, water-soaked spots on the

surface. These spots rapidly increasing in size and the tissue decomposes in a soft,

blister-like area on the surface of the fruits. Often, a slimy or watery substance oozes

from breaks in the blister. The blister margin is darker than the fruit skin. Soft rot

often follow bruising and is first white to cream colored. After it expose to the air, it

becomes brown and it turns to black until it will result to a foul odor (Jones, 2000).

With correct usage, in the case of higher dosage, involving a viable probiotic

at the appropriate time after an intensive anti-microbial intervention and a cessation

thereof, at least temporarily with garlic (colloidal silver) whilst the beneficial

organisms are established in a non competitive milieu, colon ecology can be

effective correction and maintained the used of these useful substances.same as

urethritis, vaginitis and oral thrush are caused by yeast by fungus of the candida

group. Because of solid scientific research, garlic is the number one natural

treatment for candida infections, used of thousands of holistic physicians in America.

It is not quite true to say that garlic selects only the unfriendly organisms for attack, it

does tend to attack foe rather than live naturally in the gut soon recover, while the

invaders do not. At lower dosage, garlic does not kill invaders but simply strops their

multiplication affording the body an opportunity to marshal its own defenses. Garlic


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is the only antibiotic which at the same time as fighting microbial invaders, also

protects the body against poisons produced by infection (Stephen, 2001).

Beef Extract

Beef extract support the growth of microorganism when combined with

peptone. This contains peptides and amino acids, nucleotide functions, organic

acids, minerals and some vitamins. Beef extract functions to complement the

nutritive property of peptone by contributing materials, phosphates, energy

resources and those essential factors missing in peptone. The liquid medium will be

transferred into a bottle (Dalmacio, Raymundo and Zamora, 2002) .

RELATED STUDIES

Since 2004, Cutler and Wilson have studied the effect of Allicin on these

bacteria. They come up with a conclusion that the effect of allicin on these bacteria

on these bacteria may be due to the hydrogen bonding of water to the reactive

oxygen atom in allicin and the dilution of these components in water that helps to

preserve the Allicin molecule.

Cytotoxicity of fresh juice and alcohol extract from garlic were studied by

Ames' test, Rec assay, Micronucleus test and the check of the influence to HEp 2

and Chinese hamster embryo (CHE) primary cultured cells. No evidence of

mutagenicity of these samples were observed in Ames' test and Rec assay, while

there was dose dependent increase of micronucleated cells and polychromatocytes

on the bone marrow cells of mice and chinese hamsters treated with garlic juice.

There were severe damages, e.g. growth inhibition and morphological changes of


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both cultured cells due to garlic juice, but no or slightly cytotoxic signs were

observed even in high concentration of garlic extract. A higher sensitivity to the

cytotoxic effects of garlic was seen by the present findings with CHE primary cells

than HEp 2 cell line (Yoshida, Hirao, Nakagawa, 2001).

Another study was conducted about the influence of garlic extract on the

chronic toxicity test. The said tests were examined orally in Wistar rats for 6 months.

There were no toxic symptoms due to garlic extract even at dose level of 2000

mg/kg for 5 times a week during 6 months. High dose of garlic extract did not inhibit

the body weight gain, while the food consumption decreased slightly for the

nutritional effects of it in both male and female rats. There were no significant

differences in urinary, hematological and serological examinations compared each

groups. In the histopathological findings, no toxic signs were observed on any of the

tissues and organs examined (Sumiyoshi H et al. 2001).

Also a study was conducted about the effect of garlic in suppressing the

growth of E. carotovora. The results revealed that the different concentration of the

said extract were able to suppress the said bacteria growth. The result tells that the

degree of suppression on the growth ability of E. carotovora depends on the

increment levels of garlic extract. As the increment level of concentration is higher

the suppressing ability of the said bacteria increasing (Golindo, 2010).
http://www.ncbi.nlm.nih.gov/pubmed?term=%22Sumiyoshi%20H%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Sumiyoshi%20H%22%5BAuthor%5D


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Null Hypothesis

There is no significant difference in the concentration of the garlic extract in

treating the tomato soft rot.

Definitions of terms

For the reader, the different terms that are not common are provided with the

definition for this to make this study clearer and more comprehensive. The following

terms are both conceptually and operationally defined.

Tomato (Lycopersicum esculentum)- a vegetable with a scientific name

Lycopersicum esculentum. It serves as the plant or the fruit that is to be implanted

by the disease E. Carotovora and is to be treated by the garlic extract.

Erwinia carotovora- a pathogen that is common in plant, this is a kind of

bacteria that promotes tomato soft rot.

Garlic ( Allium sativum)- It will serve as the plant that will suppress the E.

Carotovoragrowth.

Tomato soft rot- a postharvest disease that commonly attack tomato fruits. It

is cause by the bacteria Erwinia carotavora.

Allicin- a compound responsible for the hot sensation of raw garlic. Together

with the garlic extract it will be used as the suppressor of the Erwinia carotovora

growth.


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Beef Extract- an extract that is to be taken from the beef broth. It will be the

medium for the support of the growth of Erwinia carotovora. It will be injected to the

tomato to produce the E. carotovora.

Autoclave- an enclosed container used for decontaminating the culture

media.


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CHAPTER III

METHODOLOGY

This chapter provides the information prior to the methods, materials and type

of data gathering that is essential to the fulfilment of this study.

Research design

Figure 1. Research Design

Bioassay

T4

T5

R1

R2

R1

R3

R3

R1

R2

R3

R1

R2


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A complete randomized design or CRD will be used in this study. The said

research design has five treatments namely the: T1-control (distilled water only); T2-

25% of garlic extract; T3- 50% of garlic extract; T4- 75% of the garlic extract; and T5-

100% of garlic extract. The punchered sterile filter paper disc will serve as a test

medium in the different treatment and will be planted on the pure culture media. the

filter paper disc will be soaked in the garlic extract and will be planted equidistantly

to the culture media. the zone of inhibition will be observe every 24 hours

consecutively, until 72 hours will be reached. The following treatments will be

replicated three times. These treatments will be applied to the culture media which is

the tomato soft rot (E. carotovora).

Research locale

This study will be conducted at Mindanao State University, Barangay Fatima,

and General Santos City. It is a tertiary school in General Santos City. It has eight

(8) colleges, namely; the college of Agriculture, the college of Business

Administration and Accountancy, the college of Education, the college of

Engineering, college of Fisheries, college of Natural Sciences and Mathematics, the

college of Social Sciences and Humanities, and the college of Law. It is also known

as one of the best school in Mindanao for producing well educated citizen and

globally competitive students of this country.

Materials and Equipments

The materials that will be used in this study are the following: garlic bulbs,

beef broth, tomato fruits, distilled water, transfer needle, ruler, forceps, beaker,


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graduated cylinder, cups, casserole, puncher, alcohol lamp, autoclave, stirring rod,

knives, cheese cloth, mortar and pestle, tape, notebook,ballpen, and pentel pen.

Procedure

Below a schematic diagram shows the different steps in the whole procedure

that will be observed in doing this research.

Figure 2. Research Procedure Flow Chart

Preparation of the materials

that will be used

Preparation of nutrient agar Preparation of culture media

Application of treatmentData gathering

Preparation of garlic extract Preparation of filter paper disc


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Gathering and Preparation of the Tomato Fruit

The researcher will buy 15 tomato fruits from the market. the tomatoes must

be fresh and free from disease. After gathering, it will be washed and dried, and put

inside the plastic wrapper to isolate the fruits and to prevent the fruit from the

bacteria that can cause contamination.

Preparation of Culture Media

A 5-10 ml of the nutrient agar media will be dispensed in sterile petri dishes

for the growth of Erwinia carotovora.

Preparation of Nutrient Agar

To every liter of the nutrient broth, 20 grams of agar will be added and

dissolved in boiling water bath and strained. A 5 ml of the solutions will then sterilize

for 15 minutes at 15 psi. Bacterial oozes will be pour-plated in petri dish with culture

media. And it will incubate at the room temperature inside the isolation chamber.

Preparation of garlic extract

Newly harvested garlic cloves will be chopped into small pieces using sharp

knives and then will be macerated using the mortar and pestle. A 50 ml of extracts

will be prepared for each treatment.

Preparation of Filter Paper

Punchered sterile filter paper will serve as a test discs for different

treatments.


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Application of Treatments

The filter paper discs will be soaked for 15 minutes in each treatment and will

be planted equidistantly at the pure culture of Erwinia carotovoraafter 24 hours of

incubation.

Data Gathering

The data will be gathered in every 24 hours subsequently until 72 hours

based on the growth of bacteria Erwinia carotovora. Its growth pattern will be

determined base on the zone of inhibition that will be applied on it. The researcher

will use a ruler in measuring its growth from the center of its growth in every

concentration.

Statistical treatment

This study will be carried out using the CRD or Complete Randomized

Design. The researcher will provide different tables in collecting and specifying the

data. In determining the statistics (Statement of the Problem 1) of this study the

researcher will use the ANOVA (Analysis of Variance). Analysis of Variance is the

statistical tool chose by the researcher because this type of statistical tool can give

credible results and more understandable statistical interpretation in the data. In the

Statement of the Problem 2, qualitative approach will be used.


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Volume of the solution x percentage of garlic extract = volume of the garlic

extract

Volume of Solution Percentage of Garlic Extracts Volume of Garlic Extracts

100 ml 25% 25 ml

100 ml 50% 50 ml

100 ml 75% 75 ml

100 ml 100% 100 ml

Figure 3. Determination of Garlic Extracts Volume in ml


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BIBLIOGRAPHY

Books and Published Magazines

Dalamacio, I., A. Raymundo. 1986, general microbiology. UPLB Microbiology

Laboratory Academe publishing house. 30 p.

Gould, Wilbur A. 1992. Tomato Production, processing and Technology. CTI

Publishing. p. 2

Jones, J, Benton, Jr. 1999. Tomato plant culture in the Field, greeenhouse and

home garden CRC press.

Marteneou, b. 2001. First fruit: the Creation of The flavor tomato and the birth of the

biotech food, Mc Graw Hill.

Maulana, Rajab.March 2008. Bioassay of different concentrations of tabacco leaf

extracts against helminthos phorium leafspot (Helminthosporium Haydis

Nish and Miy.) of corn. 18 p.

Tortora, G., B.R. Turne, C.L, case. 2005 Microbiology: an introduction, 8th

edition

Pearson Education South Asia PTE LTD. P.15.

Taylor, D. 2000. Manila Bulletin. P. B-16

Walker. J.T. 1924. Fungicide. Encyclopedia America corporation. 170 p.

Steiner M, Lin RS (June 1998). "Changes in platelet function and susceptibility of

ipoproteins to oxidation associated with administration of aged garlic

extract". J Cardiovasc Pharmacol31 (6): 9048.


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Website:

file:///E:/Bioassay.htm, modified last September 20, 2011

http;//en.wikipedia.org/wiki/allicin. February 15, 2010

http://www.bionewsonline.com/m/what_is_bioassay.htm. 2005

http://www.mged.org/Workgroups/MAGE/bioassay.html. Last modified: 17 Jul, 2006

http:/www.pfaf.org/database/plants.php.allium+sativum. January 8, 2010.

www.arrdc.org/pdf/tomato/soft.pdf. Retrieved on March 29, 2009

Wikipedia. Org/ wiki/Erwinia. Retrieved on March 29, 2009
http://e/Bioassay.htmhttp://www.bionewsonline.com/m/what_is_bioassay.htmhttp://www.mged.org/Workgroups/MAGE/bioassay.htmlhttp://www.arrdc.org/pdf/tomato/soft.pdfhttp://www.arrdc.org/pdf/tomato/soft.pdfhttp://www.mged.org/Workgroups/MAGE/bioassay.htmlhttp://www.bionewsonline.com/m/what_is_bioassay.htmhttp://e/Bioassay.htm


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CURRICULUM VITAE

PERSONAL DATA

Name: Arjan A. Otero

Address: MSU Compound Fatima, General Santos city

Date of Birth: September 22, 1991

Place of Birth: Poblacion, Norala, South Cotabato

Fathers Name: Armando E. Otero

Mothers Name: Jocelyn A. Otero

EDUCATIONAL BACKGROUND

Elementary: San Jose Elementary School

Secondary: Malandag National High school

Tertiary: Mindanao State University General Santos City

Course: Bachelor of Secondary Education

Major: Biology


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APPENDICES

Table 1. Zone of inhibition (mm) ofE. carotovora as affected by the different

concentration of garlic extracts after 24 hours under the laboratory condition

Treatment Replication Total Mean

I II III

T1

T2

T3

T4

T5

Grand total

Grand mean

CV (%):


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Proposed Budgetary Expenses

Materials Quantity Amount

Garlic cloves 35 cloves (P5.00/clove) P175.00

Tomato fruit 15 fruit (P1.00/fruit) P15.00

Distilled water 1 liter P50.00

Beef kilogram (P160.00/kilo P40.00

Others ---------- ----------

Total amount: P280.00

original to be printed

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