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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
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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