DETERMINATION OF ANTIMICROBIAL ACTIVITY OF (Allium sativum
L.) EXTRACT AGAINST SELECTED AGROBACTERIUM
TUMEFACIENS OF HASS AVOCADO.
EVANS KIPKOSGEI
BTMB/065J/2016
This project proposal submitted to the Department of Pure and Applied
Sciences is part of the fulfillment requirement for Bachelor’s programme in
Technology in Industrial Microbiology and Biotechnology of Technical
University of Mombasa.
August, 2021.
DECLARATION
This project is my original work and has not been presented for a degree in any other university.
Signature………………………………………Date…………………….
EVANS KIPKOSGEI
BTMB/065J/2016
This project proposal has been submitted for examination with approval of university supervisor.
Signature………………………………………...Date…………………………
DR. DANIEL MUNGA
(Technical University of Mombasa).
ii
DEDICATION
I dedicate this work to Mr Hoseah Kimeli Metto and the late Mrs Wilfrida Metto, my Guardians
who has contributed so much to my academic life. Mrs Eunice Jelagat my mother who stood with
me in all odds of education life. My siblings Brian, Cynthia, shaleen and Bravin Kyeva, let this be
an inspiration to you.
iii
ACKNOWLEDGEMENT
I want to thank the Almighty for having brought me this far since the journey commenced. I am
grateful for His guidance and protection all through. I want to thank my family for their support
and dedication throughout my study. I thank my supervisor Dr. Daniel Munga for having guided
me throughout the period of writing my proposal and may God bless him. To the course
lecturers, staff in the department of Pure and Applied science, your invaluable support enabled
me to successfully go through this course. I wish to thank my colleagues who were a source of
encouragement thus enabling me to go through this course.
iv
TABLE OF CONTENTS
DECLARATION ............................................................................................................................ ii
DEDICATION ............................................................................................................................... iii
ACKNOWLEDGEMENT ............................................................................................................. iv
LIST OF ABBREVIATIONS ....................................................................................................... vii
LIST OF FIGURES ..................................................................................................................... viii
ABSTRACT ................................................................................................................................... ix
CHAPTER I .................................................................................................................................... 1
1.0 INTRODUCTION .................................................................................................................... 1
1.1 Background information ....................................................................................................... 1
1.2 Statement of the problem. ..................................................................................................... 2
1.3 Justification. .......................................................................................................................... 2
1.4 General objective .................................................................................................................. 2
1.4.1 Specific objective .......................................................................................................... 2
1.5 Research questions. ............................................................................................................... 3
1.6 Hypotheses ............................................................................................................................ 3
1.7 Limitation of study. ............................................................................................................... 3
CHAPTER 2 ................................................................................................................................... 4
LITERATURE REVIEW ............................................................................................................... 4
2.0 Introduction ........................................................................................................................... 4
2.1 Theoretical review of Allium sativum l. ............................................................................... 4
2.1.1 Botanical description ...................................................................................................... 4
2.1.2 Biochemical description ................................................................................................. 6
2.2 Critiques of the existing literature....................................................................................... 10
2.3 Summary ............................................................................................................................. 10
2.4 Research gap. ...................................................................................................................... 10
v
CHAPTER 3 ................................................................................................................................. 12
MATERIALS AND METHODS .................................................................................................. 12
3.0 The research Design ............................................................................................................ 12
3.1 Target population ................................................................................................................ 12
3.2 Sampling techniques and illustrations ................................................................................ 12
3.3 Media Preparation and Screening for Antibiotic Susceptibility ......................................... 13
3.4 Extraction method ............................................................................................................... 13
3.5 Phytochemical screening this should come immediately after extraction. ......................... 14
3.5.1 Determination of Alkaloids .......................................................................................... 14
3.5.2 Determination of Flavonoids ........................................................................................ 14
3.5.3 Determination of Steroids ............................................................................................. 14
3.5.4 Determination of Tannins ............................................................................................. 14
3.5.5 Determination of Terpenoids ........................................................................................ 14
3.6 Determination of Minimum Inhibitory Concentration (MIC) ............................................ 15
3.7 Determination of the Minimum Bactericidal Concentration (MBC) .................................. 15
3.8 Data processing and analysis .............................................................................................. 15
WORK PLAN ............................................................................................................................... 16
BUDGET ...................................................................................................................................... 17
REFERENCES ............................................................................................................................. 18
vi
LIST OF ABBREVIATIONS
DADS
diallyl disulfide
DAS
diallyl sulfide
DATS
diallyl trisulfide
E. coli
Escherichia coli
GBM CSC
Glioblastoma Multiforme Cancer Stem Cells
HCI
Hydrochloric acid
MDA
Mueller-Hinton dextrose agar
MIC
Minimum inhibitory concentration
vii
LIST OF FIGURES
Figure 1: Garlic, (Allium sativum), plant. ..................................................................................... 6
viii
ABSTRACT
Agrobacterium tumefaciens is a soil-borne bacterium that, in nature, is capable of inserting a
defined fragment of its DNA into the genome of dicotyledonous plants (Tzfira and Citovsky,
2002; Gelvin, 2003). It has been the focus of research for a wide spectrum of biologists, from
bacteriologists to molecular biologists to botanists, for a number of years. Moreover, the unique
process by which it delivers trans-kingdom DNA into host plant cells has become a staple in almost
all undergraduate plant science courses. However, it is important to remember that A.
tumefaciens remains at the cutting edge of plant science. Our understanding of the molecular
mechanisms of DNA integration has grown immeasurably in recent years, and in a climate of
intense political debate surrounding the future of transgenic crops, the future role of A.
tumefaciens in the controlled creation of transgenic crops or simply as the model type IV secretion
system and as a research tool would appear to hang in the balance.
The aim of this research proposal will determine the in-vitro antibacterial activity of Allium
sativum L. (garlic) against Agrobacterium tumefaciens on hass avocado.
ix
CHAPTER I
1.0 INTRODUCTION
1.1 Background information
Crown gall is a plant disease that affects a large variety of broad-leaved (dicotyledonous) plants,
including tomatoes, apple, pear, cherry, almond, raspberry, and rose plants leading to several
losses. Agrobacterium tumefaciens cleverly transfers a genetic principle to plant host cells and
integrates it into their chromosomes. Therefore, the focus of crown gall disease control has
typically been the elimination of A. tumefaciens through soil fumigation. Although successful in
several cases, these measures have not provided consistent and effective disease control in many
crops, including English walnut. Observations by nursery operators and walnut growers suggest
fumigation with methyl bromide (MeBr) is inconsistent in reducing crown gall incidence and, in
some cases, actually increases crown gall incidence. One potential method of controlling this plant
disease could be the use of biological substances found in plants such as Allium sativum. Allium
sativum L., garlic, is one of the most representative species of the genus, used as food, spice and
medicinal plant since ancient times. The medicinal value of the plant is consistent with various
biological properties such as its antimicrobial, cardiovascular and anticancer effects. Various garlic
preparations have been shown to exhibit a wide spectrum of antibacterial activity against Gramnegative
and
Gram-positive
bacteria
including
species
of
Escherichia,
Salmonella,
Staphylococcus, Streptococcus, Klebsiella, Proteus, Bacillus, and Clostridium. Even acid-fast
bacteria such as Mycobacterium tuberculosis are sensitive to garlic. Although there has been much
research on the effect that garlic has on pathogenic bacteria, there are very few studies on its effect
on plant bacteria including inhibitory effects of garlic on plant-associated microbes. It was
observed by Timonin and Thexton that applying juice from crushed garlic tissues to soil led to a
drastic drop in counts of microorganisms in the soil around the roots of plants. The activity of
garlic extracts against the seed-spoilage organisms Aspergillus niger and Fusarium pallidorosum
was also reported by (Arya et al.2004). A study by (Wei et al,2011). also indicated that crude
Allium sativum extract inhibited the growth of Fulvia fulva, a tomato fungus. Therefore, this study
will determine the in-vitro antibacterial activity of Allium sativum L. (garlic) against
Agrobacterium tumefaciens on hass avocado.
1
1.2 Statement of the problem.
Hass avocado plants are massively infected by crown gall disease in Nandi county.This has
reduced the size, quality and the number of avocado fruits being produced in the area. Avocado
fruits are food to this community and with the rising rate of Agrobacterium tumefaciens infection,
the avocado plants will lose its value as a source of food and to this community.The control of
Agrobacterium tumefaciens on large avocado tree is usually by pruning and and use of creosote
based chemical compounds which are toxic and contaminate soil.This study reviews laboratory
methods that can be employed to inhibit the growth of Agrobacterium tumefaciens. There is also
need to minimize the cost of chemical compounds used in treatment.
1.3 Justification.
With increasing rapid spread of agrobacterium tumefaciens, crown gall disease on Avocado trees
and neighboring trees, there is need to find a remedy that will help reduce the spread and inhibit
the disease. The remedy should not contaminate the soil or nearby plants and also should not be
expensive so that all the farmers can afford. Allium sativum L is able to thrive in tropical
environment and its method of chemical compound extraction is easy. In addition to this, Allium
sativum is cheap and readily available and many people use is as a flavoring ingredient in food
preparations and promoted as a DS for patients with hypertension and hyperlipidemia, and for
preventing cardiovascular disease. Garlic is used to prevent various cancers, including colorectal,
gastric, breast, and prostate cancers.
1.4 General objective
To determine antimicrobial activity of allium sativum l. extract against selected agrobacterium
tumefaciens of hass avocado.
1.4.1 Specific objective
i. To determine the phytochemicals present in the Allium sativum L extract.
ii.
To determine the minimum inhibitory concentration(MIC) of garlic extract on
agrobacterium tumefaciens.
iii.
To determine the minimum inhibitory concentration(MIC) and Minimum Bactericidal
Concentration (MBC) of allium sativum L. extract against selected agrobacterium
tumefaciens.
2
1.5 Research questions.
i. What is the effect of Allium sativum L extracts on agrobacterium tumefaciens ?
ii.
What is the minimum inhibitory concentration of Allium sativum L extract?
iii.
What are the phytochemicals present in Allium sativum L extract?
1.6 Hypotheses
Agrobacterium tumefaciens (tumor) are susceptible to allium sativum L extract
1.7 Limitation of study.
Lack of previous study on the spread, infection and contamination of fruit plants especially on
Agrobacterium tumefaciens, so there is limited information in the research world.
3
CHAPTER 2
LITERATURE REVIEW
2.0 Introduction
Garlic (Allium sativum L.) has acquired a reputation in different traditions as a prophylactic as
well as therapeutic medicinal plant. Garlic has played important dietary and medicinal roles
throughout the history. Some of the earliest references to this medicinal plant were found in A
vesta, a collection of Zoroastrian holy writings that was probably compiled during the sixth century
BC (Dannesteter, 2003). Garlic has also played as an important medicine to Sumerian and the
ancient Egyptians. There is some evidence that during the earliest Olympics in Greece, garlic was
fed to the athletes for increasing stamina (Lawson and Bauer, 1998). Ancient Chinese and Indian
medicine recommended garlic to aid respiration and digestion and to treat leprosy and parasitic
infestation (Rivlrn, 1998). In the medieval period, garlic was also played an important role in the
treatment of different diseases. Avicenna (1988), in his well-known book, Al Qanoon Fil Tib (The
Canon of Medicine), recommended garlic as a useful compound in treatment of arthritis,
toothache, chronic cough, constipation, parasitic infestation, snake and insect bites, gynecologic
diseases, as well as in infectious diseases (as antibiotic). With the onset of Renaissance, special
attention was paid in Europe to the health benefits of garlic. Garlic has attracted particular attention
of modern medicine because of widespread belief about its effects in maintaining good health. In
some Western countries, the sale of garlic preparations ranks with those of leading prescription
drugs. There is appreciable epidemiologic evidence that demonstrates therapeutic and preventive
roles for garlic. Several experimental and clinical investigations suggest many favorable effects of
garlic and its preparations. These effects have been largely attributed to reduction of risk factors
for cardiovascular diseases ,reduction of cancer risk ,antioxidant effect ,antimicrobial effect and
enhancement of detoxification foreign compound and hepatoprotection (ColínGonzález, 2012;
Aviello, 2009).
2.1 Theoretical review of Allium sativum l.
2.1.1 Botanical description
Garlic, (Allium sativum), perennial plant of the amaryllis family (Amaryllidaceae), grown for its
flavourful bulbs. The plant is native to central Asia but grows wild in Italy and southern France
and is a classic ingredient in many national cuisines. The bulbs have a powerful onionlike aroma
4
and pungent taste and are not usually eaten raw.Garlic comes in two varieties: hardneck
(designated as Allium sativum var. ophioscorodon) and softneck (designated as Allium sativum
var. sativum). Hardneck varieties typically produce a flower stalk, with flowers giving way to a
seed cap. Each bulb typically contains 4-10 cloves (bulb is smaller but cloves are larger than those
of softneck). Hardneck varieties come in three different types: Rocambole, Poreclain and Purple
Stripe. By contrast, softneck garlic plants typically do not produce a flower stalk. They produce
bulbs with smaller but more numerous cloves than hardneck garlic plants. Each bulb typically
contains 12-20 cloves. Softneck plants are also braidable (bulbs can be braided together into
attractive chains by weaving the soft grass-like tops together). Softneck garlic varieties are the
ones most often commonly sold in supermarkets because they typically have a much longer shelf
life than hardnecks. Softneck varieties come in two different types: Silverskin and Artichoke. In
ancient and medieval times, garlic was prized for its medicinal properties and was carried as a
charm against vampires and other evils. The plant is used in traditional and folk medicine in many
places, and there is some evidence that it may help prevent heart disease. Garlic contains about 0.1
percent essential oil, the principal components of which are diallyl disulfide, diallyl trisulfide, and
allyl propyl disulfide.
5
Figure 1: Garlic, (Allium sativum), plant.
2.1.2 Biochemical description
Garlic has a variety of bioactive compounds, including organosulfur compounds, saponins,
phenolic compounds, and polysaccharides. The major active components of garlic are its
organosulfur compounds, such as diallyl thiosulfonate (allicin), diallyl sulfide (DAS), diallyl
disulfide (DADS), diallyl trisulfide (DATS), E/Z-ajoene, S-allyl-cysteine (SAC), and S-allylcysteine sulfoxide (alliin). In general, organosulfur compounds in raw garlic have higher
digestibility than those in cooked garlic. In addition, saponins were found to be more stable in the
cooking process. The total amount of saponin in purple garlic was almost 40 times higher than that
in white garlic, and several saponin compounds were only found to exist in purple garlic, such as
desgalactotigonin-rhamnose,
proto-desgalactotigonin,
proto-desgalactotigonin-
rhamnose,
voghieroside D1, sativoside B1-rhamnose, and sativoside R1. Moreover, garlic contained more
than 20 phenolic compounds, with higher contents than many common vegetables. The main
phenolic compound was β-resorcylic acid, followed by pyrogallol, gallic acid, rutin,
6
protocatechuic acid, as well as quercetin . Furthermore, garlic polysaccharides were reported to
contain 85% fructose, 14% glucose, and 1% galactose
Antioxidant Activity
The antioxidant activities of natural products have been widely evaluated, such as fruits,
vegetables, mushrooms, cereal, flowers, and wild fruits. Accumulating studies have found that
garlic has strong antioxidant properties. A study evaluated the antioxidant capacities of both raw
and cooked garlic, and found that the raw garlic exhibited stronger antioxidant activity (by 1,1diphenyl-2-picrilhydrazyl
(DPPH)
radical
scavenging
assay,
2,2’-Azino-bis(3-ethyl-
benzothiazoline-6-sulfonic acid) (ABTS) radical scavenging assay, and ferric ion reducing
antioxidant power (FRAP) assay). Stir-fried garlic was also shown to have stronger antioxidant
capacities (by β-carotene bleaching), indicating that the processing could affect the antioxidant
property of garlic. In another study, the results of DPPH and oxygen radical absorption capacity
(ORAC) assays showed that the ethanolic extract of garlic sprouts exhibited stronger antioxidant
activities than the ethanolic extract of raw garlic. In addition, the antioxidant properties of aged
garlic were found to be higher than fresh garlic by DPPH, ABTS, FRAP, H2O2 scavenging, and
Fe2+ chelating assays. Compared with multi clove garlic extract, single clove garlic extract had a
higher number of phenolic compounds and showed stronger antioxidant activity. Moreover, the
antioxidant activity of black garlic increased with thermal treatment, and the highest antioxidant
activity was obtained on the 21st day of processing. Also, the increased pressure improved the
antioxidant activity of garlic paste. However, the antioxidant activity of “Laba” garlic, a traditional
Chinese garlic product, decreased during fermentation. The aged garlic extract (AGE) induced the
expression of several antioxidant enzymes, such as heme oxygenase-1 (HO-1) and the glutamatecysteine ligase modifier (GCLM) subunit through the nuclear factor erythrobia-2 related factor 2
(Nrf2)-antioxidant response element (ARE) pathway, which protected human endothelial cells
against oxidative stress. Garlic saponins were reported to protect mouse-derived C2C12 myoblasts
against growth inhibition and DNA damage induced by H2O2, and to scavenge intracellular
reactive oxygen species (ROS). In summary, garlic and its active ingredients (such as phenols and
saponins) have certain antioxidant effects. Different processing methods also affected the
antioxidant activity of garlic. Usually, raw garlic had a stronger antioxidant activity than cooked
garlic, and the antioxidant activity of fermented garlic, such as black garlic, was stronger than that
7
of crude garlic. In addition, the cellular experiment showed that the mechanism of antioxidative
action of garlic might be involved with the enhancement of antioxidant enzyme activities and the
regulation of the Nrf2-ARE pathway
Antibacterial Activity
The antimicrobial activity of garlic is attributed to allicin activity that was reported toward a wide
variety of microorganisms including antibiotic-resistant, Gram-positive and Gram-negative
bacteria such as Shigella, Escherichia coli , Staphylococcus aureus, Pseudomonas aeruginosa,
Streptococcus mutans, S. faecalis, S. pyogenes, Salmonella enterica, Klebsiella aerogenes , Vibrio,
Mycobacteria, Proteus vulgaris, and Enterococcus faecalis . Various garlic extracts (aqueous,
chloroform, methanolic, and ethanolic extracts) were reported to inhibit the growth of several
pathogenic bacteria with varying degrees of susceptibility. For instance, a study revealed that
ethanolic garlic extract showed higher inhibitory effect against E. coli and Sal. typhi than the
aqueous extract that showed little or no inhibition effect. (Meriga et al.) reported that aqueous
garlic extract showed antibacterial activity toward Gram-negative (Kl. pneumoniae and E. coli) as
well as Gram-positive (e.g., Bacillus subtilis and S. aureus) strains, whereas methanolic garlic
extract showed antimicrobial activity against all tested strains except S. aureus. However, hexane,
ethyl acetate, and chloroform extracts did not show any antibacterial effect. Moreover, garlic
extracts prevented the growth of enterotoxigenic E. coli strains and other pathogenic intestinal
bacteria, which are the main cause of diarrhea in humans and animals. Besides the antibacterial
activity of garlic, it was reported to prevent the toxins produced by bacterial infection . Allicin also
showed effectiveness toward methicillin-resistant S. aureus (MRSA). Allicin’s antimicrobial
activity is due to its chemical interaction with enzymes containing thiol e.g., thioredoxin reductase,
RNA polymerase, and alcohol dehydrogenase by oxidizing protein cysteine or glutathione residues
under physiological conditions. Allicin is a dose-related biocide that can influence essential
metabolism of cysteine proteinase, and thus, kill all eukaryotic cells due to the presence of thiol
groups in all living cells.
Antifungal Activity
Garlic extracts showed a broad spectrum fungicidal effect against a wide range of fungi including
Candida, Torulopsis, Trichophyton, Cryptococcus, Aspergillus, Trichosporon, and Rhodotorula
species. Recently, garlic extract was found to inhibit the Meyerozyma guilliermondii and
8
Rhodotorula mucilaginosa germination and growth. Another study reported the antifungal activity
of various A. sativum extracts namely aqueous, ethanolic, methanolic, and petroleum ether against
human pathogenic fungi such are Trichophyton verrucosum, T. mentagrophytes, T. rubrum,
Botrytis cinerea, Candida species, Epidermophyton floccosum, Aspergillus niger, A. flavus,
Rhizopus stolonifera, Microsporum gypseum, M. audouinii, Alternaria alternate, Neofabraea alba,
and Penicillium expansum [56]. The garlic extract acted by affecting the fungal cell wall and
causing irreversible ultrastructural changes in the fungal cells, which lead to loss of structural
integrity and affected the germination ability. These changes in the cytoplasmic content lead to
nucleus and cell organelles damage that ultimately leads to cell death. Moreover, allicin and garlic
oil showed potent antifungal effects against Candida albicans, Ascosphaera apisin, and A. niger
and they acted by penetrating the cellular membrane as well as organelles membranes like the
mitochondria and leading to organelles destruction and cell death. DADS and DATS separated
from garlic essential oil showed antifungal activity against a number of fungi (C. albicans, C.
tropicalis, and Blastoschizomyces capitatus). In addition to that, saponins extracted from A.
sativum exhibited antifungal activity against Botrytis cinerea and Trichoderma harzianum.
Anticancer Activity
Raw garlic extract was found to be the most effective and highly specific anticancer drug when
compared with 33 raw vegetable extracts against different cancer cells without affecting the noncancerous cells.( Shang et al. ) reported that the anticancer mechanisms of garlic extracts were
attributed to the inhibition of cell growth and proliferation, regulation of carcinogen metabolism,
stimulation of apoptosis, prevention of angiogenesis, invasion, and migration and thus reducing
the anticancer agent’s negative effects. Interestingly, in 1960, tumor cells were reported to be killed
when incubated in an allicin solution. Allicin isolated from garlic was reported to suppress
colorectal cancer metastasis through enhancing the immune function and preventing the formation
of tumor vessels as well as survivin gene expression to enhance the cancer cell’s apoptosis. It also
can enhance the treatment of pancreatic cancer thereby invert gene silencing and restrain cancer
cell proliferation. Furthermore, (Zhang et al.) revealed that allicin can prevent gastrointestinal
cancer cells MGC 803 proliferation and induce apoptosis, which can be accomplished through
enhancing p38 expression and cleaved caspase 3. Allicin-derived polysulfanes have been reported
9
to target microtubules, which lead to interruption of the cell-cycle and finally to apoptosis. Several
studies reported the activity of allicin in preventing cell proliferation by targeting tubulin that
shapes the mitotic spindle and thus inhibits cell division ( Iciek et al.) have reported the anti-tumor
properties of organo-sulfur compounds (OSC) including allicin, DADS, alliin, DAS, allyl
mercaptan (AM), and S-allyl cysteine (SAC), isolated from garlic. In addition, garlic powders
inhibited the DNA damage caused by N-nitrosodimethylamine in the liver when administered to
rats by 35% and this effect was due to the high concentration of alliin up to 60% in the sample.
Notably, (Fleischauer and Arab) reported that continuous garlic intake could decrease different
kinds of cancer propagation such as lung, colon, stomach, breast, and prostate. (Piscitelli et al.)
reported that garlic reduced the plasma concentrations of saquinavir by about 50% in healthy
participants, after 3-week of a garlic supplement uptake, in addition to this, many researchers
evaluated the antitumor and cytotoxic actions of garlic and its related constituents in vitro and in
vivo. Moreover, Z-ajoene has shown anti-proliferative activities against different types of cancers
and it inhibits the growth of human breast cancer cells and glioblastoma multiforme cancer stem
cells (GBM CSC) . It was found to stimulate apoptosis in human leukemic cells by promoting the
peroxide production, caspase-3-like and caspase-8 activities.
2.2 Critiques of the existing literature
Most researchers mainly focus on the cooking properties of from Allium sativum extract rather
than the medicinal properties of the plant extract. Many parts of the world refers differently to the
plant i.e. common name, this makes it difficult to identify the correct name for the plant.
2.3 Summary
Garlic, fresh shape, powder state and oil of garlic have been used all around the world, especially
in Far East for centuries. It is scientifically proven that garlic is effectively used in cardiovascular
diseases as a regulator of blood pressure, with dropper effects on glycaemia and high blood
cholesterol, against bacterial, viral, mycotic and parasitic infections. It's also known that garlic is
a wonderful plant having the properties of empowering immune system, anti-tumour and
antioxidant effects.
2.4 Research gap.
A lot of research done on allium sativum l. clove extract has been based on the studies on the
antioxidants, anticancer, anti-inflammatory, antibacterial, antifungal and anti-protozoal activities.
A recent increase in the popularity of alternative medicine and natural products has renewed
10
interest in garlic and their derivatives as potential natural remedies. This review may be useful to
increase our knowledge of garlic therapeutic effects and improve our future experimental and
clinical research plans. Although it is shown that garlic may have a significant clinical potential
either in their own right or as adjuvant therapy in different disorders, however, due to some issues,
such as methodological inadequacies, small sample sizes, lack of information regarding dose
rationale, variation between efficacy and effectiveness trials, the absence of a placebo comparator,
or lack of control groups more standard experiments and researches are needed to confirm the
beneficial effect of garlic in various diseases. Future trials on the effect of garlic should include
information on the dosage of active ingredients of standardized garlic preparations for better
comparison of trials. It would also be interesting to explore the effect of different forms of garlic
extract on inhibiting plant diseases.
11
CHAPTER 3
MATERIALS AND METHODS
3.0 The research Design
The research design that will be used is Experimental design and will be carried out at the TUM
microbiology laboratory which is a certified government research institute dealing with scientific
researches thus recognized of its biosafety level II laboratories.Agar disc diffusion method will
be used to screen for antibiotic susceptibility. Indole test will be used for the determination of
minimum inhibitory concentration (MIC) of the garlic extracts. The significant antibacterial
activity of the active extract will be compared with the standard antibiotic, methyl bromide. Thus,
methyl bromide will be the positive control and distilled water will be the negative control.
Strains of virus (Agrobacterium tumefaciens) will be collected from culture collection Centre.
3.1 Target population
This research will mainly focus on the avocado trees infected with crown gall(tumors).
3.2 Sampling techniques and illustrations
Fresh A. sativum bulbs will be purchased from Kongowea market,Mombasa county. The bulbs
were separated into cloves, cleaned with distilled water to remove any surface contaminants from
harvesting and handling and the skin peeled off. The cloves will be cut into small pieces and ground
in order to increase the surface area for extraction. One hundred grams of the ground cloves is
placed in a cornical flask. Four milliliters of absolute ethanol (99.9%) is added and the flask
vigorously agitated for 20 minutes. After agitation, the solutions is placed in a dark cupboard for
maceration. Maceration will last 72 hours (3 days) with frequent agitation. Agitation will be done
three times a day to ensure even mixing of the contents. After maceration, the solution will be
filtered using filter paper (WHATMAN NO. 3) and the filtrate collected in a clean conical flask.
The filtrate will be concentrated using a rotary evaporator (BÜCHI, ROTAVAPOR R205,
SWITZERLAND) to remove excess solvent. After reported by (Arya et al), A study by (Wei et
al.)also indicated that crude Allium sativum extract inhibited the growth of Fulvia fulva, a tomato
fungus. and concentration, the extract will be pasted using a water bath set at 40°C. The paste will
be packed in a small laboratory bottle and kept in a fridge at 4°C suspension in 2 ml of broth and
12
incubated for 4 - hours in a 29 -30 water bath. Using an indelible marker, each of the petri dishes
will be divided into four quadrants. Four well isolated colonies of A. tumefaciens will be harvested
from a culture plate using a sterile platinum wire, and used to prepare a turbid. All chemicals and
reagents used in preparation and extraction will be of analytical grade.
3.3 Media Preparation and Screening for Antibiotic Susceptibility
The antibiotic susceptibility test will be carried out using the disc diffusion method, on Mueller
Hinton Agar (MHA) media. Thirty-eight grams of Mueller Hinton Agar (MHA) will be weighed
and mixed with 1L of sterile distilled water then sterilised by autoclaving at 120°C for 20 minutes.
The media will be dispensed into three presterilised petri dishes to a uniform depth of 4 mm,
covered and allowed to cool at 56°C undisturbed. After cooling, the dishes will be allowed to set
at the suspension were pipetted onto the MHA plates and distributed evenly over the media surface.
Readily prepared 5 mm diameter discs will be soaked, some in the garlic extract, some in methyl
bromide suspension (standard), some in distilled water and others in ethanol, leaving them to
absorb the substances. They will be later transferred to the inoculated plates, placing the disc from
different substances in the center of a separate quadrant from the other. The quadrants will then
be labeled with the letters G, MB, W, and E, where; G represented quadrant with disc soaked with
garlic extract, MB represented quadrant with disc soaked in methyl bromide, W represented
quadrant with disc soaked with sterile distilled water, and E represented quadrant with disc soaked
with ethanol. Methyl bromide will be used as a positive control, distilled water as a negative
control, and ethanol will be used to be sure that the activity of the extract is entirely due to the
active compound in garlic and not the solvent. The plates will be kept in an upright position for 8
hours to allow the extracts sufficient time to diffuse, after which they will be aerobically incubated
for 24 hours. The diameters of the inhibition zones were then measured.
3.4 Extraction method
Allium sativum L will be air dried for four weeks in an enclosed room away from direct sunlight.
Afterwards, it will be crushed using pestle and mortar for the purpose of increasing surface area
for reaction. Fifty grams (50 g) of the crushed clove sample will be extracted with 100 ml of
ethanol and kept on a rotary shaker for 12 hours. The extract will then be filtered and centrifuged
13
at 500 rpm for 5 minutes on a centrifuge and the supernatant will be collected. The supernatant
will then be filtered with a No 3 Whatman filter paper and evaporated to dryness. The crude leave
sample extract obtained will be maintained at 4⁰C until further use (Kibiti and Afolayan, 2015)
3.5 Phytochemical screening this should come immediately after extraction.
The extracts of Allium sativum L will be analyzed for the presence of the following
phytochemicals.
3.5.1 Determination of Alkaloids
Each 2 ml of extract will be put in test tubes and 2 drops of Meyer’s reagent and dragendorff
reagent will be added on each test tube of extract. The formation of orange-red and buff-colored
precipitate indicates a positive results for alkaloids.
3.5.2 Determination of Flavonoids
Each 5ml dilute ammonia will be added to 2ml of the extracts. After which H2SO4 will be added
to each and shaked. The layers will be allowed to separate. A yellow coloration at the ammonia
layer indicates the presence of flavonoid.
3.5.3 Determination of Steroids
Each 2ml of extract will be put in a test tube.2ml of chloroform is added. After which 2ml
concentrated H2SO4 is added.
3.5.4 Determination of Tannins
Each 2ml of extract will be put in a test tube, and then diluted with distilled water. After which, 2
drops of ferric chloride solution is added. A transient greenish to black color indicates the presence
of tannins.
3.5.5 Determination of Terpenoids
0.05mls of both acetone and ethanol extract will be mixed with 2mls of chloroform and shaked
and afterwards concentrated hydrogen sulphate will be added to form a layer, a reddish brown
colour will appear on that layer of both extracts which confirmed the presence of terpenoids
14
3.6 Determination of Minimum Inhibitory Concentration (MIC)
The MIC is determined using the indole test as by (Kumar et al.) Thirteen grams of nutrient broth
are weighed and mixed in I L of sterile distilled water. 0.005 g of phenol red indicator is added to
the media, sterilised by autoclaving at 120°C for 20 minutes, and allowed to cool in a 50°C water
bath. Serial concentrations of the garlic extract are prepared by first dissolving 1 g of dried A.
sativum extract in 10 ml of sterile distilled water to give a concentration of 100 mg/ml (this was
the stock solution). Serial dilutions of the stock solutions are then made by successively doubling
the volume measured from the stock with distilled water to give concentrations 100, 50, 25, 12.5,
and 6.25mg/ml. Eight milliliters of the melted and cooled medium are dispensed into adequate
number (17) of sterile universal bottles, and 0.5 ml of bacterial suspension added to each. Two
milliliters of serial concentrations of extracts are added in each concentration in triplicate, and
mixed gently by inverting the bottles several times. The bottles are labelled D1 - D5 in the order of
decreasing concentration. Two universal bottles with inoculated medium are set aside, and 2 ml of
methyl bromide added to one of them, while to the other, 2 ml of distilled water are added to act
as the positive and negative controls respectively. The bottles are then incubated at 29
for 24
hours after which the lowest concentration of extract at which no growth occurred is visually
detected by no colour change.
3.7 Determination of the Minimum Bactericidal Concentration (MBC)
10-fold dilutions of the culture medium in the tube in which no apparent growth was observed
(50 mg/ml) are made, and incubated at 29 for 24 hours on agar plates. The concentration of the
extract at which the subculture from the test dilution method yields no viable microorganisms is
detected. This is the MBC.
3.8 Data processing and analysis
The data obtained will be entered into Microsoft excel spreadsheets where it will be subjected to
exploratory data analysis using one way ANOVA using SPSS for windows. This will be based on
two variables being measured i.e. Chemical composition and antimicrobial activity (mic). The
results will be presented in table form and graphs
15
ACTIVITY
APRIL MAY
WORK PLAN
JUNE JULY AUG
PROPOSAL
WRITING
PROPOSAL
PRESENTATION
FIELD WORK
LAB WORK
DATA
ANALYSIS
REPORT
WRITING
REPORT
PRESENTATION
16
OCT
NOV
DEC
ACTIVITY
BUDGET
COST
PROJECT MATERIAL
7000
TRANSPORT
3000
CHEMICAL AND REAGENT
10000
EQUIPMENT HIRE
10000
DOCUMENTATION
5000
TOTAL
35000
17
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