A

PROJECT REPORT
ON
"Effect of Azadirachta indica L. (Neem) against anti-fungal
activity of Aspergillus niger"

SUBMITTED IN PARTIAL FULFILLMENT

FOR THE DEGREE
OF

Master of Science
In
Microbiology
(2020-2021)

Miet
GROUP OF INSTITUTIONS

BY
ARZOO
Under the Guidance of

Miss. Shipra Chaudhary

Lecturer
Department of Microbiology
Meerut Institute of Engineering and Technology,Meerut
C. C. S University, Meerut

1
 Acknowledgement
I am thankful to Prof. Shalini principal, Biotechnology and Microbiology Programme,
Meerut Institute of Engineering and Technology(M.I.E.T.) Meerut, for providing me the
entire necessary requirement during the whole period of M.Sc.

I am also very grateful to my Supervisor Miss. Shipra Chaudhary, Sr. Lecturer, Department
of Microbiology, M.I.E.T. for his kind and invaluable support and suggestions in completing
this work properly.

I would also like to thank my teacher Mrs. Alka Chaudhary (Sr. Lecturer M.I.E.T.) for her
invaluable support throughout the dissertation work.

I also want to pay my gratitude to all the staff of M.I.E.T. for supporting me directly or
indirectly.

This acknowledgement will not complete until I pay my respectful homage to my family,
whose enthusiasm to see the work complete was as infectious as their inspiration.I am
grateful to my parents and my friends for their support.

Last but not the least I convey my regard to all those who directly or indirectly contribute to
the success of this project work.

Arzoo Hudda

2
 Candidate's declaration
This is to certify that the thesis work entitled " Effect of Azadirachta indica against
anti-fungal activity of aspergillus niger" is submitted by me in partial fulfillment of the
requirement for the completion of Master of Microbiology submitted in Department of
Microbiology, M.I.E.T. Meerut(U.P) is an authentic record of my own carried out work
during the final year of the M.Sc degree under the Supervision of Miss.Shipra Chaudhary,
Senior Lecturer, Department of Microbiology,M.I.E.T., Meerut, comprises only my original
work and due acknowledgement has been made in the text to all other material used.

Project By:
Arzoo Hudda
Roll No.

This is to certify that the above statement made by the candidate is true to the best of my
knowledge.

Miss. Shipra Chaudhary

Senior Lecturer,
Department of Microbiology
M.I.E.T., Meerut

3
 TABLE OF CONTENT

CHAPTER PAGE NO.

1.INTRODUCTION 5-20

2.REVIEW OF LITERATURE 21-31

3.MATERIAL AND METHOD 31-38

4. OBSERVATION 39-48

5.RESULT AND DISCUSSION 48-49

6.CONCLUSION 49-50

7.REFERENCE 50-55

4
CHAPTER-1

Introduction
Ayurveda, a natural system of medicine, originated in India more than 3,000 years ago. The
term Ayurveda is derived from the Sanskrit words ayur (life) and veda (science or
knowledge). Thus, Ayurveda translates to knowledge of life. Based on the idea that disease is
due to an imbalance or stress in a person's consciousness, Ayurveda encourages certain
lifestyle interventions and natural therapies to regain a balance between the body, mind,
spirit, and the environment.
In India, Ayurveda is considered a form of Traditional medical care.

Traditional medicine is “the knowledge, skills and practices based on the theories, beliefs and
experiences indigenous to different cultures, used in the maintenance of health and in the
prevention, diagnosis, improvement or treatment of physical and mental illness”. (World
Health Organization, http://www.who.int/topics/traditional_medicine/en/).
There are many different systems of traditional medicine, and the philosophy and practices
of each are influenced by the prevailing conditions, environment, and geographic area within
which it first evolved (WHO 2005), however, a common philosophy is a holistic approach to
life, equilibrium of the mind, body, and the environment, and an emphasis on health rather
than on disease. Generally, the focus is on the overall condition of the individual, rather than
on the particular ailment or disease from which the patient is suffering, and the use of herbs is
a core part of all systems of traditional medicine (Engebretson 2002; Conboy et al. 2007;
Rishton 2008; Schmidt et al. 2008).

Traditional Medicinal use

In India, the plant is variously known as "Sacred Tree", " Heal All", "Nature's Drugstore", "
Village Pharmacy " and "Panacea for all diseases". Products made from neem trees have been
used in India for over 2 millennia for their medicinal properties. Neem products are believed
to be anti-helminthic, anti-fungal, anti-diabetic, anti-bacterial, anti-viral, contraceptive and
sedative. It is considered a major component in Ayurvedic and Urani medicine and is
particularly prescribed for skin diseases.

5
 Figure 1- some Medicinal Plants

Medicinal plants

Table 1.1
S. No. Botanical Family Parts used Medicinal uses
Name

1. Azadiracht Meliaceae Leaves/Twigs Jaundice, skin

a indica diseases, antiseptic,
boils, chronic ulcers,
small pox

2. Curcuma Zingiberaceae Rhizome Antioxidant and

longa natural dye

3. Aegle Rutaceae Leaves Diarrhea, dysentery

marmelos

● Tulsi:- Closely related to culinary basil, tulsi (Ocimum sanctum, a.k.a. holy basil) is
a medicinal herb used in Ayurveda that is native to India and Southeast Asia. Tulsi is
considered an adaptogenic herb, meaning it can help adapt the body to stress and
boost energy. In alternative medicine, tulsi is typically used for anxiety, stress, and
fatigue, and may be used in herbal formulations to help treat asthma, bronchitis, colds,
and the flu.

6
 Figure 2: Tulsi Plant

● Neem:- Azadirachta indica, commonly known as neem, has attracted worldwide

prominence in recent years, owing to its wide range of medicinal properties. Neem
has been referred to as a “wonder herb” for the many beneficial properties it
possesses. It has as many as 130 different biologically-active compounds that enhance
the well-being of several body parts such as skin, hair and blood, among others.

Figure 3: Neem Plant

● Cinnamon:- Cinnamon is the name for several species of trees and the commercial
spice products that some of them produce.Cinnamon bark is used for gastrointestinal
(GI) upset, diarrhea, and gas. It is also used for stimulating appetite; for infections
caused by bacteria and parasitic worms; and for menstrual cramps, the common cold,
and the flu (influenza).

Figure 4: Cinnamon

● Lavender:- Lavender is an herb. The flower and the oil of lavender are used to
make medicine.Lavender is used for restlessness, insomnia, nervousness, and
depression. It is also used for a variety of digestive complaints including meteorism
(abdominal swelling from gas in the intestinal or peritoneal cavity), loss of appetite,
vomiting, nausea, intestinal gas (flatulence), and upset stomach.

7
 Figure 5: Lavender flower

Azadirachta indica
Kingdom :Plantae
Division:Magnoliophyta
Class:Magnoliopsida
Order :Sapindales
Family :Meliaceae
Genus :Azadirachta
Species :A. indica
Scientific Name :Azadirachta indica

Figure 6: Neem Leaves

Azadirachta indica, commonly known as neem, has attracted worldwide prominence in recent
years, owing to its wide range of medicinal properties. Neem has been extensively used in
Ayurveda, Unani and Homoeopathic medicine and has become a cynosure of modern
medicine. Neem elaborates a vast array of biologically active compounds that are chemically

8
diverse and structurally complex. More than 140 compounds have been isolated from
different parts of neem. All parts of the neem tree- leaves, flowers, seeds, fruits, roots and
bark have been used traditionally for the treatment of inflammation, infections, fever, skin
diseases and dental disorders. The medicinal utilities have been described especially for neem
leaves. (Arumugam et al., 2014, Omóbòwálé et al., 2016, Patel et al., 2016).
Neem is a fast-growing tree that can reach a height of 15–20 metres (49–66 ft), and rarely
35–40 m (115–131 ft).Neem fruit, seeds, leaves, stems, and bark contain diverse
phytochemicals, some of which were first discovered in azadirachta seed extracts, such as
azadirachtin established in the 1960s as an insect antifeedant, growth disruptor, and
insecticide. The yield of azadirachtin from crushing 2 kg of seeds is about 5 g.
Neem trees, including the leaves, bark, fruit, flowers, oil, and gum are associated with the
aforementioned medical folklore in the treatment of certain medical conditions such as
cancer, hypertension, heart diseases, and diabetes. The potential effects that are seen when
using these extracts can certainly be attributed cellular and molecular mechanisms, these
mechanisms include free radical scavenging, detoxification, DNA repair,cell cycle alteration,
programmed cell death mitigation and autophagy, immune surveillance, anti-inflammatory,
anti-angiogenic, and anti-metastatic activities and the ability to modulate of various signaling
pathways(Arumugam et al., 2014, Omóbòwálé et al., 2016, Patel et al., 2016).

Genome and transcriptomes

Neem genome and transcriptomes from various organs have been sequenced, analyzed, and
published by Ganit Labs in Bangalore, India.ESTs were identified by generation of
subtractive hybridization libraries of neem fruit, leaf, fruit mesocarp, and fruit endocarp by
CSIR-CIMAP Lucknow.

Traditional medicine
Products made from neem trees have been used in the traditional medicine of India for
centuries, but there is insufficient clinical evidence to indicate any benefits of using neem for
medicinal purposes. In adults, no specific doses have been established, and short-term use of
neem appears to be safe, while long-term use may harm the kidneys or liver; in small
children, neem oil is toxic and can lead to death.Neem may also cause miscarriages,
infertility, and low blood sugar.

9
Medicinal properties of Azadirachta indica
Neem leaf is used for leprosy, eye disorders, bloody nose, intestinal worms, stomach upset,
loss of appetite, skin ulcers, diseases of the heart and blood vessels (cardiovascular disease),
fever, diabetes, gum (gingivitis), and liver problems. The leaf is also used for birth control
and to cause abortions.

Treats Acne
Neem has an anti-inflammatory property which helps reduce acne. Azadirachta Indica also
helps reduce skin blemishes.
Neem is a rich source of Vitamin E which helps repair damaged skin cells.

Treats Infections
Neem has scientifically proven antifungal property which helps treat fungal infections.
Plant diseases are caused by bacteria, fungi, or viruses and can range in severity from mild
leaf or fruit damage to death. Plant diseases reduce the production and quality of food. Fungi
are among the dominant causal agents of plant diseases. Considerable post-harvest losses
of plant produce are brought about by decay caused by fungal pathogens and such
postharvest pathogens also produce toxins that create serious health problems for
consumers. These aspects make it difficult to support the food needs of the rapidly
growing global population. Hence inhibiting the growth of fungi, in field as well as during
storage, has great significance in agriculture. Microbial biodegradation, mainly by fungi,
causes heavy losses of wood and their products. Until recently, the chemical method of plant
disease control was prevalent in agriculture. However, environmental concerns due to
resistance of pathogens to these chemicals, high inputs, residual toxicity problems,
environmental pollution and carcinogenic effects, has led to seek alternatives to synthetic
chemicals (Girish et al., 2009; Goel et al., 2016). Currently attention is focused on the
use of natural products of plant origin as an alternative to synthetic fungicides, which are
not only effective, but are also safe to human health and are biodegradable (Wang et al.,
2010). There is also a trend to minimize the usage of chemical pesticides by amending it
with fungicidal components of biological origin (Deepa et al., 2013). Presently, higher
plant products have attracted the attention to search for phytochemicals having
anti-microbial activity. Neem plant extracts provide environmentally suitable fungicides and
wood preservatives for the effective management of plant pathogenic fungi, storage fungi
and wood destroying fungi that will help in reducing food loss as well as in preserving wood.

Useful in Detoxification
Neem can prove useful in detoxification both internally and externally. Consumption of neem
leaves or powder stimulates kidneys and liver increasing the metabolism and eliminating the
toxins out of the body. Externally, neem scrubs or paste can be used to remove germs,
bacteria, dirt, etc from your skin preventing rashes and skin diseases.

10
Increases Immunity
Neem is known for its antimicrobial and antibacterial effects. These properties play a huge
role in boosting immunity.

Prevents Gastrointestinal Diseases

Neem's anti-inflammatory properties help reduce inflammation of the gastrointestinal tract
which helps reduce a series of diseases like constipation, stomach ulcer, flatulence, etc. Try
out these home remedies for constipation.

Treats Wounds
Neem leaves have an antiseptic property that is why it is used to heal wounds.

Reduces Dandruff
Neem is extensively used in shampoos and conditioners. Azadirachta Indica has antifungal
and antibacterial properties which help eliminate dandruff and strengthen your hair.

Reduces Joint Pain

Application of neem oil or extract on the affected area can help reduce pain and discomfort.
Hence it is widely used for treating arthritis.

Exfoliates skin
Neem is an excellent exfoliant. It helps remove dead cells from the surface of the skin which
will help prevent the growth of blemishes.

Dental plaque
Early research suggests that applying neem leaf extract gel to the teeth and gums twice daily
for 6 weeks might reduce plaque formation. It also might reduce the number of bacteria in the
mouth that can cause plaque. However, using a mouth rinse containing neem extract for 2
weeks does not appear to reduce plaque or gingivitis.

11
Active Compounds of Azadirachta indica L. (Neem)
Azadirachta indica L. (neem) shows therapeutics role in health management due to the rich
source of various types of ingredients. The most important active constituent is azadirachtin
and the others are nimbolinin, nimbin, nimbidin, nimbidol, sodium nimbinate, gedunin,
salannin, and quercetin. Leaves contain ingredients such as nimbin, nimbanene, 6-desacetyl
nimbinene, nimbandiol, nimbolide, ascorbic acid, n-hexacosanol and amino acid,
7-desacetyl-7-benzoyl azadiradione, 7-desacetyl-7-benzoyl gedunin, 17-hydroxy
azadiradione, and nimbiol. Quercetin and ß-sitosterol, polyphenolic flavonoids, were purified
from neem fresh leaves and were known to have antibacterial and antifungal properties and
seeds hold valuable constituents including gedunin and azadirachtin. (Ali Textbook of
Pharmacognosy. New Delhi, India: Publication and Information Directorate; 1993.)

Mechanism of Action of Active Compounds

Neem (Azadirachta indica), a member of the Meliaceae family, has therapeutic implication in
the disease's prevention and treatment. But the exact molecular mechanism in the prevention
of pathogenesis is not understood entirely. It is considered that Azadirachta indica shows
therapeutic role due to the rich source of antioxidant and other valuable active compounds
such as azadirachtin, nimbolinin, nimbin, nimbidin, nimbidol, salannin, and
quercetin.Mordue (Luntz)., Nisbet Azadirachtin from the neem tree Azadirachta indica:
its action against insects. Anais da Sociedade Entomológica do Brasil. 2000).

Possible mechanism of action of Azadirachta indica is presented as

follows:-
Neem (Azadirachta indica) plant parts shows antimicrobial role through inhibitory effect on
microbial growth/potentiality of cell wall breakdown. Azadirachtin, a complex
tetranortriterpenoid limonoid present in seeds, is the key constituent responsible for both
antiferedant and toxic effects in insects. Experiments suggest that the ethanol extract of neem
leaves showed in vitro antibacterial activity against both Staphylococcus aureus and MRSA
with greatest zones of inhibition noted at 100% concentration. (Sarmientos Marimba
,Gonzales.An in-vitro study on the antibacterial effect of neem (Azadirachta indica) leaf
extracts on methicillin-sensitive and methicillin-resistant Staphylococcus aureus .
PIDSP Journal. 2011).
● Neem plays the role of free radical scavenging properties due to its rich source of
antioxidants. Azadirachtin and nimbolide showed concentration-dependent antiradical
scavenging activity and reductive potential in the following order: nimbolide >
azadirachtin > ascorbate.
● Neem ingredient shows an effective role in the management of cancer through the
regulation of cell signaling pathways. Neem modulates the activity of various tumour
suppressor genes , angiogenesis, transcription factors and apoptosis.

12
 ● Neem also plays a role as anti-inflammatory via regulation of pro-inflammatory
enzyme activities including cyclo-oxygenase (COX), and lipo-oxygenase (LOX)
enzymes.

Botanical Family Plant used Active Medicinal uses

Name compound

Azadirachta Meliaceae Leaves/Twigs margesic Jaundice, skin diseases,

indica acid discutient, antiseptic, boils,
chronic ulcers, small pox,
glandular.

Therapeutic Implications of Neem and Its Various Ingredients in Health

Management
Active constituents of neem play an important role to cure different diseases via activation of
antioxidative enzymes, rupture the cell wall of bacteria and play a role as chemopreventive
through the regulation of cellular pathways. Pharmacological activities of neem are
discussed.

Antioxidant Activity
Free radical or reactive oxygen species are one of the main culprits in the genesis of various
diseases. However, neutralization of free radical activity is one of the important steps in
disease prevention. Antioxidants stabilize/deactivate free radicals, often before they attack
targets in biological cells and also play a role in the activation of antioxidative enzymes that
play a role in the control of damage caused by free radicals/reactive oxygen species. Neem
plants have been reported to have antioxidant activity. Plants fruits, seeds, oil, leaves, bark,
and roots show an important role in diseases prevention due to the rich source of
antioxidant.Nunes, Silva, Guedes, Almeida;Phytochemicals as Nutraceuticals—Global
Approaches to Their Role in Nutrition and Health. InTech; 2012. Biological oxidations
and antioxidant activity of natural products.
Leaf and bark extracts of A. indica have been studied for their antioxidant activity and results
of the study clearly indicated that all the tested leaf and bark extracts/fractions of neem grown
in the foothills have significant antioxidant properties.

13
Effect of Neem as Anti-Inflammatory
A study result has confirmed that extract of A. indica leaves at a dose of 200 mg/kg,showed
significant anti-inflammatory activity in cotton pellet granuloma assay in rats. Other study
results revealed that neem leaf extract showed significant anti-inflammatory effect but it is
less efficacious than that of dexamethasone and study results suggest that nimbidin
suppresses the functions of macrophages and neutrophils relevant to inflammation. (Tidjani,
Dupont, 1989).Indian Journal of Experimental Biology. 1998.

Hepatoprotective Effect
Medicinal plants and their ingredients play a pivotal role as hepatoprotective without any
adverse complications. A study was performed to investigate the hepatoprotective role of
azadirachtin-A in carbon tetrachloride (CCl4) induced hepatotoxicity in rats and histology
and ultrastructure results confirmed that pretreatment with azadirachtin-A dose-dependently
reduced hepatocellular necrosis. Furthermore results of the study show that pretreatment with
azadirachtin-A at the higher dose levels moderately restores the rat liver to normal. (Baligar,
Aladakatti, Ahmed, Hiremath;Hepatoprotective activity of the neem-based constituent
azadirachtin-A in carbon tetrachloride intoxicated Wistar rats. Canadian Journal of
Physiology and Pharmacology. 2014.

Wound Healing Effect

In an incision wound, tensile strength of the healing tissue of neem plants was found to be
significantly higher as compared to the other plants. Other results showed that leaf extracts of
Azadirachta indica promote wound healing activity through increased inflammatory response
and neovascularization. (Evaluation of the wound healing activity of methanolic extract
of Azadirachta Indica (Neem) and Tinospora cordifolia (Guduchi) in rats.
Pharmacologyonline. 2010.)

Antimicrobial Effect
Neem and its ingredients play an important role in the inhibition of growth of numerous
microbes such as viruses, bacteria, and pathogenic fungi. The role of neem in the prevention
of microbial growth is described individually as follows.

1. Antibacterial Activity
A study was performed to evaluate antimicrobial efficiency of herbal alternatives as
endodontic irrigants and compared with the standard irrigant sodium hypochlorite and finding
confirmed that Neem leaf extracts and seed extracts showed zones of inhibition suggesting
that they had antimicrobial properties.Furthermore, leaf extracts showed significantly greater
zones of inhibition than 3% sodium hypochlorite.(Comparison of the antibacterial
efficiency of neem leaf extracts, grape seed extracts and 3% sodium hypochlorite
against E. faecalis—an in vitro study. Journal of International Oral Health. 2013.

14
Ongoing research over the past 45 year recognizes these traditional uses of neem, but
researchers typically list them as" Knows to be" rather than reporting on their action. More
recent reports focus on anti-bacterial activities in the mouth, specially gum disease and
cavity, as well as preventing sexually transmitted diseases as a vaginal contraceptive.
(Siddiqui,Current. Sci., 1942).

2.Antiviral Activity
Results showed that neem bark (NBE) extract significantly blocked HSV-1 entry into cells at
concentrations ranging from 50 to 100 μg/mL. Furthermore, blocking activity of NBE was
noticed when the extract was preincubated with the virus but not with the target cells
suggesting a direct anti-HSV-1 property of the neem bark. (In vitro antiviral activity of
neem (Azadirachta indica L.) bark extract against herpes simplex virus type-1 infection.
Phytotherapy Research. 2010).
Leaves extract of neem (Azadirachta indica A. Juss.) (NCL-11) has shown virucidal activity
as suggested via virus inactivation and yield reduction assay besides interfering at an early
event of its replication cycle. ( Rao,Kumar, Res., 1969).

3. Antifungal Activity
Experiment was made to evaluate the efficacy of various extracts of neem leaf on seed borne
fungi Aspergillus and Rhizopus and results confirmed that growth of both the fungal species
was significantly inhibited and controlled with both alcoholic and water extract. Furthermore,
alcoholic extract of neem leaf was most effective as compared to aqueous extract for
retarding the growth of both fungal species.( khan, Wassilew, Eschborn, Germany,
1987).(Antifungal activities and chemical characterization of Neem leaf extracts on the
growth of some selected fungal species in vitro culture medium. Journal of Applied
Sciences and Environmental Management. 2009.)

4. Antimalarial Activity
Experiment was made to evaluate the antimalarial activity of extracts using Plasmodium
berghei infected albino mice and results revealed that neem leaf and stem bark extracts
reduced the level of parasitemia in infected mice by about 51–80% and 56–87%, respectively,
and other studies showed that azadirachtin and other limonoids available in neem extracts are
active on malaria vectors. (Khalid, Duddect, Nat). (Nathan,, Kalaivani, Murugan, Effects
of neem limonoids on the malaria vector Anopheles stephensi Liston (Diptera:
Culicidae) Acta Tropica. 2005.)

5. Neuroprotective Effects
A study was performed to investigate the neuroprotective effects of Azadirachta indica leaves
against cisplatin- (CP-) induced neurotoxicity and results showed that morphological findings
of neem before and after CP injection implied a well-preserved brain tissue. No changes, in
biochemical parameters, were observed with neem treated groups.( Abdel

15
Moneim,Azadirachta indica attenuates cisplatin-induced neurotoxicity in rats. Indian
Journal of Pharmacology. 2014.)

6.Immunomodulatory and Growth Promoting Effect

Experiment was performed to investigate growth promoting and immunomodulatory effects
of neem leaves infusion on broiler chicks and results showed that neem infusion successfully
improved antibody titre, growth performance, and gross return at the level of 50 mL/liter of
fresh drinking water. (Durrani., Chand., Jan, Sultan, Durrani , Akhtar;
Immunomodulatory and growth promoting effects of neem leaves infusion in broiler
chicks. Sarhad Journal of Agriculture. 2008.)

16
Microbes found in Soil
There are five different types of soil microbes: bacteria, actinomycetes, fungi, protozoa and
nematodes. Each of these microbe types has a different job to boost soil and plant health.

Bacteria
Bacteria are the crucial workforce of soils. They are the final stage of breaking down
nutrients and releasing them to the root zone for the plant. In fact, the Food and
Agriculture Organization once said “Bacteria may well be the most valuable of life
forms in the soil.”

Actinomycetes
Actinomycetes were once classified as fungi, and act similarly in the soil. However,
some actinomycetes are predators and will harm the plant while others living in the
soil can act as antibiotics for the plant.

Fungi
Like bacteria, fungi also live in the rootzone and help make nutrients available to
plants. For example, Mycorrhizae is a fungi that facilitates water and nutrient uptake
by the roots and plants to provide sugars, amino acids and other nutrients.

Protozoa
Protozoa are larger microbes that love to consume and be surrounded by bacteria. In
fact, nutrients that are eaten by bacteria are released when protozoa in turn eat the
bacteria.

Nematodes
Nematodes are microscopic worms that live around or inside the plant. Some
nematodes are predators while others are beneficial, eating pathogenic nematodes and
secreting nutrients to the plant.

17
Disease caused by different Fungi

Table 1.2
Fungi Main host Disease Infected Reference
caused tissue/organ

Hemi-biotrophic

1. Colletotrichum Carica papaya Papaya Fruit Cia et al.

gloeosporioides anthracnose (2007)

2. Fusarium More than Vascular wilt Root Agrios(2005)

oxysporum 100 species of and root rot
plants

3. Fusarium solani Glycine max, Root crown Tap root and Fu and
Phaseolus rot, dieback Hypocotyls Change(1999
vulgaris and wilt ) ;Iqbal et al.
(2005);
Abeysinghe(2
007).

Necrotrophic

1. Rhizoctonia Oryza sativa, Sheath blight Hypocotyls Datta et

solani Phaseolus and roots al.(1999)
vulgaris ;Abeysinghe
(2007)

2. Neurospora sp. Anacardium Green mould Fruit Andrade et

occidentale al. (1990)

3. Aspergillus niger Vitis vinifera, Black mould Fruit Samson et al.

Allium cepa, (2001)
Arachis
hypogaea

18
Microbe effect on soil

Table 1.3
Microorganisms Characteristics Beneficial role

Prokaryotic

1. Bacteria Rigid cell wall, divide by Recycle biomass, control

binary fission, some capable of atmospheric composition,
photosynthesis components of phytoplankton
and soil microbial population.

2. Archaea Rigid cell wall, unusual Produce and consume low

membrane structure, molecular weight components,
photosynthetic membrane, lack aid bacteria in recycling dead
chlorophyll biomass, some are
extremophiles

Eukaryotic

1. Fungi Rigid cell wall, single cell Recycling biomass, stimulate

form(yeast), reproduction by plant growth
budding, multicellular
form(hyphae, mycelium), no
photosynthetic membrane

2. Algae Rigid cell wall, photosynthetic Important components of

phytoplankton.

19
Uses of different Medical Plants in Curing different disease

Table1. 4
Scientific Name Common Family Part used Medical uses
Name

1. Punica Pomegranate Lythraceae Dried Cancer,

granatum pericarp osteoarthritis,
and other
disease

2. Curcuma longa Turmeric Zingiberaceae Stem Arthritis, peptic

L. ulcers, cough,
diabetes,
dermatological
conditions,
respiratory
problems,
cardiovascular
and
hepatobiliary
disease

3. Ocimum Tulsi Lamiaceae Leaves Bronchitis,

tenuiflorum bronchial
asthma, malaria,
diarrhea, skin
disease.

4. Cinnamomum Cinnamon Lauraceae Wood Gastrointestinal

verum upset, diarrhea,
gas, menstrual
cramps,
common cold,
influenza

5. Lavandula Lavender Lamiaceae Flowers Anti-convulsant

,
anti-depressive,
sedative, and
calming
property

6. Elaeagnus Russian Elaeagnaceae Petal, gum, Anti-pyretic,

angustifolia olive/ seed and anti-diarrheal
oleaster leaves and kidney
disorder

20
CHAPTER-2

Review of Literature
Over time, research has shown that Azadirachta indica is rich in a wide range of compounds,
of which several have pharmacological potential. Out of all these compounds, triterpenes
lead the way in having therapeutic use. In particular, Nimbin (triterpene) has shown to have
antipyretic, fungicidal, antihistamine and antiseptic properties. Also Nimbin is associated
with anti-inflammatory and antioxidant effects, therefore reducing damage by mitigating the
production of reactive oxygen species. (Naik et al., 2014, Schumacher et al., 2011).
Flavonoids are also found in neem , which function as inhibitors of prosta-glandin
biosynthesis, and endoperoxides and the enzymes like protein kinases and
phosphodiesterases, all involved in inflammation (Batista et al., 2018, Hernández-Aquino
and Muriel, 2018, Naik et al., 2014).
Oil extracts are the most typical used form of Neem and its in-depth phytochemical analysis
has confirmed the presence in high amounts of triterpenes, flavonoids and saponins, while
other components such as catechins and nimbins, seem to be present in lower amounts (Naik
et al., 2014, Schumacher et al., 2011). Other metabolites found in Neem extracts are:
limonoids, tannins, alkaloids, terpenoids, reducing sugar, catechins, sterols and gallic acid
(Naik et al., 2014, Roma et al., 2015, Saleem et al., 2018, Schumacher et al., 2011).

The leaf of the Neem tree appears to have developed a particular set of glycoproteins named
as neem leaf glycoprotein (NLGP) that when tested on mammalian subjects, showed
immune-modulatory activity, providing the potential to restrict tumor growth by modulating
local and systemic immunity (Banerjee et al., 2014, Dayakar et al., 2015, Durrani et al.,
2008, Kundu et al., 2018). Recently, Dash, Dixit, and Sahoo (2017) conducted an analysis
involving leaf extracts (aqueous and methanoic indicating high levels of saponins, tannins
and glycosides in the aqueous extracts. While methanolic extracts showed top levels of
alkaloids, tannins, and flavonoids (Dash et al., 2017).

Previous studies (non-methanoic) reported glycosides nimbanene, 6-desacetyl nimbinene,

nimbandiol, nimbolide, ascorbic acid, n-hexacosanol and amino acid,
7-desacetyl-7-benzoyl azadiradione, 7-desacetyl-7-benzoyl gedunin, 17-hydroxy
azadiradione, and nimbioland in leaf extracts (Alzohairy, 2016), which shows the high
variety of compounds available, but interestingly place much stress on the extraction process.
Biochemical analysis done on leaf extracts has revealed high presence of proline, which is a
current treatment for neurodegenerative diseases like Alzheimer’s and Parkinson’s disease,
Type 2 Diabetes Mellitus and Polycythemia (Dash et al., 2017, Gladkevich et al., 2007,
Mesgari-abbasi et al., 2019, Yenkoyan et al., 2018).

21
Other recent studies by Hossaińs group was able to characterize, leaf extracts, by up to 5
different extraction methods: hexane, ethyl-acetate, chloroform, butanol, methanol, and test
for their antioxidant capacity, given that all these solvent have different polarities, each one
showed interesting differences. Most importantly, chloroform extracts were deemed as having
the highest antioxidant effect, mostly containing (2E)-3,7,11,15-tetramethyl-
2-hexadecen-1-ol, methyl 14-methyl pentadecanoate, linoleoyl chloride, phytol, methyl
isoheptadecanoate and nonacosane.

On the other side of the spectrum, the methanolic extracts show the lowest antioxidant effect.
These extracts mainly contained: m-Toluylaldehyde, methyl 14-methyl pentadecanoate,
Linoleoyl chloride, Methyl isoheptadecanoate.Hexane-derived extract had the highest
biologically active compounds: (2E)-3,7,11,15-Tetramethyl-2-hexadecen-1-ol, Methyl
petroselinate, Phytol, Methyl isoheptadecanoate, Hexadecamethylcyclooctasiloxane, Butyl
palmitate, 2,6,10,14-Tetramethyl Heptadecane, Nonadecane, Isobutyl Stearate, Oxalic acid,
2-ethylhexyl tetradecyl est, Heptacosane, Eicosane, 7-hexyl- Heptacosane, 7-hexyl, and
Octacosane.

Scientists determined that butanol had the highest total phenolics (107.3 GA/g) and hexane
had the lowest concentration (20.8 GA/g). Next, quantified total flavonoids by UV over dry
samples.It was found that highest concentration of total flavonoids to be in the methanol
extract (529.5 mg/100 g) and the lowest was in the butanol extract (63.0 mg//100 g)
(Al-Hashemi and Hossain, 2016, Hussain et al., 2013, Khamis Al-Jadidi and Hossain,
2015).

Antioxidant effect
Free radicals or reactive oxygen species (ROS) are a major source of inflammation, as they
act upon many biological molecules, exerting damage by taking out electrons as a way of
entering a stable state, unleashing in the cell a state of oxidative stress (Alzohairy, 2016,
Kiranmai et al., 2011). Therefore, there is a need for providing adequate compounds (termed
antioxidants) to stabilize or neutralize these radicals as a step in preventing or blocking an
exacerbation of oxidative stress, which can lead to many diseases. These antioxidant
molecules will supplement the work of the body’s natural antioxidant defenses: superoxide
dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione (GSH), nitric
oxide dioxygenase (NOD) found in neem. (Basir and Shailey, 2012, Gautam et al., 2015).

Anti-inflammatory effect

An important property found in Neem extracts is their ability to work as anti-inflammatory

agents (Rupani and Chavez, 2018, Soares et al., 2014). Inflammation is a
pathophysiological condition involved in a plethora of diseases like cancer and diabetes, as

22
well as in other states such as alcohol consumption and food digestion (Eldeene et al., 2016).
A main bioactive compound found in Neem is limonoid. Limonoid is a furanolactone, known
for its inhibitory properties in the production of inflammatory mediators, it is also known as a
pain anesthetizer, as it stimulates the activation of endogenous opioid pathways (Naik et al.,
2014, Schumacher et al., 2011, Soares et al., 2014). Limonoid extracted from Neem, can
inhibit edema and fibrovascular tissue growth when tested on damaged rat paws. He
concluded that this was most effective at a dosage of 120 mg/kg, showing particular
inhibitory effect over major inflammatory molecules such as tumor necrosis factor
alpha(TNF-α) and interleukins (Soares et al., 2014). Over time, several other studies have
corroborated and investigated in more detail the mechanism of the anti-inflammatory activity
of limonoids (Chen et al., 2018, Kumar et al., 2010, Tapanelli et al., 2016, Zhu et al.,
2017).

Figure 7

Anticancer effect
Studies over the past several decades, on medicinal plants and phytochemicals (typically
present in the diet) continue, in order to determine their anti-cancerous activity
(Abdelbaset-Ismail et al., 2016, Arumugam et al., 2014, Cruz-Vega et al., 2009, Hao et
al., 2014, Nagini, 2014, Patel et al., 2016, Sengupta et al., 2017, Wu et al., 2014). One
such study was conducted by Pramanik et al. (2016), they tested for the chemical

23
compounds found in Neem, like azadirachtin, nimbolide and limonoid enrich extracts, over
models of buccal carcinogenesis in hamsters. They established that Neem extracts gave
positive results such as the suppression of the NF-κβ pathway. They further showed the
expression profile of proliferating cell nuclear antigen (PCNA), p21, cyclin D1, glutathione
S-transferase pi (GST-P), NF-κβ, inhibitor of κβ (Iκβ), p53, Fas, Bcl-2, Bax, Bid, Apaf-1,
cytochrome C, survivin, caspases-3, -6, -8 and -9 where all tested and results showed their
overall reduction (Manikandan, Letchoumy, Gopalakrishnan, & Nagini, 2008).Two more
cytotoxic compounds are nimbolide and azadirone, both acts to induce ROS mediated
apoptosis by inhibiting PI3K/Akt signaling and upregulating reversion-inducing cysteine-rich
proteins with Kazal motifs (Hao et al., 2014, Zhu et al., 2017)

Anti-diabetic effect

Diabetes or the lack of control over glucose concentration in the blood is rapidly rising as one
of the major chronic degenerative disorders (Hieronymus and Griffin, 2015, Joshi et al.,
2010, Shori and Baba, 2013, Upret et al., 2013). Conservatively by 2030 there is an
expectancy for diabetes to be the 11th leading cause of death worldwide (Mathers &
Loncar, 2006). As the disease progresses, it becomes a lifelong burden (physical and
economical) over the patient, therefore lower cost treatments become necessary. Among the
various methods and pharmacotherapies being developed, the use of Neem extracts has
steadily grown in interest (Al Akeel et al., 2017, Joshi et al., 2010, Mathers and Loncar,
2006).

24
 Figure 8: Neem as Anti-diabetic

Toxicity studies
As for many other traditional extracts, in those derived from Azadirachta indica, antioxidants
seem to be at the forefront as the primary providers of medical properties (Al Akeel et al.,
2017, Basir and Shailey, 2012, Shori and Baba, 2013).

Industrialized applications
Through this work, I have mentioned some beneficial roles of Azadirachta indica studied in
models of heart disease, cancer, and diabetes. Such work, along with others beyond this
review, has given rise to the development of patentable technology for both clinical and
commercial application.
Amongst the various uses of Azadirachta indica, we begin with its properties as a male
contraceptive, as an alternative to vasectomy. A 50ml injection of Azadirachta indica oil
extracted from seeds and applied to the vas deferens in rats shows to block fertility without
the loss of libido or androgens. A single dose injection reported to be effective to block
fertility over the 9 months of observation (Talwar, Upadhyay, & Dhawan, 1996). In a
second instance, Azadirachta indica oil extracts used as vaginal creams, with spermicide
effects, as an effective form of preventing unwanted pregnancies. These creams comprise a
mixture of 89:10:1 organic carrier-Azadirachta indica oil-reetha extract, wherein animals
studied showed that 2 ml of cream where enough to prevent pregnancy for up to 3 months
with no effect on ovulating cycle. Bonnet Monkey experiments showed similar results 1 in 6
females became pregnant after 50 cycles of mating. Azadirachta indica creams give rise to
immune modulation of TNF-α and γ-interferon primarily affecting placental implantation
(Asif, 2013, Talwar et al., 1993).

It's important to emphasize that unlike traditional pill formulations, the use of oil, since
applied externally, does not interfere with hormonal cycle regulation. Khillar et al., proved
that a dosage of 3 mg sufficient to kill 100% of sperm (1 million in vitro). By direct
histological cuts of testis potent spermicide effects are seen, when 100 mg of dried leaf
powder suspended in 1 ml of distilled water for a 24 h period. Lower dosages seem to affect
the motility of the sperm, hence acting as an ATPase inhibitor (Khillare & Shrivastav,
2003).
Another interesting application is the use of extracts as fungicide (Barnette & Walter, 1997)
and insecticide and antibacterial (Barnette and Walter, 1997, Locke et al., 1993, Locke et
al., 1995).

25
Figure 9: Main components of Azadirachta indica

26
FUNGI
Fungi can be single celled or very complex multicellular organisms. They are found in just
about any habitat but most live on the land, mainly in soil or on plant material rather than in
sea or fresh water. A group called the decomposers grow in the soil or on dead plant matter
where they play an important role in the cycling of carbon and other elements. Some are
parasites of plants causing diseases such as mildews, rusts, scabs or canker. In crops fungal
diseases can lead to significant monetary loss for the farmer. A very small number of fungi
cause diseases in animals. In humans these include skin diseases such as athletes’ foot,
ringworm and thrush.(Nature Microbiology. 2 (8): 17120. 25 July 2017.)

Fungi Characteristics
Some fungi are single-celled, while others are multicellular. Single-celled fungi are called
yeast. Some fungi alternate between single-celled yeast and multicellular forms depending on
what stage of the life cycle they are in. Fungi cells have a nucleus and organelles, like plant
and animal cells do. The cell walls of fungi contain chitin, which is a hard substance also
found in the exoskeletons of insects and arthropods such as crustaceans. They do not contain
cellulose, which commonly makes up plant cell walls.

Classification of Fungi
Kingdom Fungi are classified based on different modes. The different classification of fungi
is as follows:

1.Based on Mode of nutrition

On the basis of nutrition, kingdom fungi can be classified into 3  groups.

● Saprophytic – The fungi obtain their nutrition by feeding on dead organic substances.
Examples: Rhizopus, Penicillium and Aspergillus.
● Parasitic – The fungi obtain their nutrition by living on other living organisms (plants
or animals) and absorb nutrients from their host. Examples: Taphrina and Puccinia.
● Symbiotic – These fungi live by having an interdependent relationship association
with other species in which both are mutually benefited. Examples: Lichens and
mycorrhiza. Lichens are the symbiotic association between algae and fungi. Here both
algae and fungi are mutually benefited as fungi provide shelter for algae and in
reverse algae synthesis carbohydrates for fungi.
2.Based on Spore Formation
Kingdom Fungi are classified into the following based on the formation of spores:

● Zygomycetes – These are formed by the fusion of two different cells. The sexual
spores are known as zygospores while the asexual spores are known as
sporangiospores. The hyphae are without the septa.

27
 ● Ascomycetes – They are also called as sac fungi. They can be coprophilous,
decomposers, parasitic or saprophytic. The sexual spores are called ascospores.
Asexual reproduction occurs by conidiospores. Example – Saccharomyces
● Basidiomycetes – Mushrooms are the most commonly found basidiomycetes and
mostly live as parasites. Sexual reproduction occurs by basidiospores. Asexual
reproduction occurs by conidia, budding or fragmentation. Example- Agaricus
● Deuteromycetes – They are otherwise called imperfect fungi as they do not follow
the regular reproduction cycle as the other fungi. They do not reproduce sexually.
Asexual reproduction occurs by conidia. Example – Trichoderma.

Aspergillus niger
It causes a disease called "black mold" on certain fruits and vegetables such as grapes,
apricots, onions, and peanuts, and is a common contaminant of food. It is ubiquitous in soil
and is commonly reported from indoor environments, where its black colonies can be
confused with those of Stachybotrys (species of which have also been called "black
mold").Samson RA, Houbraken ,Summerbell ,Flannigan, Miller(2001). "Common and
important species of fungi and actinomycetes in indoor environments". Microorganisms
in Home and Indoor Work Environments 2001.

Some strains of A. niger have been reported to produce potent mycotoxins called ochratoxins,
other sources disagree, claiming this report is based upon misidentification of the fungal
species.[citation needed] Recent evidence suggests some true A. niger strains do produce
ochratoxin A. It also produces the isoflavone orobol."Ochratoxin A production by strains
of Aspergillus niger var. niger"

Figure 10: Aspergillus niger fungus

Plant Disease caused by Aspergillus niger

28
Aspergillus niger causes sooty mold of onions and ornamental plants. Infection of onion
seedlings by A. niger can become systemic, manifesting only when conditions are conducive.
A. niger causes a common postharvest disease of onions, in which the black conidia can be
observed between the scales of the bulb. The fungus also causes disease in peanuts and in
grapes.(spectrum of disease, diagnosis, and treatment external icon).

Human and animal disease caused by Aspergillus niger

Aspergillus niger is less likely to cause human disease than some other Aspergillus species.
In extremely rare instances, humans may become ill, but this is due to a serious lung disease,
aspergillosis, that can occur. Aspergillosis is, in particular, frequent among horticultural
workers who inhale peat dust, which can be rich in Aspergillus spores. The fungus has also
been found in the mummies of ancient Egyptian tombs and can be inhaled when they are
disturbed.
A. niger is one of the most common causes of otomycosis (fungal ear infections), which can
cause pain, temporary hearing loss, and, only in severe cases, damage to the ear canal and
tympanic membrane.Handwerk, Brian (May 6, 2005) Egypt's "King Tut Curse" Caused
by Tomb Toxins.

Figure 11:Aspergillus niger growth on PDA

29
Economically Important Fungi

Table 2.1
Fungi/strain/species Plant Type Fungi Beneficial Reference
mediated effect on
Response plant species

1. Trichoderma Arabidopsis sp. Auxin Higher Contreras

sp. dependent biomass Cornejo et al.
Agriculturally mechanism production (2009);
important sp. Biocontrol and increase Chalot and
lateral root Brun(1998);
formation Harman and
Crop Mastouri(201
management 0)

2. Ectomycorrhiz Higher plant Phenolic Plant Ha(2010)

al fungi species compound protection
degradation

3. Navisporum Wood Wood decay Nutrients Phillips et al.

floccosus mobilization (2012)

4. Perisporiopsis Leaves of Leaves decay Nutrients Chaverri and

lateritia Hevea sp. mobilization Gazis(2010)

5. Phanerochaete Wood Decompose Phosphorus Wells et al.

velutina wood translocation (1998)

Table 2.2
Microorganisms Products Agriculture application

1. Fusarium Gibberellins Plant growth hormone

moniliforme

2. Fusarium Zearalenone Growth promoter in cattle

graminearum

3. Chondrostereum Chontral Control of hardwood

purpureum

4. Phanerochaete Rotstop Control of butt rot of conifers

gigantea

30
Ecological plant-microbe interactions
The microbes and plants alone regulate several soil processes as well as the carbon cycle and
nutrient utilization. Plant diversity and abundance might modify the complete soil scheme
through the discharge of root exudates that attract or inhibit the expansion of specific
organisms.

Economic advantage of fungi

The saprophytic fungi of decay maintain the never-ending cycle of greenhouse emission that
could be the most significant staple for plant chemical processes in nature. They additionally
cause rot, decay, and decomposition of animal and plant remains emotional plant nutrients in
an exceedingly type offered to inexperienced.

There are types of fungi that serve to suppress fungi inflicting the sickness disease of the
seedlings and thereby influence favorably the expansion of crops.

Some fungi like Empusa sepulchralis, Metarhizium anisopliae, and Cordyceps melodic can
be used to control some insect pests. Others parasitic to some insects particularly, some
spore-forming ones. The fungi spores sprayed on the crop cuss to regulate them. Colorado
potato beetles, citrus rust mites, and spittle-bugs of insect cuss that may be controlled by
exploitation fungi. These types of fungi form loops on their mycelium which traps and
strangle nematodes as they attempt to pass through. They later absorb nutrition from the
nematodes.

31
CHAPTER-3

Aim and objective

The present Experiment were designed to achieve the following objective

1. Isolated, Identification, Purification of Aspergillus niger from soil using Soil Plate
Method.
2. To prepare solvent extract of Azadirachta indica.
3. To test anti-fungal activities of Azadirachta indica.

Materials and methods

Materials

Apparatus:-
1. Incubator
2. Laminar air flow
3. Bunsen burner
4. Hot air oven
5. Autoclave
6. Refrigerator
7. Electronic balance
8. Rotator
9. Centrifuge

Glassware:-
1. Beaker
2. Test tube
3. Conical flask
4. Petri plates
5. Test tube stand
6. Cork borer
7. Micropipette
8. Measuring cylinder
9. Tip box
10. Inoculating loop

32
Methods
Isolation of fungi using soil plate methods
This method, adapted from the direct-inoculation technique of Waksman, 1960,was designed
to study the ecological distribution of various species of fungi in soil. The soil-plate method
permits isolation of fungi embedded in or attached to mineral particles that might be
discarded in the residue when dilution plates are prepared. Spore masses tend to remain more
intact in soil plates than in dilution plates, thereby favoring more valid data on the
distribution of fungi in soil.

Many limitations are common to both methods and they present essentially the same
ecological picture of the fungal flora of the soil, but a greater number of species of fungi are
usually isolated on soil plates than on dilution plates. Several Basidiomycetes occasionally
appear on soil plates. Root pathogen and medium to fast growing fungi present in soil in
relatively low numbers appear more often on soil plates than on dilution plates. The
procedure is easier to follow, since it is less time consuming and requires fewer laboratory
materials than the dilution-plate method.
The amount of soil used to prepare a soil plate with the soil investigated, and is determined
by trial. With many natural surface soils about 0.005-0.015 g results in a convenient number
of colonies in each dish. A microspatula, made by flattening the ends of a nichrome needle, is
useful for transferring the microsample to petri dishes and for crushing soil aggregates. There
is a small variation in weight among transfers from the same soil sample.
8-10ml of melted and cooled agar medium is added, and the soil particles are dispersed
throughout the agar. With sandy soils, adequate dispersal may be obtained by general shaking
and rotating the plate before the agar solidifies. If the soil is very dry, or contains a high
proportion of clay, it is preferable to mix the soil particles with a drop of sterile water in the
dish before the medium is added.
It is suggested that for purposes of studying the ecological distribution of fungi in soil,
presence or absence of species in each soil plate be recorded, without regard to the number of
colonies per plate.

1. All the procedures should be carried out aseptically in the laminar air flow chamber.
2. 5 mg, 10 mg and 15 mg of soil samples are taken.
3. The soil samples and PDA were mixed thoroughly by simply rotating the petri dishes
containing PDA medium.
4. The petri dishes were incubated at 25 degree celsius for 6 to 8 days.
5. The petri dishes were observed at an interval of 2 days for fungal growth.

33
Solvent extraction method
Liquid–liquid extraction (LLE), also known as solvent extraction and partitioning, is a
method to separate compounds or metal complexes, based on their relative solubilities in two
different immiscible liquids, usually water (polar) and an organic solvent (non-polar). There
is a net transfer of one or more species from one liquid into another liquid phase, generally
from aqueous to organic. The transfer is driven by chemical potential, i.e. once the transfer is
complete, the overall system of chemical components that make up the solutes and the
solvents are in a more stable configuration (lower free energy). The solvent that is enriched in
solute(s) is called extract. The feed solution that is depleted in solute(s) is called the raffinate.
LLE is a basic technique in chemical laboratories, where it is performed using a variety of
apparatus, from separatory funnels to countercurrent distribution equipment called as mixer
settlers.[not verified in body] This type of process is commonly performed after a chemical
reaction as part of the work-up, often including an acidic work-up.
The term partitioning is commonly used to refer to the underlying chemical and physical
processes involved in liquid–liquid extraction, but on another reading may be fully
synonymous with it. The term solvent extraction can also refer to the separation of a
substance from a mixture by preferentially dissolving that substance in a suitable solvent. In
that case, a soluble compound is separated from an insoluble compound or a complex matrix.

Inhibition zone
The Zone of inhibition is a circular area around the spot of the antibiotic in which the
microbial colonies do not grow. The zone of inhibition can be used to measure the
susceptibility of the microbe towards antibiotics. The process of measuring the diameter of
this Zone of Inhibition can be automated using Image processing. In this work an algorithm is
developed, using Computer Vision, which will detect the zones of inhibition of the
microorganisms. This work demonstrates an effective approach of measuring the Zone of
Inhibition by calculating the radius of the zone by drawing contours and setting the right
value of threshold. This work also determines if a particular microbe is susceptible or
resistant to the applied antibiotic using the calculated Zone of Inhibition and the prescribed
standard values.

34
Figure 12

Potato Dextrose Agar (PDA)

Potato Dextrose Agar (PDA) is used for the cultivation of fungi. Potato Dextrose Agar (PDA)
is a general purpose medium for yeasts and molds that can be supplemented with acid or
antibiotics to inhibit bacterial growth. It is recommended for plate count methods for foods,
dairy products and testing cosmetics. PDA can be used for growing clinically significant
yeast and molds. The nutritionally rich base (potato infusion) encourages mold sporulation
and pigment production in some dermatophytes.

Principle of Potato Dextrose Agar (PDA)

Potato Dextrose Agar is composed of dehydrated Potato Infusion and Dextrose that
encourage luxuriant fungal growth. Agar is added as the solidifying agent. Many standard
procedures use a specified amount of sterile tartaric acid (10%) to lower the pH of this
medium to 3.5 +/- 0.1, inhibiting bacterial growth. Chloramphenicol acts as a selective agent
to inhibit bacterial overgrowth of competing microorganisms from mixed specimens, while
permitting the selective isolation of fungi.

Uses of potato dextrose agar

Potato Dextrose Agar is used for the detection of yeasts and molds in dairy products
and prepared foods.
It may also be used for the cultivation of yeasts and molds from clinical specimens.
Potato Dextrose Agar with TA (Tartaric Acid) is recommended for the microbial
examination of food and dairy products.
Potato Dextrose Agar with Chlortetracycline is recommended for the microbial
enumeration of yeast and mold from cosmetics.
Potato Dextrose Agar with Chloramphenicol is recommended for the selective
cultivation of fungi from mixed samples.

35
Figure 13: Potato Dextrose Agar

Purification
Purification is often the First step in any microbial laboratory study and is a component of
most undergraduate microbiology curriculum, furthermore, learning to isolate purified
colonies requires using expensive and biohazardous material, physical laboratory space and
specialized equipment.
Steps:-
1. With the help of an inoculating needle.
2. Take some material from the edge of an actively growing fungal colony.
3. Inoculate it on another media plate.
4. Inoculate it for 2-3 days.
5. Pure culture of the organism will appear.

Figure 14: axenic culture of the fungi (test fungi) i.e Aspergillus niger

Identification of microflora
Reasons:-
Since serial dilution plate method in case of fungi favours the strains which produce abundant
spores, each composite sample was analyzed by Soil Plate Method(Warcup, 1950).
Method
For this, 5 mg of a given composite sample and PDA were used, the soil samples and PDA
were mixed thoroughly by simply rotating the Petri Plates. The Petri-dishes were incubated at

36
25+_1°C for 6-8 days. The fungal strains were transferred to the petri plates containing fresh
medium to facilitate identification and for preparing axenic culture.
PDA making procedure:-
● PDA medium was used for isolation of fungi from each composite soil sample.
● Preparing 1L of PDA, 20 gram of peeled and sliced potatoes were boiled in 500ml of
distilled water.
● The broth was filtered through a plastic sieve and the filtrate was made upto 500ml
with distilled water.
● At the same time, 20 gram of agar-2 were melted in 500 ml of distilled water by
heating.
● The potato broth and agar solution were mixed together.
● 20 gram of dextrose were also added and dissolved.
● The pH of the mixture was adjusted to 6-7 and 0.03 gram of rose bengal were added
to the medium.
● The total volume of the medium was made upto 1000 ml using distilled water.
● Then autoclave at 15lb pressure (121°C) for 15 minutes.
● Sterilized the Petri dishes and glassware in a hot air oven at 160°C for 3 hours before
use.

Plant material extraction

Preparation of plant leaf powder

The leaves of Azadirachta indica were collected and thoroughly washed with tap water and
then rinsed with sterile distilled water. The leaf of Azadirachta indica leaves are washed ,
dried and grind in an electric mixer. The powder material of Azadirachta indica was kept in
airtight glass bottles.

Figure 15: Plant material extract

37
Ten gram of air dried powder was placed in 100ml of organic solvent (methanol) in a conical
flask plugged with cotton and then kept on a rotatory shaker at 190 to 220rpm for 24hrs . It
was filtered through 8 layers of muslin cloth and centrifuged at 5000rpm for 15 min. The
supernatant was collected.
Extract was then stored at room temperature before weighing geometrically to determine the
yields after that prepared various dilution via 10%, 20%, 30%, 40%and 50% concentration of
extracts for inhibition of growth of fungal species. Control treatment was done without any
plant extract in petri plates.

Well in Agar method

A loopful of the inoculum suspension of pure culture identified fungal organism was spread
uniformly on the solidified sterile culture media (PDA) in the petri plates for uniform
distribution of organism.
Using a sterile cork borer a well of 0.5cm was made in the media and in each well, plant
extract was filled so, as to follow the diffusion of plant extract in the media. The petri plate
was incubated for 24 hrs at 30 degree celcius temperature and the observations were recorded
as diameter of inhibitory zone in mm. Well in agar plate were sterile distilled water was used
as control in all the experiments.

Figure 16: Azadirachta indica extract

38
CHAPTER-4
Observation
Table No. 4.1
Effect of Azadirachta indica Extract using 10% concentration

39
S. No Time (in days) Radial growth of
the test fungus(in
mm)

1. 0 0.5

2. 2 1.3

3. 4 4.0

4. 6 6.5

5. 8 8.0

40
 Table No. 4.2
Effect of Azadirachta indica Extract using 20% concentration

41
S. No Time(in days) Radial growth of
the test fungus(in
mm)

1. 0 0.5

2. 2 1.2

3. 4 9.0

4. 6 9.0

5. 8 9.0

42
Table No. 4.3
Effect of Azadirachta indica Extract using 30% concentration

43
S. No Time ( in days) Radial growth of
the test fungus ( in
mm)

1. 0 0.5

2. 2 1.0

3. 4 3.0

4. 6 5.0

5. 8 6.0

44
Table No. 4.4
Effect of Azadirachta indica Extract using 40% concentration

45
S. No Time ( in days) Radial growth of
the test fungus( in
mm)

1. 0 0.5

2. 2 1.1

3. 4 2.5

4. 6 4.5

5. 8 5.0

46
Table No. 4.5
Effect of Azadirachta indica Extract using 50% concentration

47
S. No Time(in Radial growth of the
days) test fungus ( in mm)

1. 0 0.5

2. 2 1.0

3. 4 2.0

4. 6 4.0

5. 8 3.0

48
CHAPTER-5
Result and discussion
Herbal medicines already form the basis of therapeutics used in the developing countries but
in recent time, there has been an increase in the use of herbal medicines in the developing
world (De and Ifeoma, 2002). Many medicinal plants possess the ability to prevent bacterial
and fungal growth. Plants possess certain chemical entities which have the ability to modify
the physiological function of a cell and hence act as anti-bacterial and anti-fungal drugs to
prevent the bacterial and fungal growth.
The phytochemicals components of the Azadirachta indica has been established in previous
studies and these include Alkaloids, Carbohydrates, Glycosidases, Flavonoids, Saponins,
Tennins and Triterpenes (Biswas et al 2002).
The objective of the current study is to analyse the anti-fungal effect of Azadirachta indica. In
this study we screened the plant Azadirachta indica for its anti-fungal properties. The dried
powdered plants were extracted using different organic solvent by using the solvent
extraction method.
In the recent study we can find out that all plants show anti-fungal activities against
Azadirachta indica species. Maximum inhibition percentage was recorded in Azadirachta
indica, Eucalyptus camaldulensis and Crotalaria juncea with 95%,90% and 87% respectively,
Suppressing the mycelium growth of Aspergillus niger.
As compared to bacteria, the methanol extract of Azadirachta indica did not show any
anti-bacterial activities against M. luteus at the concentration of 100ugl/ml (0.45cm),
methanol shows anti-fungal activities against Azadirachta indica (Azadirachta indica) at the
concentration of 10%,20%,30%,40% and 50% and inhibition zone are 8mm in 10%, 9mm in
20%, 6mm in 30%, 5mm in 40%, 3mm in 50% concentration respectively.
The current result demonstrate that selected medicinal plant (Azadirachta indica) extract
effectively suppress the radial growth of all species and these findings confirm the result of
Satish et al. (2007) ; Bobbarala et al. (2009) ; Gujar and Tulwankar (2012).

49
CHAPTER-6

Conclusion
The present study revealed that the Azadirachta indica could effectively inhibit the growth of
fungal culture. Solvent extract was most effective against fungus where methanol showed
low anti-fungal activities.
In summary this study shows that the extract of Azadirachta indica in solvent possesses
effective anti-fungal activities which are exhibited on various fungal strains. Hence the
further purification of the crude extract may lead to a potent anti-fungal compound.

50
CHAPTER-7

Reference
● Therapeutics Role of Azadirachta indica (Azadirachta indica) and Their Active
Constituents in Diseases Prevention and Treatment.

● Azadirachta indica A.Juss". Plants of the World Online. Board of Trustees of the
Royal Botanic Gardens, Kew. 2017. Retrieved 19 November 2020.

● "Azadirachta indica". Germplasm Resources Information Network (GRIN).

Agricultural Research Service (ARS), United States Department of Agriculture
(USDA). Retrieved 9 June 2017.

● The Tree. National Academies Press (US). 1992.

● Compact Oxford English Dictionary (2013), Neem, page 679, Third Edition 2008
reprinted with corrections 2013, Oxford University Press.

● Abdel-Moneim et al., 2014

○ A.E. Abdel-Moneim, M.S. Othman, A.M. Aref
○ Azadirachta indica attenuates cisplatin-induced nephrotoxicity and oxidative
stress
○ BioMed Research International, 2014 (2014), pp. 1-19, 10.1155/2014/647131
○ View Record in ScopusGoogle Scholar
○ Abdelbaset-Ismail et al., 2016.

● A. Abdelbaset-Ismail, D. Pedziwiatr, E. Suszyńska, S. Słuczanowska-Głąbowska, G.

Schneider, S.S. Kakar, M.Z. Ratajczak
○ Vitamin D3 stimulates embryonic stem cells but inhibits migration and growth
of ovarian cancer and teratocarcinoma cell lines
○ Journal of Ovarian Research, 9 (1) (2016), pp. 1-12,
10.1186/s13048-016-0235-x
○ Google Scholar
○ Akter et al., 2013

● R. Akter, M. Mahabub-Uz-Zaman, S. Rahman, M. Afroza Khatun, A.M. Abdullah,

N.U. Ahmed, F. Islam
○ Comparative studies on antidiabetic effect with phytochemical screening of
Azadirachta indica and Andrographis paniculata

51
 ○ IOSR Journal of Pharmacy and Biological Sciences, 5 (2) (2013), pp. 122-128
○ CrossRefView Record in ScopusGoogle Scholar.

● Farjana et al., 2014

○ A. Farjana, N. Zerin, M.S. Kabir
○ Antimicrobial activity of medicinal plant leaf extracts against pathogenic
bacteria
○ Asian Pacific Journal of Tropical Disease, 4 (S2) (2014), pp. S920-S923,
10.1016/S2222-1808(14)60758-1
○ ArticleDownload PDFView Record in ScopusGoogle Scholar

● Forum and Fernandes Da, 2017

○ Forim, M. R., Fernandes Da, S. M. F. D. G., Fernandes, J. B., & Vieira, P. C.
(2017). Process for obtaining biopolymeric nanoparticles containing
Azadirachta indica A. Juss. (neem.) oil and extracts, biopolymeric
nanoparticles, and powder microparticles (Patent No. US9668473 B2).
USPTO.
○ Google Scholar

● Gautam et al., 2015

○ M.K. Gautam, M. Gangwar, S.K. Singh, R.K. Goel
○ Effects of Azadirachta indica on vascular endothelial growth factor and
cytokines in diabetic deep wound
○ Planta Medica, 81 (9) (2015), pp. 713-721, 10.1055/s-0035-1545917
○ View Record in ScopusGoogle Scholar

● Gautam et al., 2013

○ M.K. Gautam, R.R. Ghatule, A. Singh, V. Purohit, M. Gangwar, M. Kumar,
R.K. Goel
○ Healing effects of Aegle marmelos (L.) Correa fruit extract on experimental
colitis
○ Indian Journal of Experimental Biology, 51 (2) (2013), pp. 157-164
○ View Record in ScopusGoogle Scholar

● Gaule et al., 2012

○ R.R. Ghatule, G. Shalini, M.K. Gautam, A. Singh, V.K. Joshi, R.K. Goel
○ Effect of Azadirachta indica leaves extract on acetic acid-induced colitis in
rats: Role of antioxidants, free radicals and myeloperoxidase
○ Asian Pacific Journal of Tropical Disease, 2 (SUPPL2) (2012), pp.
S651-S657, 10.1016/S2222-1808(12)60238-2
○ ArticleDownload PDFView Record in ScopusGoogle Scholar

52
● Godmode et al., 2013
○ W.N. Ghonmode, O.D. Balsaraf, V.H. Tambe, K.P. Saujanya, A.K. Patil, D.D.
Kakde
○ Comparison of the antibacterial efficiency of neem leaf extracts, grape seed
extracts and 3% sodium hypochlorite against E. faecalis – An in vitro study
○ Journal of International Oral Health : JIOH, 5 (6) (2013), pp. 61-66
○ View Record in ScopusGoogle Scholar

● Ghosh, Bose, Haque, & Baral, 2009

○ D. Ghosh, A. Bose, E. Haque, R. Baral
○ Neem (azadirachta indica) leaf preparation prevents leukocyte apoptosis
mediated by cisplatin plus 5-fluorouracil treatment in swiss mice
○ Chemotherapy, 55 (3) (2009), pp. 137-144, 10.1159/000211558
○ CrossRefView Record in ScopusGoogle Scholar

● Gladkevich et al., 2007

○ A. Gladkevich, F. Bosker, J. Korf, K. Yenkoyan, H. Vahradyan, M. Aghajanov
○ Proline-rich polypeptides in Alzheimer’s disease and neurodegenerative
disorders – Therapeutic potential or a mirage?
○ Progress in Neuro-Psychopharmacology and Biological Psychiatry (2007),
10.1016/j.pnpbp.2007.06.005
○ Google Scholar

● GLOBE NEWSWIRE, 2019

○ GLOBE NEWSWIRE (2019). Pest Control Market Projected to Reach $27.5
Billion by 2025 – Report by MarketsandMarkets. MarketsandMarkets.
https://www.globenewswire.com/news-release/2019/10/07/1925966/0/en/Pest-
Control-Market-Projected-to-Reach-27-5-Billion-by-2025-Report-by-Markets
andMarkets.html.
○ Google Scholar

● Gupta et al., 2019

○ Gupta, A., Ansari, S., Gupta, S., & Narwani, M. (2019). Therapeutics role of
neem and its bioactive constituents in disease prevention and treatment 8(3),
680–691.
○ Google Scholar

● Hao et al., 2014

○ F. Hao, S. Kumar, N. Yadav, D. Chandra
○ Neem components as potential agents for cancer prevention and treatment
○ Biochimica et Biophysica Acta – Reviews on Cancer, 1846 (1) (2014), pp.
247-257, 10.1016/j.bbcan.2014.07.002

53
 ○ ArticleDownload PDFView Record in ScopusGoogle Scholar.

● https://www.intechopen.com/chapters/69577

● Muthuraman Yuvaraj and Murugaragavan Ramasamy (May 13th 2020). Role of

Fungi in Agriculture, Biostimulants in Plant Science, Seyed Mahyar Mirmajlessi and
Ramalingam Radhakrishnan, IntechOpen, DOI: 10.5772/intechopen.89718. Available
from: https://www.intechopen.com/chapters/69577
● Abbasi PA, Riga E, Conn KL, Lazarovits G. Effect of neem cake soil amendment on
reduction of damping-off severity and population densities of plant-parasitic
nematodes and soil-borne plant pathogens. Can. J Plant Pathol. 2005; 27:38-45.

● . Aboellil AH. Trilogy, a product of neem (Azadirachta indica) induces resistance in

cucumber against Podosphaera xanthii. Res. J Microbiol. 2007; 2:402-414.

● Adepoju AO, Ogunkunle ATJ, Femi-Adepoju AG. Antifungal activities of seed oil of
neem (Azadirachta indica A. Juss.). Global J Biol. Agric. Health Sci. 2014;
3(1):106-109.

● Al-Hazmi RHM. Effect of neem (Azadirachta indica) leaves and seeds extract on the
growth of six of the plant disease causing fungi. Global Adv. Res. J Microbiol. 2013;
2(5):89-98.

● Alzohairy MA. Therapeutics role of Azadirachta indica (neem) and their active
constituents in disease prevention and treatment. Evid. Based Complement.
Alternate. Med., 2016, 11. Article ID 7382506,
http://dx.doi.org/10.1155/2016/7382506.

● Amadioha AC. Controlling rice blast in vitro and in vivo with extracts of Azadirachta
indica. Crop Protect. 2000; 19(5):287-290.

● Amadioha AC. Fungitoxic effects of some leaf extracts against Rhizopus oryzae
causing tuber root of potato. Arch. Phytopath. Plant Protect. 2001;
33(6):499-507.

● Amadioha AC. Fungitoxic effects of extracts of Azadirachta indica against

Cochliobolus miyabeanus causing brown spot disease of rice. Arch. Phytopath.
Plant Protect. 2002; 35(1):37-42.

● Anjali Kumari, Ritesh K, Sudarshan M, Jaipal SC, Kumar S. Antifungal efficacy of

aqueous extract of neem cake, karanj cake and vermicompost against some
phytopathogenic fungi. The Bioscan. 2013; 8:671-674.

54
● Arumugam PA, Mohamad I, Salim R, Mohamed Z. Antifungal effect of Malaysian
neem leaf extract on selected fungal species causing otomycosis in in-vitro
culture medium. Malaysian J Med. Health Sci. 2015; 11(2):69-84.

● Ashfaq Ahmed M, Gayathri R, Vishnupriya V. Anti-fungal activity of neem oil. Int. J

Pharm. Sci. Rev. Res. 2016; 39(1):200-202.

● "SSRL Publications & Reports | Stanford Synchrotron Radiation Lightsource" (PDF).

Archived from the original (PDF) on March 9, 2008. Retrieved January 21, 2006.

● ^ pnj rose. "Solvent Extraction Notes".

● ^ "7.7: Liquid–Liquid Extractions". Chemistry LibreTexts. 2013-10-25. Retrieved

2017-03-26.

● ^http://courses.chem.psu.edu/chem36/Experiments/PDF's_for_techniques/Liquid_Liq
uid.pdf.

● ^"Basic Technology and Tools in Chemical Engineering Field - S. Wesley -

Documents".

● ^"Archived copy" (PDF). Archived from the original (PDF) on 2015-09-29. Retrieved
2015-09-28.

● ^ a b Rezaee, Mohammad; Assadi, Yaghoub; Milani Hosseini, Mohammad-Reza;

Aghaee, Elham; Ahmadi, Fardin; Berjani, Sana (2006). "Determination of organic
compounds in water using dispersive liquid–liquid microextraction". Journal of
Chromatography A. 1116 (1–2): 1–9. doi:10.1016/j.chroma.2006.03.007.
ISSN 0021-9673. PMID 16574135.

● (PDF) Measurement of the Zone of Inhibition of an Antibiotic.

● anti-fungal effect of malaysian neem leaf extract... - Google Scholar.

● https://microbiologyinfo.com/potato-dextrose-agar-pda-principle-uses-compositi
on-procedure-and-colony-characteristics/

● aspergillus niger on pda - Google Search

○ black mold (Aspergillus niger ) on Potato dextrose agar (Fungal
Non-selective Media PDA ) - 5473758

55
56