CONTENTS

INTRODUCTION:

1. OVERVIEW OF BIOTECH PARK.

2. INTRODUCTION OF MICROBIOLOGY.

3. WHAT ARE BACTERIA?

4. WHAT ARE FUNGI?

5. BASIC RULES OF MICROBIOLOGY LABORATORY

6. REQUIREMENT OF MICROBIOLOGY LABORATORY

7. CLEANING OF GLASSWARES

8. PREPARATION OF STERILISATION

9. MEDIA PREPARATIONS

10. PREPARATION OF PDA MEDIA & PDA PLATES

11. SOIL BORNE DISEASES IN HORTICULTURE PLANTS CAUSED BY

FUSARIUM

12. GINGER, POTATO, TOMATO AND ITS DISEASES.

13. FUSARIUM

14. TRICHODERMA

15. ASPERGILLUS NIGER

16. ESSENTIAL OILS

17. LEMON GRASS

18. REVIEW OF LITERATURE

19. COMMON METHOD OF MEDIA PREPARATION

20. COMMON METHOD OF PREPARATION OF PURE CULTURE

EXPERIMENTS:

1. PREPARATION OF PURE CULTURE OF TRICHODERMA AND

ASPERGILLUS NIGER

2. EXTRACTION OF ESSENTIAL OIL FROM THUJA

3. ANTIFUNGAL ACTIVITY OF ESSENTIAL OIL OF THUJA AGAINST

ASPERGILLUS NIGER

4. EXTRACTION OF ESSENTIAL OIL FROM LEMON GRASS

5. SOIL ANALYSIS OF SOIL FOUND NEAR LEMON GRASS

6. ANTIFUNGAL ACTIVITY OF ESSENTIAL OIL OF LEMON GRASS

AGAINST ASPERGILLUS NIGER

7. OIL ANALYSIS OF ESSENTIAL OIL OBTAINED FROM LEMON GRASS

APPARATUS:

1. ANALYTICAL BALANCE

2. LAMINAR AIR FLOW

3. AUTOCLAVE

4. HOT AIR OVEN

5. MILLIPOLE WATER PURIFICATION SYSTEM

6. BOD INCUBATOR

7. BIOSAFETY CABINET

8. HOT PLATE

9. VORTEX MIXTURE

10. pH METER

11. CLEVENGER APPARATUS

12. POLARIMETER

13. WATER BATH

REFERENCES
 OVER VIEW OF BIOTECH PARK

Biotech Park, Lucknow

The State of Uttar Pradesh, a vital hub of scientific activities endowed with rich
biological resources and biodiversity has the unique distinction of having a number
of research institutions, which have expertise and capabilities in biotechnology. In
view of this, Lucknow was declared as the Biotechnology City of India during the
89th session of the Indian Science Congress on January 3, 2002. These institutions
are providing a great impetus and support in the development of Biotech Park.

THE BIOTECH PARK

The Biotech Park has been set up on 8 acres of land provided by the Department of
Science and Technology, Government of Uttar Pradesh. The thrust areas identified
for the initial phase of the Park are:

• Health Care

• Agriculture

• Environment
Biotech Park is divided into following blocks:

BLOCK I: BIO-BUSINESS BLOCK

The Bio-Business Block houses the following:

• Business Support Facilities

• Bioinformatics Centre

• Conference Hall

• Cafeteria

• Laboratory Space

BIOINFORMATICS CENTRE

A Bioinformatics Centre has been setup to establish

a close network with various institutions and
provide information to industries regarding
technological advancements.

BLOCK II: SOLVENT EXTRACTION BLOCK

The extraction unit comprises of solid liquid solvent extraction system with solvent
recovery system for extraction of
Photochemical/Lead molecules from high value
medicinal plants and multipurpose reaction cum
hydrolysis and solvent recovery unit along with
chromatography.

Extraction unit has the following infrastructural

facilities:

Oil fired steam boiler, evaporation capacity: 500

kg/hr and 150 psi steam pressure for basic heating requirements to main extraction
unit.
Hot air tray dryer, High capacity hammer mill and vacuum oven for drying,
grinding/pulverizing of raw materials/finished herbs/medicinal plants

Refrigerated brine chilling circulation unit for carrying out reactions and chilling
of condenser water.

The solid liquid extraction unit consists of two drug holders with agitators of 125 -
150 kg/batch capacity depending on plant bulk density with efficient solvent
recovery system from extract and spent marc.

Silica gel chromatography column is for chromatographic separation over silica gel
with solvent recycling system, for enriching and isolation of the photochemical in
high purity. Stainless steel reaction vessel with agitator and solvent reflux/recovery
system is for production of semi synthetic drug molecules and for chemical
transformations of the photochemical.

Falling film evaporator is for concentrating herbal/aqueous extracts for separation

of the extracts from the plant biomass.

Minor equipments like vacuum pumps, metering pumps, weighing machine,

weighing balance, trolleys etc for auxiliary infrastructure, process and material
handling etc.

Capacity: 250 kg biomass/batch

Block III: Bio-fertilizer, Tissue Culture & Centre

Support Block

Bio-fertilizer unit – Ground floor

Plant Tissue culture unit – First floor

Molecular and Analytical laboratory – Second

floor

BIOFERTILIZER UNIT

This unit has facilities for perfecting the technology and production of bacterial
fertilizers and pesticides, phosphate solubilizing bacteria (PSB), Azotobacter unit
(with a capacity of producing about 240 tones/annum biofertilizer), Trichoderma
unit (with a capacity of producing about 500 tones/annum biofertilizer).

TISSUE CULTURE AND HARDENING FACILITY

Tissue culture facility at Biotech Park, Lucknow is spread over 2000 sq ft. Area that
possess the capacity to raise and multiply banana, potato, Jatropha seedlings. The
culture media used in the unit is completely biodegradable and non-toxic.
ADVANTAGES OF PROPAGATION BY TISSUE CULTURE

• The elimination of diseases and the production of disease free plantlets.

• The rapid production of large numbers of genetically identical plantlets.

• Introduction of new varieties and or genotypes.

• Preservation of germplasm.

• Production of haploid plants which can be used for plant breeding.

CAPACITY:

10000 to 100000 plants / batch and it can produce about 2 million plants/ annum

MOLECULAR BIOLOGY AND ANALYTICAL FACILITY

The facility is equipped with High Performance Liquid Chromatography (HPLC),

Gas chromatography (GC), High Performance Thin Layer Chromatography
(HPTLC), Atomic absorption spectrophotometer (AAS), Nanodrop-
spectrophotometer, Gel Electrophoresis, Elisa, Polari meter, and other supportive
equipments.

DISTILLATION UNIT

The distillation unit has been set up for obtaining essential oil
from aromatic plants such as Mentha arvensis, Mentha piperata,
Mentha cardiaca, Lemon grass, Palmarosa, Citronella, Basil,
Vetiver, and Geranium etc. The recovery of essential oil from
different aromatic plants has been found to be relatively high as
compared to conventional distillation unit used by the farmers at
their site. About 1000 kg fresh herbs per batch can be distilled in
the unit. They are quantitatively analyzed using HPLC and
HPTLC.
VERMICOMPOSTING UNIT

This is the fastest and effective way of recycling of organic

waste with the help of earthworms for the production of
useful compost. To utilize the large quantity of agro-waste
generated from distillation and solvent extraction units the
vermin-composting unit has been set up at the Park. The
unit will serve as demonstration-cum-training facility for
the farmers.

FACILITIES BEING OPERATED UNDER PUBLIC-PRIVATE PARTNERSHIP

MODE

Distillation and vermin-composting unit, biofertilizer unit and tissue culture and
hardening facilities are being run under public-private partnership mode as given
below:

• Distillation and vermicomposting units.

• Hindustan Bio-energy Pvt. Ltd.

• Bio-fertilizer Unit

• Hindustan Bio-energy Pvt. Ltd.

• Tissue culture and hardening facility.

ENTREPRENEURS IN THE PARK

The biotech park in turn provides incubator facilities to these institutes and
entrepreneurs to perfect the facility, up scale the products and even produce small
quantity for testing and quality control-

1. AquaBioChip Genomics (India) Pvt. Ltd., Lucknow

2. Chandan Health Care Ltd., Lucknow

3. Clintech Research India Pvt. Ltd., Lucknow

4. Cognate Bioservices Inc., USA

5. Deva Biofuels, Indore

6. Environmental Biotech Pvt. Ltd., Indore

7. HH Biotechnologies Pvt. Ltd

8. IQRA Biotech Services, Lucknow

9. Life Care Innovations Pvt. Ltd., Gurgaon

10. Sheel Biotech Ltd., Manesar

11. Software Technology Parks of India, Lucknow

12. Sepragen Corporation, Haryard, USA

INTRODUCTION OF MICROBIOLOGY
Micro mean very small and biology is the study of living things, so microbiology is the
study of very small living things normally too small not to be seen with the naked eye.

Microbiology (from Greek μῑκρος, mīkros, "small"; βίος, bios, "life"; and -λογία,
-logia) is the study of microorganisms, which are unicellular or cell-cluster microscopic
organisms. This includes eukaryotes such as fungi and protists, and prokaryotes.
Viruses, though not strictly classed as living organisms, are also studied. In short;
microbiology refers to the study of life and organisms that are too small to be seen with
the naked eye. Microbiology typically includes the study of the immune system, or
Immunology. Generally, immune systems interact with pathogenic microbes; these two
disciplines often intersect which is why many colleges offer a paired degree such as
"Microbiology and Immunology".

Microbiology is a broad term which includes virology, mycology, parasitology,

bacteriology and other branches. A microbiologist is a specialist in microbiology.

Microbiology is researched actively, and the field is advancing continually. It is

estimated only about one percent of all of the microbe species on Earth have been
studied. Although microbes were directly observed over three hundred years ago, the
field of microbiology can be said to be in its infancy relative to older biological
disciplines such as zoology and botany.

History

Ancient

The existence of microorganisms was hypothesized for many centuries before their
actual discovery. The Roman Marcus Terentius Varro made the first extant reference
to microbes when he warned against locating a homestead in the vicinity of swamps
"because there are bred certain minute creatures which cannot be seen by the eyes,
which float in the air and enter the body through the mouth and nose and there cause
serious diseases.

In 1546 Girolamo Fracastoro proposed that epidemic diseases were caused by

transferable seed like entities that could transmit infection by direct or indirect contact
or even without contact over long distances.
However, early claims about the existence of microorganisms were speculative, and not
based on any data or observation. Actual observation and discovery of microbes had to
await the invention of the microscope in the 17th century.

Modern
Antonie van Leeuwenhoek, known as the "father of microbiology", was the first to
observe microorganisms using a microscope. In 1676, Leeuwenhoek first observed
bacteria and other microorganisms, using a single-lens microscope of his own design.
While Van Leeuwenhoek is often cited as the first microbiologist, Robert Hooke made
the first recorded microbiological observation, of the fruiting bodies of molds, in 1665.

The field of bacteriology (later a sub discipline of microbiology) was founded in the 19th
century by Ferdinand Cohn, a botanist whose studies on algae and photosynthetic
bacteria led him to describe several bacteria including Bacillus and Beggiatoa. Cohn
was also the first to formulate a scheme for the taxonomic classification of bacteria.
Louis Pasteur and Robert Koch were contemporaries of Cohn’s and are often
considered to be the founders of medical microbiology. Pasteur is most famous for his
series of experiments designed to disprove the then widely held theory of spontaneous
generation, thereby solidifying microbiology’s identity as a biological science. Pasteur
also designed methods for food preservation (pasteurization) and vaccines against
several diseases such as anthrax, fowl cholera and rabies. Koch is best known for his
contributions to the germ theory of disease, proving that specific diseases were caused
by specific pathogenic microorganisms. He developed a series of criteria that have
become known as the Koch's postulates. Koch was one of the first scientists to focus on
the isolation of bacteria in pure culture resulting in his description of several novel
bacteria including Mycobacterium tuberculosis, the causative agent of tuberculosis.

While Pasteur and Koch are often considered the founders of microbiology, their work
did not accurately reflect the true diversity of the microbial world because of their
exclusive focus on microorganisms having direct medical relevance. It was not until the
late 19th century and the work of Martinus Beijerinck and Sergei Winogradsky, the
founders of general microbiology (an older term encompassing aspects of microbial
physiology, diversity and ecology), that the true breadth of microbiology was revealed.
Beijerinck made two major contributions to microbiology: the discovery of viruses and
the development of enrichment culture techniques. While his work on the Tobacco
mosaic virus established the basic principles of virology, it was his development of
enrichment culturing that had the most immediate impact on microbiology by allowing
for the cultivation of a wide range of microbes with wildly different physiologies.
Winogradsky was the first to develop the concept of Chemolithotrophy and to thereby
reveal the essential role played by microorganisms in geochemical processes. He was
responsible for the first isolation and description of both nitrifying and nitrogen-fixing
bacteria
The scope and Relevance of Microbiology

In the 20th century microbiology contributed greatly to the field of biochemistry

and genetics. It also helped stimulate the rise of molecular biology. There is wide
variety of fields in microbiology and many have a great impact on society. Include
the more applied discipline such as medical, public health, industry, food and dairy
microbiology. Microbiology, ecology, physiology, biochemistry and genetics are
examples of basic microbiology research fields.

The Future of Microbiology

Microbiologist will be faced with many exciting and important future challenges
such as finding new ways to combat disease, reduce pollution, and feed the World’s
populations. As the study of gene structure, the control of diseases and the industrial
processes based on the phenomenal ability of microorganism to decompose and
synthesized complex organic molecules. Microbiology is one of the most rewarding
of profession because it gives its practitioners the opportunity to be in contact with
all the other natural sciences and thus to contribute in many different ways to the
betterment of human life.

Types of Microbiology

Medical Microbiology:

It includes the study of the role of microbes in human illness. Includes the study of
microbial pathogenesis and epidemiology and is related to the study of disease
pathology and immunology.

Environmental Microbiology:

This is the study of the function and diversity of microbes in their natural
environmental. It includes the study microbial ecology, microbial-mediated nutrient
cycling, geo microbiology, microbial diversity and bioremediation. Characterization
of key bacterial habitats such as the rhizosphere and phyllosphere, soil and ground
water ecosystems, open oceans or extreme environments (extremophiles).

Industrial Microbiology:

This includes the exploitation of microbes for use in industrial processes. Examples
include industrial fermentation and waste water treatment. It is closely linked to the
biotechnology industry. This field also includes brewing, an important application of
microbiology.
Food Microbiology:
Food microbiology is the study of microorganisms causing food spoilage.

Pharmaceutical Microbiology:

This is the study of microorganisms causing pharmaceutical contamination and

spoilage.

What are Bacteria?

Bacteria are microscopic organism whose single cells have neither a membrane-
bounded nucleus nor other membrane - bounded organelles like mitochondria and
chloroplast. Another group of microbes, the archaea, meet these criteria but are so
different from the bacteria in other ways that must have had a long, independent
evolutionary history since close to the dawn of life. In fact, there is considerable
evidence that you are more closely related to the archaea than they are to the
bacteria.

FIGURE OF BACTERIA
What are fungi?

Fungi are a group of organisms and micro-organisms that are classified within their
own kingdom, the fungal kingdom, as they are neither plant nor animal. Fungi draw
their nutrition from decaying organic matters, living plants and even animals. They
do not photosynthesize as they totally lack the green pigment chlorophyll, present in
green plants. Many play an important role in the natural cycle as decomposers and
return nutrients to the soil, they are not all destructive. Fungi are even used for
medical purposes, such as species within the Penicillium genus which provide
antibiotics, e.g. penicillin.

FIGURE OF FUNGI
RULES OF MICROBIOLOGY LABORATORY

A microbiological laboratory is a place of working with a variety of microorganisms.

A wide range of precautions are taken during work. Some of the precautions are as
follows:
• Always wear an apron in the laboratory.
• Cut nails regularly.
• The long hair must be tied back to avoid contamination and fire hazard.
• Keep your working laboratory bench clean of everything.
• Never keep books and bags etc. on the working bench.
• Clean your working bench with ethanol (70%) or phenol (1:100).
• Always wash your hands with soap in running water.
• Never spit and smoke in the laboratory.
• Do not put things from the laboratory into the mouth.
• Do not eat or drunk or talk while working with microorganisms.
• Do not mishandle chemical solutions or electricity.
• Do not pour the contents of any tubes down the drain.
• Do not leave any cultures in the incubator once you have finished using them.
• When the work is finished, place the Petri-plates containing cultures in the
bins in washroom.
• Record your result at time.
• After completion of work, keep your slide, pipette, culture tube, plate in
container and steam sterilize before washing.
• Clean lens of objective with tissue paper.
• Never leave your culture on working table or seat.
• After completion of work always label the culture with name or code with
date of work.
• While working the broth culture do not suck the suspension with mouth.
• Open the culture tube near the burner.
• Always use the flame sterilized inoculation needle or loop.
• Keep the stain, reagent, stock culture to their respective place when the work
is completed.
 STERILISATION

METHODS OF STERILISATION:

Sterilization is a process of making an article surface or medium free from any type
of microorganisms. Through sterilization any kind of microorganisms can be
removed. However sterilization is one of the most steps for cultivation, isolation and
study of microorganisms in the laboratory. There are few other methods for
destroying the microorganisms such as disinfection and incineration.

TYPES OF STERILISATION:

There are two types of sterilization:

a) Physical

b) Chemical

(a) PHYSICAL METHODS OF STERILISATION:

There are many kinds of physical methods used to sterilize the materials:

1) MOIST HEAT: Culture media are sterilized by using moist heat i.e. steam under
pressure. It is done through water vapor and also by using pressure cooker.

2) DRY HEAT: Dry heat is produced by a hot air oven. Glassware like pipettes,
Petri-dishes, test tubes etc. is sterilized in an oven at 150 0C for one hour and 250 C
for 30 minutes. Care should be taken to remove these instruments only when the
temperature cools down otherwise the glassware will be broken. In addition sterilize
the mouth of culture tubes, glass slides etc. through flaming i.e. bringing these near
the vicinity of flame of the burner only for a second. Perform incineration of
inoculation needle, inoculation loop and points of forceps by keeping their points in
the flames until they turn red hot.

3) RADIATION: Normally UV Radiation is used in inoculation chamber or laminar

air flow. Expose the working area under UV Radiation before half an hour or so to
start the work. The source of UV Radiation is generally UV lamp or UV tubes
enclosed in quartz because glass will not transmit UV Radiation. The radiation
emitted by UV source cause damage to the cells by hydration and thymine dimer
formation and produce ethyl effect. Care should be taken not to see the UV
Radiation with naked eyes.

(b) CHEMICAL METHODS OF STERILISATION:

There are several methods used for sterilization of glassware working table hands
etc. for microbiological work.

1) ALCOHOL: Ethanol 70% or isopropanol 70% is used to sterilize the working

table top of the inoculation chamber etc.

2) ALDEHYDE: Generally lab is fumigated with Formaldehyde when

contamination increases.
a) PREPARATION OF PDA MEDIUM & PDA PLATES:

REQUIREMENTS:

Potato tubers : 200gm

Dextrose : 20gm

Agar : 15gm

Distilled water : 1 liter

HCl : 1N

NaOH : 1N

Knife

Muslin cloth

Heater

Beaker (1 liter Capacity)

Erlenmeyer flasks (2) (500ml capacity)

PROCEDURE:

PREPARATION OF PDA MEDIUM:

1. Take potato tubers peel off & weigh 200gm.

2. Chop the tubers into small pieces with the help of a knife.

3. Transfer the chopped potatoes into a beaker containing about 100ml of

distilled water.

4. Boil the contents with the help of a heater for about 20 minutes.

5. Decant supernatant, filter with 4 fold of the muslin cloth & collect the filtrate
into a beaker. This filtrate is called potato extract.

6. Transfer dextrose (20g) and agar (15g) into the extract and gently heat and
shake to dissolve the ingredients.

7. Finally transfer this medium into a measuring cylinder of 1 liter capacity and
make the volume to 1 liter by adding more distilled water.

8. Measure the pH of the medium and adjust to 5-6 by using 1N HCl or NaOH.
 9. Pour the medium into two or more Erlenmeyer flasks, put cotton plug, and
cover the plug with aluminium foil/paper and autoclave at 121 degree Celsius
for 20 minutes.

10. When temperature cools down take out the flasks and use if required or store
at room temperature.

PREPARATION OF PDA PLATES:

REQUIREMENTS:

PDA medium

Culture tubes (smooth mouth or screw-capped)

Test tube stand

Heater

Aluminium foil or paper

PROCEDURE:

1. Before starting autoclaving place few Petri-dishes into an oven and sterilize
them at 2000C for about half an hour.

2. When temperature cools down, transfer them into the cabin of the laminar
air flow or inoculation chamber built for inoculation work. However, these
must be sterilized by UV light 30 minutes before start of the work.

3. Bring the flask containing PDA medium and pour about 15-20 ml medium
aseptically into the bottom half of the Petri-dishes when temperature remains
to about 400C (care should be taken not to pour too hot medium otherwise it
will cause condensation of vapor into water droplets and help
contamination).

4. Place the plates in tiers and wait for about 20-30 minutes to solidify the
medium.

5. These plates containing solidified medium are called PDA plates (agar
plates).

6. Use the plates immediately for cultivation of fungi or store for further use.

7. PDA plates, NAM plates and other agar plates are also prepared following
the same procedure.
SOIL BORNE DISEASES IN HORTICULTURE PLANTS CAUSED BY
FUSARIUM

Se
ed rot-seedling blight

Fusarium avenaceum
Fusarium culmorum
Fusarium moniliforme
Fusarium graminearum

IN POTATO:
Fusarium Dry Rot - F. sambucinum, F. solani, F. avenaceum

Fusarium Wilt - F. oxysporum, F. sp. Melongenae

Vascular Wilt Diseases - F. eumartii, F. oxysporum, F. avenaceum, F. solani

IN TOMATO:

Fusarium Wilt - F. oxysporum, F. sp. Lycopersici

Fusarium crown rot - Fusarium oxysporum

Root rot - F. oxysporum

GINGER

Ginger is a tuber that is consumed as a delicacy, a medicine or a spice. It is the

rhizome of the plant Zingiber officinale. It lends its name to its genus and family
(Zingiberaceae).

Scientific classification

Kingdom: Plantae

Division: Magnoliophyta

(unranked): Angiosperms

(unranked): Monocots

(unranked): Commelinids

Order: Zingiberales

Family: Zingiberaceae

Genus: Zingiber

Species: officinale

Binomial name

Zingiber officinale

Ginger cultivation began in Asia and has

since spread to West Africa and the Caribbean.[2] It is sometimes called root ginger
to distinguish it from other things that share the name ginger. The English name
ginger comes from the French gingimbre, from medieval Latin gingiber, from Greek
zingiberis, from Pali siṅgivera, ultimately of Dravidian origin from Tamil injiver
(meaning root of inji).

The characteristic odor and flavor of ginger is caused by a mixture of zingerone,

shogaols and gingerols, volatile oils that compose one to three percent of the weight
of fresh ginger

Ginger oil has been shown to prevent skin cancer in mice and a study at the
University of Michigan demonstrated that gingerols can kill ovarian cancer cells.
Ginger has a sialagogue action, stimulating the production of saliva, which makes
swallowing easier.

Young ginger rhizomes are juicy and fleshy with a very mild taste. They are often
pickled in vinegar or sherry as a snack or just cooked as an ingredient in many
dishes. They can also be stewed in boiling water to make ginger tea, to which honey
is often added; sliced orange or lemon fruit may also be added

DISEASES IN GINGER

Ginger is the second most important cash crop of Sikkim. Diseases are important
production constraints and often associated with Ralstonia (Pseudomonas)
solanacearum, Pythium spp., Fusarium oxysporum and Pratylenchus coffeae.

Pathogenicity experiments conducted, showed the involvement of Pythium spp. (soft

rot), Fusarium oxysporum (dry rot) and R.solanacearum (wilt) and also noticed that,
Pratylenchus coffeae increased the severity of infection along with F. oxysporum.

RHIZOME ROT MANAGEMENT

Rhizome rot is a complex problem caused by multiple factors. Beside pathogens, the
acidic soil condition of the soil is another important factor for the disease.

September onwards there is little loss since by then temperature goes down and the
rainfall is almost stopped.

Bacterial Wilt:

The bacterial wilt is the most important diseases of all and is very serious. 95% of
the ginger growing areas are infected with this disease in both Darjeeling and
Sikkim Hills.

Fungal Diseases:

The fungal or yellowing of ginger is another important disease and found to occur in
both Darjeeling and Sikkim Hills. Like bacterial wilt it also spreads very fast. The
plant infected with this disease looks yellow which starts from the lowermost leaf on
the leaf margins that progress very fast to the upper leaves.

Usually it occurs with bacterial wilt but can be easily identified with that of wilting.
Like bacterial wilt, it cannot be identified in the seed rhizome.
 The disease is caused by fungi Fusarium oxysporium and Pythium spp. usually
appears along with the bacterial wilt causing soft rot.

Fusarium is invariably associated with nematode Pratylenchus and results in storage

losses.

.
TOMATO

Cross-section and full view of a

ripe tomato

Scientific classification

Kingdom: Plantae

(unranked): Angiosperms

(unranked): Eudicots

Order: Solanales

Family: Solanaceae

Genus: Solanum

Species: S. lycopersicum

Binomial name

Solanum lycopersicum
L.

Synonyms

Lycopersicon lycopersicum
Lycopersicon esculentum

The tomato (Solanum lycopersicum) is an herbaceous, usually sprawling plant in the

nightshade family widely cultivated for its edible fruit. Savory in flavor, the fruit of
most varieties ripens to a distinctive red color. Tomato plants typically reach to 1–3
metres (3–10 ft) in height, and have a weak, woody stem that often vines over other
plants. The leaves are 10–25 centimetres (4–10 in) long, odd pinnate, with 5–9 leaflets on
petioles, [2] each leaflet up to 8 centimetres (3 in) long, with a serrated margin; both the
stem and leaves are densely glandular-hairy. The flowers are 1–2 centimetres (0.4–
0.8 in) across, yellow, with five pointed lobes on the corolla; they are borne in a cyme of
3–12 together. It is a perennial, often grown outdoors in temperate climates as an
annual.
Fungal diseases of Tomato

Leaf Mold

Leaf mold is caused by the fungus, Fulvia fulva, also known as Cladosporium fulverum.
This is a disease of tomatoes only. The disease occurs all over the world, but it is
primarily a greenhouse disease in Connecticut. It causes tomato leaves to fall off, which
will lower yield. High humidity is required for this fungus to grow successfully.

Identification of Disease: Fungal growth is seen on undersides of leaves only. The color
of the fungal growth is unusual, especially the deeper color in center.

Late Blight

Late Blight is a very devastating disease of tomato, potato, and eggplant. It is caused by
the fungus Phytophthora infestans. Growth Stages Affected/ Time of Season. Leaves,
green and ripe fruit and stems are affected.

Symptoms: Fields should be scouted frequently in the early morning when the leaves
are still wet. Dark green water-soaked spots appear on leaves. The spots sometimes have
a purplish tinge and are an indefinite shape. They enlarge rapidly to green or brown
spots which can cover most of the leaf. Fluffy gray to white moldy growth appears on
undersides of the small leaf spots when lesions and a ring of moldy growth can be seen
on undersides of larger spots. This growth can be seen in early morning when leaves are
still wet. Dark brown to black spots form on stems, which can cause the portions of the
plant beyond the stem spot to dry up rapidly. This disease causes a rot of green and ripe
fruit in which dark greenish-brown greasy areas form and may enlarge until the entire
fruit is covered. Generally the fruit remains firm, although, secondary soft rot often sets
in. Favorable weather for this disease is cool nights, with warm days. High humidity for
24 to 48 hours allowing leaves to remain wet after rain.

Identification of Disease: Patchy spots and fluffy white growth on the undersides of
leaves are present in the early morning. There are stem spots. This affects green and
ripe fruit.

FUNGAL DISEASES IN POTATO

Silver Scurf

Silver scurf (Helminthosporium solani) is a ubiquitous fungal blemish disease of

potatoes. It is first visible as small silvery grey spots that enlarge into circles, which may
show a slightly darker margin.
Black Dot

Black dot (Colletotrichum coccodes) is a common superficial fungal blemish on potatoes.

The black dots are microsclerotia that are often just visible to the naked eye. They can
be found on tubers, stolons, roots and stems.

Powdery Scab

Powdery scab (Spongospora subterranea) is a fungal blemish disease of potatoes, which

first shows as small raised pimples beneath the skin. As they develop, the skin breaks
open to expose a dark brown powdery mass of cystosori or spore balls.

Black Scurf

Black scurf (Rhizoctonia solani) is an entirely superficial black fungal incrustation on

the tuber surface. It usually appears as small, irregular blemishes that are, in fact,
compacted masses of mycelium called sclerotia.

Skin Spot
Skin spot (Polyscytalum pustulans) is generally an invisible fungus until after
approximately 2 months of storage, when the infected tissue begins to show spots. They
tend to be bluish black and slightly raised.
FUSARIUM

Fusarium is a large genus of filamentous fungi widely Scientific classification

distributed in soil and in association with plants. Most Kingdom: Fungi
species are harmless saprobes and are relatively
mycotoxins in cereal crops that can affect human and Subkingdom: Dikarya
animal health if they enter the food chain. The main
Phylum: Ascomycota
toxins produced by these Fusarium species are
fumonisins and trichothecenes Subphylum: Pezizomycotina
Fusarium graminearum commonly infects barley if there Class: Sordariomycetes
is rain late in the season. It is of economic impact to the
malting and brewing industries as well as feed barley. Order: Hypocreales
Fusarium contamination in barley can result in head Family: Nectriaceae
blight and in extreme contaminations the barley can
appear pink.The genome of this wheat and maize Genus: Fusarium
pathogen has been sequenced. Fusarium graminearum
can also cause root rot and seedling blight. The total losses in the US of barley and
wheat crops between 1991 and 1996 have been estimated at $3 billion.

Fusarium verticillioides Fusarium chlamydospores Fusarium macroconidia

Some species may cause a range of opportunistic infections in humans. In humans

with normal immune systems, fusarial infections may occur in the nails
(onychomycosis) and in the cornea (keratomycosis or mycotic keratitis).

In humans whose immune systems are weakened in a particular way (neutropenia,

i.e., very low count of the white blood cell type called neutrophils), aggressive
fusarial infections penetrating the entire body and bloodstream (disseminated
infections) may be caused by members of the Fusarium solani complex, Fusarium
oxysporum, Fusarium verticillioides, Fusarium proliferatum and rarely other
fusarial species.
Fig. In vitro culture of Fusarium

TRICHODERMA

Trichoderma is a genus of fungi that is present in all soils, where they are the most
prevalent culturable fungi. Many species in this genus can be characterized as
opportunistic avirulent plant symbionts

Scientific classification

Kingdom: Fungi

Division: Ascomycota

Subdivision: Pezizomycotina

Class: Sordariomycetes

Order: Hypocreales
Trichoderma colony in nature
Family: Hypocreaceae
Cultures are typically fast growing at 25-
30°C, but will not grow at 35° C. Colonies Genus: Trichoderma
are transparent at first on media such as
cornmeal dextrose agar (CMD) or white on richer media such as potato dextrose
agar (PDA).

Mycelium are not typically obvious on CMD, conidia typically form within one week
in compact or loose tufts in shades of green or yellow or less frequently white.
A yellow pigment may be secreted into the agar, especially on PDA. Some species
produce a characteristic sweet or 'coconut' odor.

Conidiophores are highly branched and thus difficult to define or measure, loosely
or compactly tufted, often formed in distinct concentric rings or borne along the
scant aerial hyphae.

Main branches of the conidiophores produce lateral side branches that may be
paired or not, the longest branches distant from the tip and often phialides arising
directly from the main axis near the tip.

The branches may rebranch, with the secondary branches often paired and longest
secondary branches being closest to the main axis. All primary and secondary
branches arise at or near 90° with respect to the main axis.

The typical Trichoderma conidiophore, with paired branches assumes a pyramidal

aspect. Typically the conidiophore terminates in one or a few phialides. In some
species (e.g. T. polysporum) the main branches are terminated by long, simple or
branched, hooked, straight or sinuous, septate, thin-walled, sterile or terminally
fertile elongations. The main axis may be the same width as the base of the phialide
or it may be much wider.

Phialides are typically enlarged in the middle but may be cylindrical or nearly
subglobose. Phialides may be held in whorls, at an angle of 90° with respect to other
members of the whorl, or they may be variously penicillate (gliocladium-like).
Phialides may be densely clustered on wide main axis (e.g. T. polysporum, T.
hamatum) or they may be solitary (e.g. T. longibrachiatum).

Conidia typically appear dry but in some species they may be held in drops of clear
green or yellow liquid (e.g. T. virens, T. flavofuscum). Conidia of most species are
ellipsoidal, 3-5 x 2-4 µm (L/W = > 1.3); globose conidia (L/W < 1.3) are rare. Conidia
are typically smooth but tuberculate to finely warted conidia are known in a few
species.
Synanamorphs are formed by some species that also have typical Trichoderma
pustules. Synanamorphs are recognized by their solitary conidiophores that are
verticillately branched and that bear conidia in a drop of clear green liquid at the tip
of each phialide.

Chlamydospores may be produced by all species, but not all species produce
chlamydospores on CMD at 20° C within 10 days. Chlamydospores are typically
unicellular subglobose and terminate short hyphae; they may also be formed within
hyphal cells. Chlamydospores of some species are multicellular (e.g. T.
stromaticum).

Trichoderma species are frequently isolated from forest or agricultural soils at all
latitudes. Hypocrea species are most frequently found on bark or on decorticated
wood but many species grow on bracket fungi (e.g. H. pulvinata), Exidia (H.
sulphurea) or bird's nest fungi (H. latizonata) or agarics (H. avellanea).

Trichoderma, being a saprophyte adapted to thrive in diverse situations, produces a

wide array of enzymes. By selecting strains that produce a particular kind of
enzyme, and culturing these in suspension, industrial quantities of enzyme can be
produced.

T. reesei is used to produce cellulase and hemicellulase

T. longibratum is used to produce xylanase

T. harzianum is used to produce chitinase.

Synanamorphs are formed by some species that also have typical Trichoderma
pustules. Synanamorphs are recognized by their solitary conidiophores that are
verticillately branched and that bear conidia in a drop of clear green liquid at the tip
of each phialide.

ASPERGILLUS

Aspergillus is a genus of a few hundred molds found throughout much of nature

worldwide. Aspergillus was first catalogued in 1729 by the Italian priest and
biologist Pier Antonio Micheli.

Viewing the fungi under a microscope, Micheli was reminded of the shape of an
Aspergillum (holy water sprinkler), and named the genus accordingly. Today
"aspergillum" is also the name of an asexual spore-forming structure common to all
Aspergilli; around one-third of species are also known to have a sexual stage
Conidial head of Aspergillus niger Aspergillus on tomato in details

Aspergillus species are highly aerobic and are found in almost all oxygen-rich
environments, where they commonly grow as molds on the surface of a substrate, as
a result of the high oxygen tension.

Commonly, fungi grow on carbon-rich substrates such as monosaccharides (such as

glucose) and polysaccharides (such as amylose). Aspergillus species are common
contaminants of starchy foods (such as bread and potatoes), and grow in or on many
plants and trees.

In addition to growth on carbon sources, many species of Aspergillus demonstrate

oligotrophy where they are capable of growing in nutrient-depleted environments,
or environments in which there is a complete lack of key nutrients.

Aspergillus niger is a prime example of this; it can be found growing on damp walls,
as a major component of mildew.

Commercial importance

Various Penicillium, Aspergillus spp. (and some other fungi) growing in axenic
culture.

Species of Aspergillus are important medically and commercially. Some species can
cause infection in humans and other animals. Some infections found in animals have
been studied for years. Some species found in animals have been described as new
and specific to the investigated disease and others have been known as names
already in use for organisms such as saprophytes.

Other species are important in commercial microbial fermentations. For example,

alcoholic beverages such as Japanese sake are often made from rice or other starchy
ingredients (like manioc), rather than from grapes or malted barley.

Typical microorganisms used to make alcohol, such as yeasts of the genus

Saccharomyces, cannot ferment these starches, and so koji mold such as Aspergillus
oryzae is used to break down the starches into simpler sugars.
Members of the genus are also sources of natural products that can be used in the
development of medications to treat human disease.

Perhaps the most well-known application of ASPERGILLUS NIGER is as the major

source of citric acid; this organism accounts for over 99% of global citric acid
production, or more than 1.4 million tonnes per annum.

ASPERGILLUS NIGER is also commonly used for the production of native and
foreign enzymes, including glucose oxidase and hen egg white lysozyme. In these
instances, the culture is rarely grown on a solid substrate, although this is still
common practice in Japan, but is more often grown as a submerged culture in a
bioreactor.

Aspergillosis

Aspergillosis is the group of diseases caused by Aspergillus. The most

common subtype among paranasal sinus infections associated with
aspergillosis is Aspergillus fumigates. The symptoms include fever, cough,
chest pain or breathlessness, which also occur in many other illnesses so
diagnosis can be difficult. Usually, only patients with already weakened
immune systems or who suffer other lung conditions are susceptible.
ESSENTIAL OIL

INTRODUCTION TO ESSENTIAL OIL

An essential oil is a concentrated, hydrophobic liquid

containing volatile aroma compounds from plants. Essential
oils are also known as volatile or ethereal oils, or simply as the
"oil of" the plant from which they were extracted, such as oil of
clove. Oil is an "essential" in the sense that it carries a
distinctive scent, or essence, of the plant.

Essential oils do not as a group need to have any specific

chemical properties in common, beyond conveying
characteristic fragrances. They are not to be confused with
essential fatty acids.

Essential oils are generally extracted by distillation. Other

processes include expression, or solvent extraction. They are
used in perfumes, cosmetics, soap and other products, for
flavoring food and drink, and for scenting incense and
household cleaning products.

Various essential oils have been used medicinally at different

periods in history. Medical application proposed by those who
sell medicinal oils range from skin treatments to remedies for
cancer, and are often based on historical use of these oils for
these purposes. Such claims are now subject to regulation in
most countries, and have grown more vaguely to stay within
these regulations.

Interest in essential oils has revived in recent decades with the

popularity of aromatherapy, a branch of alternative medicine
which claims that the specific aromas carried by essential oils
have curative effects. Oils are volatilized or diluted in a carrier
oil and used in massage, diffused in the air by a nebulizer or by
heating over a candle flame, or burned as incense, for
example.

PLANT USED FOR THE EXTRACTION OF ESSENTIAL OILS

LEMONGRASS
 TAXONOMY:

Kingdom: Plantae

Phylum: Tracheophyta

Class: Liliopsida

Subclass: Commelinidae

Order: Poales

Family: Poaceae

Genus: Cymbopogon

Species: flexuosus

Lemongrass is an aromatic tropical plant with long, slender

blades that can grow to a height of 5 ft (1.5 m).The herb has
been used for centuries in South America and India and has
also become popular in the United States. The fresh stalks and
leaves have a clean lemon like odour because they contain an
essential oil, which is also present in lemon peel.

USES OF LEMON GRASS ESSENTIAL OIL

Analgesic: Lemongrass Essential Oil helps relieve pain in muscles,

joints, toothache and headache etc. resulting from viral infections like
cough & cold, influenza, fever, pox etc. It also Essential oil in Lemon
Grass

Fresh C. citratus grass contains about 0.4% of volatile oil. The oil contains 65% to
85% of citral (key component that gives lemony aroma n taste in lemon grass n have
found to be essential in cancer cell commit suicide)(a mixture of 2 geometric
isomers, geraniol and neral). Citral is used as a flavoring to fortify lemon oil and in
perfumes and colognes for its lemon scent. Citral isolated from C. citratus from
Laguna was found to be of good quality with 93.7% purity. GC analysis in 1 report
finds geraniol and neral, along with related geraniol, geranic acid, and nerolic acid.

Other compounds found in the oil include myrcene (12% to 25%)(lemongrass tea
has potent analgesic activity in rodents, and beta-myrcene was subsequently
identified as the active ingredient responsible for this effect.), diterpenes,
methylheptenone, citronellol, linalol, farnesol, other alcohols, aldehydes, linalool,
terpineol, and more than a dozen other minor fragrant component .Reports
concerning chemical analyses of C. citratus specific to country of origin are
available, finding some similarities to the above components. Philippine lemongrass
has been found to contain alpha and beta pinene, limonene, phellandrene, and
others, findings of 21 components such as anisaldehyde, cinnamaldehyde, catechol,
and hydroquinone from certain fractions of this species from Bangladesh, and
various constituents from this species and others (including C. winterianus, C.
jwarancusa) from China and Morocco.

Other species' chemical components have been reported. C. flexuosus grass contains
approximately 0.5% volatile oil, which in some strains contains up to 85% citral.
However, many strains have a higher concentration of geraniol (50%) with citral
(10% to 20%) and methyl eugenol as minor components. Yet another type of East
Indian lemongrass is reported to contain no citral but up to 30% borneol.
Nonvolatile components of C. citratus consist of luteolins, homo-orientin,
chlorogenic acid, caffeic acid, P-coumaric acid, fructose, sucrose, octacosanol, and
others. Flavonoids luteolin and 6-C-glucoside have also been isolated. One study
reports high concentrations of cobalt.
 PROCEDURE OF EXTRACTION

1. Separate the leaves from their inflorescence and chop them

finely.
2. Take a RBC flask of variable capacity.
3. Rinse and dry the flask properly.
4. Fill the leaves in RBC flask.
5. Fill the required amount of water in RBC flask.
6. Put the flask on heating mantle carefully.
7. Attach the extraction burette with RBC flask to complete the
Clevenger apparatus.
8. Set the temperature at 70˚C on heating mantle till boiling
starts and then set it at 50˚C.
9. Observe the entire process carefully.
10. After completion of process take a clean empty vial
weighs it.
11. Collect the oil in vial and weigh it again.

12. Subtract the weight of empty vial from the weight of vial
containing oil to obtain the oil content.
13. Calculate the % yield from the following formula:

Percent yield = Oil content (g) × 100

Weight of sample (g)

=1.707 /200×100

= 0 .8535% oil

OIL ANALYSIS
AIM: To determine the specific gravity of LEMON GRASS and
LEMON GRASS oil sample.

PROCEDURE:

 Take an empty pycnometer and weigh.

 Fill the pycnometer with1ml of oil sample and weigh.
 Rinse and dry the pycnometer properly.
 Fill the pycnometer with 1mlof distilled water and weigh.
 Calculate the specific gravity of oil from following formula:

Specific gravity of lemon grass oil = Z–X

Y-X

= 19.4637 –
19.0440/19.5446-19.0440

= 0.838

Where,

X = wt. of empty pycnometer = 19.0440gm

Z = wt. of (pycnometer + 0.5 ml oil sample) = 19.4637

gm

Y= wt. of (pycnometer + 0.5ml distilled water) =

19.5446gm
 Specific gravity of Lemon grass oil=
Z-X

=19.4942-
19.0555/19.5619-19.0555

=0.8762

Where:

X = wt. of empty pycnometer=19.0555gm

Z= wt. of (pycnometer+ 0.5 ml oil sample) =19.4942gm

Y= wt of (pycnometer+0.5 ml distilled water) = 19.5619

EXPERIMENT 2

To determine acid value and free fatty acid (FFA) content of oil
sample.

PROCEDURE
 Take an empty small beaker and weigh.
 Add 0.5ml of oil sample in the beaker & weigh.
 Add 5 ml of Isopropyl alcohol to the oil sample in beaker.

 Heat the mixture for 5min. on water bath.

 Cool and add 5 drops of phenolphthalein indicator.
 Titrate against 0.1N potassium hydroxide (KOH).
 Record the point of color change.

 Calculate acid value and free fatty acid content from

following formula:

Acid value = 56.1 × titrated value × normality

Sample weight

Acid value for lemon grass oil

=1.85163

Where,

Sample wt. = (wt of beaker+0.5ml oil sample) –

(wt. of empty beaker)

FFA = Acid value/2 =0.9258

ACID VALUE FOR LEMON GRASS OIL

Acid value = 56.1* titrated value *
normality

Sample weight

Acid value for Lemon grass oil=4.448

Where

Sample wt = (wt of beaker+0.5 ml oil sample)-(wt of

empty beaker)
 FFA =Acid value/2=2.224

Helps cure body pain resulting from sudden exercises, sports

etc.