R_PR_MSC MICRO

INTRODUCTION

Fungi are significant destroyers of foodstuffs and grains

during storage, rendering them unfit for human consumption by retarding

their nutritive value and often by producing mycotoxins (Marin et al.,

1999; Janardhana et al., 1998). A significant portion of the agricultural

produce in the country and the world over become unfit for human

consumption due to mycotoxins contamination of grains, especially those

produced by species of Aspergillus (Janardhana et al. 1999; Chandra and

Sarbhoy, 1997; Devi et al., 2001). More than 25% of the world cereals

are contaminated with known mycotoxins and more than 300 fungal

metabolites are reported to be toxic to man and animals (Galvanoet al.,

2001). The main toxic effects are carcinogenicity, genotoxicity,

terratogenicity, nephrotoxicity, hepatotoxicity, reproductive disorders and

immunosuppression (Lacey, 1988; Desjardins et al, 2000).

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.

Mites are common associate with mold as they occur in nature. Mites are

in size commonly just about at the limit of visibility by the unaided eye.

"In recent studies, increased levels of Reactive Oxygen Species (ROS)

were shown to be correlated with increased levels of aflatoxin

biosynthesis in Aspergillus pirasiticus." Commonly, fungi grow on

carbon-rich substrates such as monosaccharides (such as glucose) and

polysaccharides (such as amylose). Aspergillus species are common

(1)

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.

SCIENTIFIC CLASSIFICATION:

Domain Eukarya

Kingdom Fungi

Phylum Ascomycota

Class Eurotiomycetes

Order Eurotiales

Family Trichocomaceae

Genus Aspergillus

Aspergillus is a genus of filamentous fungi belonging to

Deuteromycetes. Fungi in this genus are easy to grow; some produce a

wide range of useful enzymes, but others are dangerous pests and

pathogens. For instance, Aspergillus niger is used to produce citric acid,

gluconic acid, amylase, proteases, and peptic enzymes, but Aspergillus

parasiticus grows on grains and produces aflatoxin, a powerful liver

(2)

carcinogen. Aspergillus is a genus of around 200 fungi (moulds) found

worldwide. Aspergillus clavatus is a species of Aspergillus with conidia

dimensions 3-4.5 x 2.5-4.5 micrometres. It is found in soil and animal

manure. Aspergillus flavus is a fungus associated with aspergillosis of the

lungs and sometimes believed to cause corneal, otomycotic, and

nasoorbital infections. It is believed to be allergenic. A mold causing

aspergillosis in birds and man. Aspergillus fumigatus is a fungus of the

genus Aspergillus. It causes Stone brood in honeybees and is classified as

one of the diseases of the honeybee. Aspergillus niger is a fungus and one

of the most common species of the genus Aspergillus. It causes black

mould on certain types of fruit and vegetables, and is a common

contaminant of food. It is the agent which ferments soybeans to produce

miso Aspergillus parasiticus is a mold known to produce aflatoxin,

although strains of it exist that do not produce this carcinogen. It is

sometimes found on black olives. Genus of common molds causing food

spoilage and some pathogenic to plants and animals.

Some Aspergillus species are important as agricultural

pathogens. Aspergillus species, cause disease on many grain crops,

especially maize, and synthesize mycotoxins including aflatoxin.

Consumption of such grains leads to many health problem i.e.

(3)

1. Allergic bronchopulmonary aspergillosis or ABPA (affects

patients with symptoms that produce significant respiratory

morbidity such as asthma, cystic fibrosis and sinusitis).

2. Cute invasive aspergillosis (risk increases if patient has weakened

immunity such as some AIDS patients and those undergoing

chemotherapy).

3. Disseminated invasive aspergillosis (widespread through body).

(27)

Knowledge of the exact location of the pathogen in seed or

the depth of seed infection by particular pathogen can be helpful in the

control of seed borne infection. Infectious diseases accounts for high

proportion of health problems in the developing countries including India.

Microorganisms have developed resistance to many antibiotics and as a

result, immense clinical problem in the treatment of infectious diseases

has been created (Davies, 1994). The resistance of the organisms

increased due to indiscriminate use of commercial antimicrobial drugs

commonly used for the treatment of infectious disease. This situation

forced the researchers to search for new antimicrobial substance from

various sources including medicinal plants (Bauer et al., 1996). There are

alarming reports of opportunistic fungal infections (Singh, 2001). The

infections caused by opportunistic fungi are included under new spectrum

of fungal pathogens. Such fungi were earlier reported from various plants

as pathogens. But now they are known to cause disease in human beings.

(4)

There is an increasing awareness amongst clinicians and

microbiologists pertaining to importance of infection caused by

opportunistic fungi. Aspergillosis is caused due to inhalation of

Aspergillus fumigatus spores. Aspergillus fumigatus is an opportunistic

pathogen which usually affects cavities that have formed in the lungs

from preexisting lung diseases. In the lungs, Aspergillus fumigatus forms

tangled mass of fungus fibers, blood clots. The fungus mass gradually

enlarges, destroying lung tissue in the process, but usually does not

spread to other areas (Alex et al., 1998).

Nature has been a source of medicinal agents for thousands

of years and an impressive number of modem drugs have been isolated

from natural sources. India is a land of rich biodiversity. The total number

of lower and higher plants in India is about 45,000 species. The plants are

potential source of medicines since ancient times. According to

World Health Organization, 80% of the populations in the world depend

on traditional medical practitioners for their medicinal needs.

(ELUMALAI et al., 2009)

Neem (Azadirachta indica)

Scientific classification

Kingdom : Plantae

Division : Magnoliophyta

Order : Sapindales

Family : Meliaceae

Genus : Azadirachta

Species : indica

(5)

(16)

Azadirachta indica. Juss. Family name:

Meliaceae ,Vernacular name: Neem distributed widespread in the world.

The Chemical constituents contain many biologically active compounds

that can be extracted from neem, including alkaloids, flavonoids,

triterpenoids , phenolic compounds , Carotenoids , steroids and

ketones ,azadirachtin is actually a mixture of seven isomeric compounds

labeled as azadirachtin A-G and azadirachtin E is more effective .Other

compounds than azadirachtin that have a biological activity are salannin,

volatile oils, meliantriol and nimbin. Neem leaf is effective in treating

eczema, ringworm, acne, anti-inflammatory, antiheperglycemic and it is

used to heal chronic wounds , diabetic foot and gangrene developing

conditions . It is believed to remove toxins from the body, neutralize free

radicals and purify the blood. It is also used in treatment of malaria.

Recently it used as anti-cancer and it has hepato-renal protective activity

and hypolipidemic effects . The juice of green neem leaves drunk with

milk increase appetite, uses as a collyrium, the juice instantly relieves

headaches and cures eyes infections. Boiled neem leaf water makes an

excellent antiseptic to clean wounds, soothes, swellings and eases skin

problems (Yagoub et al.,)

Babul (Acacia Nilotica)


Kingdom : Plantae

(unranked) : Angiosperms

(6)

(unranked) : Eudicots

(unranked) : Rosids

Order : Fabales

Family : Fabaceae

Genus : Acacia

Species : Nilotica

(14)

Nilotica may also be used for medicinal purposes, as a

demulcent or for conditions such as gonorrhoea, leucorrhoea. diarrhea,

dysentery or diabetes. It is styptic and astringent. In Siddha medicine, the

gum is used to consolidate therwise watery semen.

Tulsi (Ocimum Sanctum]


kingdom : Plantae

Division : Magnoliophyta

Class : Magnoliopsida

Order : lamiales

Family : Labiatae

Genus : Ocimum

Species : sanctum

(21)

Tulsi has been widely known for its health promoting and

medicinal value for thousands of years. Commonly called sacred or holy

(7)

basil, it is a principal herb of Ayurveda, the ancient traditional holistic

health system of India. Tulsi is known as "The Incomparable One", "The

Mother Medicine of Nature", and "The Queen of Herbs". In Ayurvedic

medicines it is used as a whole plant, the leaves, root, stem and the

essential oil. Essential oil of Tulsi have antibacterial, antifungal and

antiviral properties. It inhibits the growth of Escherichia coli,

Microbacterium tuberculosis etc. It's antitubercular activity is one-tenth

the potency of streptomycin and one-fourth that of isoniazid. Essential oil

of Tulsi has been reported to possess 100% larvicidal activity against the

Culex mosquitoes. Trials have shown excellent antimalarial activity of

Tulsi It's extracts have marked insecticide activity against mosquitoes.

Essential oil of Tulsi was found to have anti-allergic properties. When

administered to laboratory animals, the compound was found to inhibit

mast cell deregulation and histamine release in the presence of allergen.

These studies reveal the potential role of Ocimum sanctum extracts in the

management of immunological disorders including allergies and Asthma.

(24)

Heena (Lawsonia. Inermis)


Kingdom Plantae

Division Magnoliophyta

Class Magnoliopsida

Order Myrtales

Family Lythraceae

(8)

Genus Lawsonia

Species Inermis

(18)

Some benefits of Henna include treatment of burns,

congruity with nerves when used in poultices, soft moist heated mass

spread on cloth and applied to the sore, and is used for the treatment of

ulcers of the mouth, stomach, bums, and hot swellings. Applying a

decoction made of henna and rose balm mixed with melted candlelight

wax also helps in the treatment of pleurisy.

Henna also helps in the treatment of small pox in its early

stages. This is done by tingeing the bottom of the child's feet with henna,

and this will prevent its progression to his eyes. This treatment is well

known fact and a most common treatment in the case of small pox.

(26)

Nilgiri (eucalyptus globules)


Kingdom : Plantae

(unranked) : Angiosperms

(unranked) : Eudicots

(unranked) : Rosids

Order : Myrtales

Family : Myrtaceae

Genus : Eucalyptus

(9)

Species : globulus

(17)

Medicinal Action and Uses -Stimulant, antiseptic, aromatic.

The medicinal Eucalyptus Oil is probably the most powerful

antiseptic of its class, especially when it is old, as ozone is formed in it on

exposure to the air. It has decided disinfectant action, destroying the

lower forms of life. Internally, it has the typical actions of a volatile oil in

a marked degree.

Eucalyptus Oil is used as a stimulant and antiseptic gargle.

Locally applied, it impairs sensibility. It increases cardiac action. Its

antiseptic properties confer some antimalarial action, though it cannot

take the place of Cinchona.

An emulsion made by shaking up equal parts of the oil and

powdered gum-arabic with water has been used as a urethral injection,

and has also been given internally in drachm doses in pulmonary

tuberculosis and other microbic diseases of the lungs and bronchitis. In

croup and spasmodic throat troubles, the oil may be freely applied

externally.

The oil is an ingredient of 'catheder oil,' used for sterilizing

and lubricating urethral catheters. In large doses, it acts as an irritant to

the kidneys, by which it is largely excreted, and as a marked nervous

depressant ultimately arresting respiration by its action on the medullary

(10)

centre. For some years Eucalyptus-chloroform was employed as one of

the remedies in the tropics for hookworm, but it has now been almost

universally abandoned as an inefficient anthelmintic, Chenopodium Oil

having become the recognized remedy. In veterinary practice, Eucalyptus

Oil is administered to horses in influenza, to dogs in distemper, to all

animals in septicaemia. It is also used for parasitic skin affections. (22)

Effect of Eucalyptus globules Leaves :

anti-bacterial [an agent that destroys bacteria; bactericide]

antioxidant [contributing to the oxidation of free radicals which are

believed to contribute to premature aging and dementia]

antiseptic [an-agent for inhibiting the growth of microorganism on

living tissue or destroying pathogenic or putrefactive bacteria]

anti-inflammatory [an agent to ease inflammation]

expectorant [an agent that promotes the discharge of mucous and

secretions from the respiratory passages, i.e. loosens phlegm in the

respiratory passages]

(25)

Aavla (Emblica officinalis)


Kingdom Plantae

Division Angiospermae

Class Dicotyledonae

Order Geraniales

(11)

Family Euphorbiaceae

Genus Emblica

Species officinalis

The fruits are sour, astringent, bitter, acrid, sweet, cooling,

anodyne, ophthalmic, carminative, digestive, stomachic, laxative,

alterant, aphrodisiac, rejuvenative, diuretic, antipyretic and tonic. They

are useful in vitiated conditions of tridosha, diabetes, cough, asthma,

bronchitis, cephalalgia, ophthalmopathy, dyspepsia, colic, flatulence,

hyperacidity, peptic ulcer, erysipelas, skin diseases, leprosy,

haematogenesis, inflammations, anemia, emaciation, hepatopathy,

jaundice, strangury, diarrhoea, dysentery, hemorrhages, leucorrhoea,

menorrhagia, cardiac disorders, intermittent fevers and greyness of hair .

(19)

(12)

ALOE VERA


Kingdom : Plantae

Order : Asparagales

Family : Asphodelaceae

Genus : Aloe

Species : vera

(15)

Seeing so many medicinal uses of Aloe vera plant it is pretty

easy to say that Aloe vera is of great importance in our lives and solves

great purpose in medical field as well. Apart from above mentioned

medicinal uses of Aloe vera there are other more important points to be

noted about Aloe vera plant which are of more importance and critical

issues:

AIDS Cure:

Aloe vera is showing a great potential to fight against AIDS.

SO many researches are going to get the best potential result out of Aloe

vera plants for cure of AIDS.

Cancer cure:

Aloe vera plants are proving to be great help for cancer

patients by activation white blood cells which promote growth of non-

cancerous cells. Researchers have found the cancer fighting properties of

Aloe vera and are making it count.

(13)

The medicinal uses of Aloe vera plants is never ending you

can consider Aloe vera to solve every problem of your body and its pretty

reliable and without any side effects. You will be surprised to see the

much more medicinal uses of Aloe vera. (20)

Conventionally, plant fungal diseases are controlled using

synthetic fungicides, which increase agricultural costs and contaminate

the environment. A possible alternative to solve these problems is use of

plants with the ability to produce antifungal substances. . So the time

reached to study, extensively, effect of aqueous plant extracts on control

of the microbs so that better, effective low cost measure can be suggested

for healthy human life.(Charimbu M. K., 2009)

(14)

REVIEW OF LITERATURE

Kaaya et al. (2005) conducted study to establish the

inhibitory effects of neem kernels (Azadirachta indica) on mould species

commonly isolated from harvested maize in Uganda. Moulds were grown

on potato dextrose agar (PDA) with or without neem kernels. To

determine the effect of neem kernel size, different sizes of neem

fragments were used and the ratio of inhibition zone to fragment weight

was calculated. Neem kernels significantly inhibited growth of the

majority of moulds apart from Rhizopus species. Values of the inhibition

zones showed that Penicillium expansum was the most inhibited followed

by Penicillium italicum and Aspergillus fumigatus, while Aspergillus

flavus and Aspergillus niger were the least inhibited. Asimilar trend was

observed when the inhibition zone ratio values were computed. These

results indicate that if neem kernels are used by farmers or traders, they

have the potential of improving maize quality during storage.

Satish et al. (2007) tested aqueous extract of fifty-two plants

from different families for their antifungal potential against eight

important species of Aspergillus such as Aspergillus candidus,

Aspergillus columnaris, Aspergillus flavipes, Aspergillus flavus,

Aspergillus fumigatus, Aspergillus niger, Aspergillus ochraceus, and

Aspergillus tamarii which isolated from sorghum, maize and paddy seed

samples. The test fungi were mainly associated with seed biodeterioration

during storage. Among fifty-two plants tested, aqueous extract of Acacia

(15)

nilotica, Achras zapota, Datura stramonium, Emblica officinalis,

Eucalyptus globules, Lawsonia inermis, Mimusops elengi, Peltophorum

pterocarpum, Polyalthia longifolia, Prosopis juliflora, Punica granatum

and Sygigium cumini have recorded significant antifungal activity against

one or the other Aspergillus species tested.

Bansod and Rai (2008) extracted oils from fifteen medicinal

plants and screened for their activity against Aspergillus fumigatus and

Aspergillus niger by disc diffusion method. Minimum inhibitory

concentrations (MICs) of oils (%v/v) against Aspergillus fumigatus and

Aspergillus niger done by agar dilution method and minimum inhibitory

concentration (MIC) and minimum cidal concentration (MCCs) data

(%v/v) obtained by the broth micro dilution method. The results showed

that the maximum antimycotic activity was demonstrated by oils of

Cymbopogon martini, Eucalyptus globulus and Cinnamomum zylenicum

as compared to control, followed by Cymbopogon citratus which showed

activity similar to control (miconazole nitrate). The oils of Mentha

spicata, Azadirachta indica, Eugenia caryophyllata, Withania somnifera

and Zingiber officinale exhibited moderate activity. The oils of Cuminum

cyminum, Allium sativum, Ocimum sanctum, Trachyspermum copticum,

Foeniculum vulgare and Elettaria cardamomum demonstrated

comparatively low activity against Aspergillus niger and Aspergillus

fumigatus as compared to control. Mixed oils showed maximum activity

as compared to standard. These results support the plant oils can be used

(16)

to cure mycotic infections and plant oils may have role as pharmaceutical

and preservatives.

Lawsonia inermis L. is a much branched glabrous shrub or

small tree, cultivated for its leaves although stem bark, roots, flowers and

seeds have also been used in traditional medicine. The plant is reported to

contain carbohydrates, proteins, flavonoids, tannins and phenolic

compounds, alkaloids, terpenoids, quinones, coumarins, xanthones and

fatty acids. The plant has been reported to have analgesic, hypoglycemic,

hepatoprotective, immunostimulant, anti-inflammatory, antibacterial,

antimicrobial, antifungal, antiviral, antiparasitic, antitrypanosomal,

antidermatophytic, antioxidant, antifertility, tuberculostatic and

anticancer properties. It is now considered as a valuable source of unique

natural products for development of medicines against various diseases

and also for the development of industrial products. This review gives a

bird's eye view mainly on the pharmacognostic characteristics, traditional

uses, phytochemistry and pharmacological actions of the plant.

(Chaudhary et al., 2010)

The chemical compounds have been identified and scientists

feel that there are many more compounds yet to be identified in neem.

The compound and their role is given as Nimbin: anti-inflammatory,anti-

pyretic,anti-histamine,anti-fungal Nimbidin: anti-bacterial, anti-ulcer,

analgesic, anti-arrhythmic, anti-fungal. Ninbidol: anti-tubercular, anti-

protozoan, .anti-pyretic, Gedunin: vasodilator, anti-malarial, anti-fungal,

Sodium nimbinate:diuretic, spermicide, anti-arthritic, Quercetin:anti-

(17)

protozoal Salannin:insect repellent, Azadirachtin:insect repellent, anti-

feedant,anti-hormonal. (23)

As a medicinal plant, henna has been used as an astringent,

antihemorrhagic, intestinal antineoplastic, cardio-inhibitory, hypotensive,

and a sedative. It has been employed both internally and locally in

jaundice, leprosy, smallpox, and affections of the skin. The fruit is

thought to have emmenagogue properties. It has also been used as a folk

remedy against amoebiasis, headache, jaundice, ranging from beriberi to

burns and bruises ans leprosy.Henna extracts show antibacterial,

antifungal, and ultraviolet light screening activity. Henna has exhibited

antifertility activity in animals. It is used to create an instant 'Scab' on

large areas & is believed to have antiseptic properties. As a cooling agent

it is used for burning of skin. It also has great dandruff fighting ability.

Henna is also used for rheumatic and arthritic pains. Alcoholic extract of

the leaves showed mild antibacterial activity against Staph aureus and

Escherichia coli'. Antibacterial and antifungal activities have been

confirmed .The antihaemorrhagic properties are attributed to lawsone.

The Naphthoquinone has emmenogogue and oxytocic actions. (18)

(18)

AIM

To study the Antifungal activity of some plant extract

against important seed borne pathogen of Aspergillus species.

OBJECTIVES

The study was conducted with the following objective:-

1. To isolate and identify Aspergillus species from different seed

sample.

2. To find out extend of activity of plant extract against Aspergillus

species.

3. To compare the atifungal activity of plant extract with synthetic

fungicides.

4. Purification of plant extract.

5. study of antifungal activity by using terpenes,phenol,tannis

alkaloids, quinines, and flavinoids from plant extract.

(19)

MATERIALS

SAMPLES:-

• Groundnut

• Sorghum

• Maize

• Paddy

MEDIA:-

• Czapeck -Dox-Agar(CDA)

CHEMICALS :-

• Mancozeb

• Copper oxychloride

APPARATUS:-

• Centrifuge

• Autoclave

• Airtight brown bottle

• Blender

STAINING REAGENT:-

• Young fungal culture

• Mounting fluid i.e. lactophenol plus cotten blue

• Ethanol(95%)Euperol or Nail-polish

(20)

PLANT MATERIAL

List of plant species tested for antifungal activity

Sr.No. Name of the plant Family

1 Acacia nilotica Mimosaceae

2 Aloe vera Liliaceae

3 Azadirachta indica Meliaceae

4 Emblica officinalis Euphorbiaceae

5 Eucalyptus globulis Myrtaceae

6 Lawsonia inermis Lythraceae

7 Ocimum sanctum Lamiaceae

Media Composition Czapeck-Dox-Agar(CDA)

Ingredients Gms/Litre

Sucrose 30.00

Sodium nitrate 2.00

Dipotassium phosphate 1.00

Magnesium sulphate o.50

Potassium chloride 0.50

Ferrous sulphate 0.01

Agar 15.00

pH(at25°C) 7.3+/- 0.2

Distilled water 1000

The above mentioned media were prepared and sterilized

by autoclaving at 15 1bs pressure for 15-20 minutes.

(21)

METHODS

1. Collection of seed samples:-

Samples of seeds i.e. groundnut, sorghum, maize & paddy

were collected from the market of Nagpur city. Each samples

should be kept separately and maintained properly

2. Preparetion of C.D.A. media.

Principle : These media are prepared according to the

formula developed by Thom and Chruch which has a defined

chemical composition. It is a semisynthetic medium contain

sodium nitrate as the sole source of nitrogen. It is one of the most

widely used medium for the general cultivation of fungi, This

medium can be also used for chlamydospore production by

Candida albicans, It has good buffering action due to the presence

of different salts. The pH is slightly above neutral value. Czapeck-

Dox-agar supports abundant growth of almost all saprophytic

Aspergilli with characteristic mycelia and conidia formation.

3. Transfer of samples on media-

One or two seeds of each samples was directly kept on the

Czapeck-Dox-Agar medium in a Petri dish with the help of sterile

forcep. Then the plates were incubated at 25 -27° C for 7 days.

After incubation the different colonies were obtained.

(22)

4. Isolation of pure culture:

These colonies from each samples was isolated separately on

the Czapeck-Dox-Agar slant and these slant were maintained

properly for further studies.

5. Study of Aspergillus species By staining of fungi cotton blue

staining:-

Principle:

The fungal propagules either are hyaline (colourless) or of

different colours. The hyaline /mycelia/spores/conidia etc. and their

cytoplasm can be stained by using lactophenol and cotton blue

stains cytoplasm and results in light blue background.

Lactophenol acts as cleaning agent whereas phenol as fungicides

Method/Procedure

1. Procure young cultures (5-7 days old) of fungi growing on culture

medium.

2. Put a drop of mounting fluid in the centre of a glass slide.

3. Transfer a portion of mycelial mat from fungal colony into the drop

of mounting fluid with the help of flamed and cooled needle.

4. With the help of two needles gently spread the fungal propagules

so that the mycelia should be mixed with stain.

5. Repeat the process for all the fungal colonies.

(23)

6. The Aspergillus niger,Aspergllus fumigatus, Aspergillus oryzea &

Aspergillus terreus were identified on the basis of macroscopic &

microscopic study.

7. Preparetion of plant extracts:-

a. Acacia nilotica(Babul):-

Leaf samples (lOOgm) of Acacia nilotica plants were blot

dried and macerated with 100ml strile distilled water in a blender

(Waring international, New Hartford, CT, USA) for 10 min. The

macerate was first filtered through double layered muslin cloth and

then centrifuged at 4000 rpm for 30 min. The supernatant was

filtered through Whatmann No. 1 filter paper and sterilized at 120°

C for 15 min., which served as the mother extract.

b. Azadirachta indica(Neem)

c. Aloe vera

d. Emblica officinalis (Avala)

e. Eucalyptus globulis (Nilgiri)

f. Lawsonia inermis (Heena)

g. Ocimum sanctum (Tulsi)

8. Study of antifungal activity of plant extracts against

Aspergillus species.

A. Prepare the CDA media with 25% concentration of the aqueous extracts of the test plants

(24)

B. About 15ml of the medium was poured into each petriplate

and allowed to solidify.

C. 7-days old culture of the test fungi were placed at the center

of the petriplate and incubated at 25 -27° C for seven days.

D. After incubation the colony diameter was measured in

millimeter,

E. For each treatment four replicates were maintained.

F. CDA medium without the aqueous extract served as control.

G. The fungitoxicity of the extract in terms of percentage

inhibition of mycelial growth was calculated by using the

formula.

%inhibition= dc-dt/dc x100

Where dc= Average increase in mycelia growth in control

dt= Average increase in mycelia growth in treatment

(Sing & Tripathi, 1999)

H. Synthetic fungicides viz; blitox(copper oxychloride),

Dithane M-45(Macozeb) were also tested at their recommended

dosage 2gm 1-1 for antifungal activity by poisoned food technique.

(25)

RESULT

Observation table: 1 Isolation of fungas by selected samples on CDA

media

Sr. no. Name of samples

Isolated fungas Medium Colony colour

1 Groundnut Aspergillus niger

CDA Black

2 Paddy Aspergillus fumigates

CDA Grayish

3 Sorghum Aspergillus terreus

CDA Brown

4 Maize Aspergillus oryzae

CDA White centre green yellow periphery

Observation table: 2 Growth of Aspergillus species on CDA media mm

(in millimeter) (Control plate)

Sr. no.

Fungus Growth in mmA B C D Average

1 Aspergillus niger 60 60 61 59 60 mm

2 Aspergillus fumigates 33 33 32 34 33 mm

3 Aspergillus oryzae 31 30 32 31 31 mm

4 Aspergillus terreus 39 38 40 39 39 mm

From the above table it was observed that Aspergillus niger

shown the highest growth followed by Aspergillus terreus, Aspergillus

fumigates and least of Aspergillus oryzae.

(26)

Observation table 3: Antifungal activity of mixture of Acacia nilotica

and Czapeck-Dox-Agar media on Aspergillus species

Sr.no.


1 Aspergillus niger 36 38 36 36 36.5 mm

2 Aspergillus fumigates 20 20 21 19 20 mm

3 Aspergillus oryzae 25 24 24 23 24 mm

4 Aspergillus terreus 17 18 17 16 17 mm

From the above observation table it was observed that plant extract

of Acacia nilotica shown the highest antifungal activity against

Aspergillus terreus followed by Aspergillus fumigates, Aspergillus

oryzae and least of Aspergillus niger

Observation table 4:- Antifungal activity of mixture of Azadirachta

indica and Czapeck-Dox-Agar media on Aspergillus species

Sr. no.

Fungus Growth in mm

A B C D Average


2 Aspergillus fumigates 6 5 5 5 5.25 mm

3 Aspergillus oryzae 14 15 14 14 14.25 mm

4 Aspergillus terreus 7 7 6 7 6.75 mm

From the above observation table it was observed that plant

extract of Azadirachta indica shown the highest antifungal activity

against

(27)

Aspergillus fumigatus followed by Aspergillus terreus,

Aspergillus oryzae and least of Aspergillus niger

Observation table 5: Antifungal activity of mixture of Emblica

officinalis and Czapeck-Dox-Agar media on Aspergillus species

Sr.

no.

Fungus Growth in mm

A B C D Average






extract of Emblica officinalis shown the highest antifungal activity

against Aspergillus terreus followed by Aspergillus fumigatus,


(28)

Observation table 6: Antifungal activity of mixture of Eucalyptus

globulis and Czapeck-Dox-Agar media on Aspergillus species

Sr.

no.

Fungus Growth in mm

A B C D Average






extract of Eucalyptus globulis shown the highest antifungal activity

against Aspergillus fumigatus followed by Aspergillus terreus,


Observation table 7: Antifungal activity of mixture of Lawsonia inermis

and Czapeck-Dox-Agar media on Aspergillus species

Sr. no.







extract of Lawsonia inermis shown the highest antifungal activity

(29)



Observation table 8 : Antifungal activity of Mixture of Ocimum

sanctum and Czapeck-Dox-Agar media on Aspergillus species

Sr. no.

Fungus Growth in mm

A B C D Average






extract of Ocimum sanctum shown the highest antifungal activity



(30)

Observation table 9: Antifungal activity of Mixture of Mancozeb and

Czapeck-Dox-Agar media on Aspergillus species

Sr. no.

Fungus Growth in mm

A B C D Average

1 Aspergillus niger 18 18 17 19 18 mm





extract of Mancozeb shown the highest antifungal activity against

Aspergillus fumigatus followed by Aspergillus niger, Aspergillus oryzae

and least of Aspergillus terreus.

Observation table 10 : Antifungal activity of Mixture of Copper

oxychloride and Czapeck-Dox-Agar media on Aspergillus species

Sr. no. Fungus

Growth in mm

A B C D Average



3 Aspergillus oryzae 16 19 17 17 17.25mm

4 Aspergillus terreus 11 11 13 12 11.75mm


extract of Copper oxychloride shown the highest antifungal activity

(31)

against Aspergillus fumigatus followed by Aspergillus terreus,

Aspergillus oryzae and least of Aspergillus niger.

From the above all table it was observed that Copper

oxychloride (Treatment plate) effective against Aspergillus niger

followed by Mancozeb (Treatment plate), Ocimum sanctum ( Treatment

plate), Azadirachta indica(Treatment plate), Lawsonia inermis (Treatment

plate), Acacia nilotica (Treatment plate), Eucalyptus globulis (Treatment

plate) and least of Emblica officinalis (Treatment plate) as compare to the

control plate.

Similarly Azadirachta indica (Treatment plate) effective

against Aspergillus fumigates followed by Copper oxychloride

( Treatment plate), Lawsonia inermis ( Treatment plate), Ocimum

sanctum (Treatment plate), Emblica officinalis (Treatment plate),

Mancozeb (Treatment plate), Eucalyptus globulis (Treatment plate) and

least of Acacia nilotica (Treatment plate) as compare to the control plate.

Similarly Ocimum sanctum (Treatment plate) effective

against Aspergillus oryzae followed by Azadirachta indica ( Treatment

plate), Copper oxychloride (Treatment plate), Emblica officinalis

(Treatment plate), Acacia nilotica (Treatment plate) Mancozeb

(Treatment plate) Lawsonia inermis (Treatment plate) and least of

Eucalyptus globulis (Treatment plate) as compare to the control plate.

(32)

Similarly Lawsonia inermis (Treatment plate) effective

against Aspergillus terreus followed by Azadirachta indica (Treatment

plate), Emblica officinalis (Treatment plate), Ocimum sanctum

(Treatment plate) , Copper oxychloride (Treatment plate), Acacia nilotica

(Treatment plate), Eucalyptus globulis (Treatment plate), and least of

Mancozeb(Treatment plate) as compare to the control plate.

(33)

Table 11: Antifungal activity of different plant extracts at 25% (v/v)

concentration against Aspergillus species.

Sr. no

Plant extract Pathogen

Aspergillus niger

Aspergillus fumigatus

Aspergillus oryzae

Aspergillus terreus

1 Acacia nilotica 39.16% 39.39% 22.58% 56.4%

2 Azadirachat a indica 62.08% 84.09% 54.03% 82.69%

3 Emblica officinalis 32.08% 65.00% 41.93% 82.69%

4 Eucalyptus globules 32.5% 38.63% 0.80% 37.825

5 Lawsonia inermis 41.25% 68.93% 5.64% 91.66%

6 Ocimum sanctum 66.25% 62.87% 54.64% 72.43%

7 Mancozeb 70% 46.21% 18.84% 32.43%

8 Copper oxychloride 71.25% 84.09% 44.35% 69.87%

Acacia nilotica extract shown highest antifungal activity of

Aspergillus terreus followed by Aspergillus fumigatus, Aspergillus niger

and least of Aspergillus oryzae. Azadirachta indica extract shown highest

antifungal activity of Aspergilus terreus followed by Aspergillus

fumigatus, Aspergillus niger and least of Aspergillus oryzae. Emblica

officinalis extract shown highest antifungal activity against of Aspergillus

terreus followed by Aspergillus fumigatus, Aspergillus oryzae and least

of Aspergillus niger. Eucalyptus globules extract shown highest

inhibition of Aspergillus fumigatus followed by Aspergillus terreus

Aspergillus niger and least of Aspergillus oryzae. Lawsonia inermis

extract shown highest antifungal activity of Aspergillus followed by

Aspergillus fumigatus, Aspergillus niger and least of Aspergillus Oryzae.

(34)

Ocimum sanctum extract shown highest antifungal activity of Aspergillus

terreus, followed by Aspergillus niger, Aspergillus fumigatus and least of

Aspergillus oryzae.

Among the six plants extract studied, Ocimum sanctum

inhibition growth Aspergillus niger to the highest extant followed by

Azadirachta indica, Lawsonia inermis, Acacia nilotica, Eucalyptus

globulus and Emblica officinalis. Copper oxychloride shown inhibition

upto 71.25% which can be compared with plants extract of Ocimum

sanctum which inhibition 66.25% fungal growth of Aspergillus niger.

Azadirachta indica and Copper oxychloride equally effected growth of

Aspergillus fumigatus (84.09%) to the highest extant followed by

Lawsonia inermis, Emblica officinalis, Ocimum sanctum, Acacia nilotica

and Eucalyptus globulus which is showing important of Azadirachta

indica in controlling fungal activity.

Ocimum sanctum inhibition shown the antifungal activity

against of Aspergillus oryzae to the highest extant followed by

Azadirachta indica, Emblica officinalis, Acacia nilotica, Lawsonia

inermis, & Eucalyptus globulus. Copper oxychloride shown inhibition

upto 44.35% and Macozeb upto the 18.84% whereas plant extract of

Ocimum sanctum and Azadirachta indica inhibited fungal growth more

then systhetic fungicides.

(35)

Lawsonia inermis inhibited growth of Aspergillus terrreus to

the highest extant followed by Azadirachta indica, Emblica officinalis,

Ocimum sanctum, Acacia nilotica & Eucalyptus globulus. Copper

oxychloride shown anti fungal activity upto 69.87% & Mancozeb upto

the 22.43% whereas plant extract of Ocimum sanctum and Lawsonia

inermis, Azadirachta indica, Emblica officinalis & Ocimum sanctum

inhibited fungal growth more then synthetic fungicides.

(36)

DISCUSSION

The present work was carried out to study "Antifungal

activity of some plant extract against important seed borne pathogen of

Aspergillus species." Antifungal activity of seven plant extracts was

assayed the seed sample were collected from different market and were

plated on czapeck-Dox-Ager medium and the plates were incubated at

25-27 ° C for 7 days after incubation the different colonies were obtained.

The isolated Aspergillus species were tested against different plant

extracts and synthetic fungicide for their antifungal activity

When seven plant extract were screened, for there antifungal

activity six plants shown the significant role for management of fungal

growth. The finding of the present investigation is an important step

towards crop protection strategies for antifungal activity against

important seed borne species of Aspergillus. The highest antifungal

activity was shown by Lawsonia inermis (91.66%), Emblica officinalis

(82.69%) and Azadirachta indica (82.66%) against Aspergillus terreus

aqueous extract of Eucalyptus globulus was not found so much effective

against Aspergillus species highest antifungal activity was (38.63%)

recorded against Aspergillus fumigatus and lowest (0.8%) against

Aspergillus oryzae. Acacia nilotica extract was effective against

Aspergillus terreus (56.4%) and inhibited Aspergillus oryzae to lowest

(22.50%).

(37)

Azadirachta indica shown the highest antifungal activity

against all Aspergillus species followed by Copper oxychloride, Ocimum

sanctum, Lawsonia inermis, Emblica officinalis,Mancozeb, Acacia

nilotica and least of Eucalyptus globulis variety of fungi including

species of Aspergillus causing significant loss in seed quality and

nutritional quality of grains have been reported (Koirala et al., 2005).

World Health Organization (WHO) banned many agriculturally important

pesticides due to wide range of toxicity against non-target organisms

including humans, which are known to cause pollution problem (Barnard

et al., 1997). Thus, there is an urgent need to search for alternative

method for prevention of biodeterioration of grains during storage

without any toxicity to the consumer. Many higher plants produce

economically important organic compounds, Pharmaceuticals and

pesticides, plant based secondary metabolites, which have defensive role

may be exploited for the control of fungal activity. There is urgent need

for screening/evaluation of diverse plants for their antifungal potential.

Biologically active plant derived pesticides are expected to play an

increasingly significant role in crop protection strategies.

The present investigation is an important step in developing

plant pesticides which are eco-friendly for the management of the seed

borne fungi and development of commercial formulation of botanicals.

Further investigation will be done for developing commercial formulation

based on field trail and toxicological experiment.

(38)

PHOTO GALLARY

(39)

(40)

(41)

(42)

SUMMARY AND CONCLUSION

The present study deals with the isolation and identification

of seed borne pathogen of Aspergillus species. From seed sample all the

isolates colony were identify by lactophend cotten blue staining,

macroscopic and microscopic study.

Seed sample were collected from different market and were

plated on czapeck-Dox-Ager medium and the plates were incubated at

25-27° C for 7 days after incubation the different colonies were obtained.

The isolated Aspergillus species. Tested an antifungal activity of different

plant extracts and synthetic fungicides.

Among the seven plant used for performing antifungal

activity of plant extract against Aspergillus species. Maximum six plants

were shown antifungal activity and more antifungal activity was shown

by four plants Azadirachta indica, Emblica officinalis, Lawsonia inermis

and Ocimum sanctum. Among these four, Azadirachta indica and

Ocimum sanctum shown significant antifungal activity against all

Aspergillus species.

The highest antifungal activity was shown by Lawsonia

inermis (91.66%), Emblica officinalis (82.69%) and Azadirachta indica

(82.66%) against Aspergillus terreus aqueous extract of Eucalyptus

globulus was not found so much effective against Aspergillus species.

(43)

Highest inhibition was (38.63%) recorded against Aspergillus fumigatus

and lowest (0.8%) against Aspergillus oryzae. Acacia nilotica extract was

effective against Aspergillus terreus (56.4%) and inhibited Aspergillus

oryzae to lowest (22.50%).

Copper oxychloride was effective against Aspergillus niger,

Aspergillus fumigatus, Aspergillus terreus inhibited fungal growth upto

more then 60% whereas Mancozed was found effective against

Aspergillus niger only.

From the study it can be concluded that Ocimum sanctum,

Lawsonia inermis, Emblica officinalis and Azadirachta indica extract can

be utilized for effective control of seed borne pathogen of Aspergillus

species.

It can be utilized for preparation of ecofriendly , economical

and harmless biological pesticides for prosperous humen life.

(44)

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