r_pr_msc micro
TRANSCRIPT
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)
Scientific classification
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]
Scientific classification
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)
Scientific classification
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)
Scientific classification
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)
Scientific classification
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
Scientific classification
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 anti- bacterial 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.
Fungus Growth in mmA B C D Average
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
1 Aspergillus niger 23 24 23 21 22.75 mm
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
1 Aspergillus niger 40 40 42 41 40.75 mm
2 Aspergillus fumigates 13 12 13 13 12.75 mm
3 Aspergillus oryzae 19 18 18 17 18.00 mm
4 Aspergillus terreus 7 6 6 8 6.75 mm
From the above observation table it was observed that plant
extract of Emblica officinalis shown the highest antifungal activity
against Aspergillus terreus followed by Aspergillus fumigatus,
Aspergillus oryzae and least of Aspergillus niger
(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
1 Aspergillus niger 40 42 40 40 40.5 mm
2 Aspergillus fumigates 20 20 21 20 20.25 mm
3 Aspergillus oryzae 30 30 32 31 30.75 mm
4 Aspergillus terreus 24 25 24 24 24.25 mm
From the above observation table it was observed that plant
extract of Eucalyptus globulis shown the highest antifungal activity
against Aspergillus fumigatus followed by Aspergillus terreus,
Aspergillus oryzae and least of Aspergillus niger
Observation table 7: Antifungal activity of mixture of Lawsonia inermis
and Czapeck-Dox-Agar media on Aspergillus species
Sr. no.
Fungus Growth in mmA B C D Average
1 Aspergillus niger 35 36 35 35 35.25 mm
2 Aspergillus fumigates 10 10 11 10 10.25 mm
3 Aspergillus oryzae 29 30 29 29 29.25 mm
4 Aspergillus terreus 3 4 3 3 3.25 mm
From the above observation table it was observed that plant
extract of Lawsonia inermis shown the highest antifungal activity
(29)
against Aspergillus terreus followed by Aspergillus fumigatus,
Aspergillus oryzae and least of Aspergillus niger
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
1 Aspergillus niger 20 20 21 20 20.25 mm
2 Aspergillus fumigates 12 12 13 12 12.25 mm
3 Aspergillus oryzae 14 14 14 13 13.75 mm
4 Aspergillus terreus 10 10 11 10 10.75 mm
From the above observation table it was observed that plant
extract of Ocimum sanctum shown the highest antifungal activity
against Aspergillus terreus followed by Aspergillus fumigatus,
Aspergillus oryzae and least of Aspergillus niger
(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
2 Aspergillus fumigates 17 17 19 18 17.75 mm
3 Aspergillus oryzae 25 24 26 26 25.25 mm
4 Aspergillus terreus 30 31 30 30 30.25 mm
From the above observation table it was observed that plant
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
1 Aspergillus niger 18 18 16 17 17.25 mm
2 Aspergillus fumigates 6 5 4 6 5.25 mm
3 Aspergillus oryzae 16 19 17 17 17.25mm
4 Aspergillus terreus 11 11 13 12 11.75mm
From the above observation table it was observed that plant
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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