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

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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 Kingdom Phylum Class Order Family Genus Eukarya Fungi Ascomycota Eurotiomycetes Eurotiales Trichocomaceae 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
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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.

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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.

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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 Division Order Family Genus Species : : : : : : Plantae Magnoliophyta Sapindales Meliaceae Azadirachta indica
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(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 Angiosperms
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(unranked) :

(unranked) : (unranked) : Order Family Genus Species : : : :

Eudicots Rosids Fabales Fabaceae Acacia 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 Division Class Order Family Genus Species : : : : : : : Plantae Magnoliophyta Magnoliopsida lamiales Labiatae Ocimum sanctum (21) Tulsi has been widely known for its health promoting and medicinal value for thousands of years. Commonly called sacred or holy
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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 Division Class Order Family Plantae Magnoliophyta Magnoliopsida Myrtales Lythraceae

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Genus Species

Lawsonia 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 Angiosperms Eudicots Rosids Myrtales Myrtaceae Eucalyptus

(unranked) : (unranked) : (unranked) : Order Family Genus : : :

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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
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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 Division Class Order Plantae Angiospermae Dicotyledonae Geraniales
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Family Genus Species

Euphorbiaceae Emblica 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, anemia, skin diseases, leprosy,

haematogenesis,

inflammations,

emaciation,

hepatopathy,

jaundice, strangury, diarrhoea, dysentery, hemorrhages, leucorrhoea, menorrhagia, cardiac disorders, intermittent fevers and greyness of hair .(19)

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ALOE VERA Scientific classification Kingdom Order Family Genus Species : : : : : Plantae Asparagales Asphodelaceae Aloe 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 noncancerous cells. Researchers have found the cancer fighting properties of Aloe vera and are making it count.

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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)

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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
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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

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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,antipyretic,anti-histamine,anti-fungal Nimbidin: anti-bacterial, anti-ulcer, analgesic, anti-arrhythmic, anti-fungal. Ninbidol: anti-tubercular, antiprotozoan, .anti-pyretic, Gedunin: vasodilator, anti-malarial, anti-fungal, Sodium nimbinate:diuretic, spermicide, anti-arthritic, Quercetin:anti(17)

protozoal Salannin:insect repellent, Azadirachtin:insect repellent, antifeedant,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)

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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. 5. Purification of plant extract. study of antifungal activity by using terpenes,phenol,tannis alkaloids, quinines, and flavinoids from plant extract.

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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

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PLANT MATERIAL
List of plant species tested for antifungal activity Sr.No. 1 2 3 4 5 6 7 Name of the plant Acacia nilotica Aloe vera Azadirachta indica Emblica officinalis Eucalyptus globulis Lawsonia inermis Ocimum sanctum Family Mimosaceae Liliaceae Meliaceae Euphorbiaceae Myrtaceae Lythraceae Lamiaceae

Media Composition Czapeck-Dox-Agar(CDA) Ingredients Sucrose Sodium nitrate Dipotassium phosphate Magnesium sulphate Potassium chloride Ferrous sulphate Agar pH(at25C) Distilled water Gms/Litre 30.00 2.00 1.00 o.50 0.50 0.01 15.00 7.3+/- 0.2 1000

The above mentioned media were prepared and sterilized by autoclaving at 15 1bs pressure for 15-20 minutes.
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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. CzapeckDox-agar supports abundant growth of almost all saprophytic Aspergilli with characteristic mycelia and conidia formation. 3. Transfer of samples on mediaOne 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.

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

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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. F. G.

For each treatment four replicates were maintained. CDA medium without the aqueous extract served as control. 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.

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RESULT
Observation table: 1 Isolation of fungas by selected samples on CDA media Sr. no. 1 2 3 4 Name of samples Groundnut Paddy Sorghum Maize Isolated fungas Medium Aspergillus niger Aspergillus fumigates Aspergillus terreus Aspergillus oryzae CDA CDA CDA CDA Colony colour Black Grayish Brown White centre green yellow periphery

Observation table: 2 Growth of Aspergillus species on CDA media mm (in millimeter) (Control plate) Sr. Fungus no. 1 2 3 4 Aspergillus niger Aspergillus fumigates Aspergillus oryzae Aspergillus terreus Growth in mm B C D Average 60 33 30 38 61 32 32 40 59 34 31 39 60 mm 33 mm 31 mm 39 mm

A 60 33 31 39

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.

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Observation table 3: Antifungal activity of mixture of Acacia nilotica and Czapeck-Dox-Agar media on Aspergillus species
Sr. no. 1 2 3 4 Fungus A Aspergillus niger Aspergillus fumigates Aspergillus oryzae Aspergillus terreus 36 20 25 17 B 38 20 24 18 Growth in mm C D 36 21 24 17 36 19 23 16

Average 36.5 mm 20 mm 24 mm 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. 1 2 3 4 Fungus A Aspergillus niger Aspergillus fumigates Aspergillus oryzae Aspergillus terreus 23 6 14 7 B 24 5 15 7 Growth in mm C 23 5 14 6 D 21 5 14 7 Average 22.75 mm 5.25 mm 14.25 mm 6.75 mm

From the above observation table it was observed that plant extract of Azadirachta indica shown the highest antifungal activity against

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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. 1 2 3 4

Fungus A Aspergillus niger Aspergillus fumigates Aspergillus oryzae Aspergillus terreus 40 13 19 7 B 40 12 18 6

Growth in mm C 42 13 18 6 D 41 13 17 8 Average 40.75 mm 12.75 mm 18.00 mm 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

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Observation table 6:

Antifungal activity of mixture of Eucalyptus

globulis and Czapeck-Dox-Agar media on Aspergillus species Sr. no. 1 Aspergillus niger Fungus A 40 20 30 24 B 42 20 30 25 Growth in mm C 40 21 32 24 D 40 20 31 24 Average 40.5 mm 20.25 mm 30.75 mm 24.25 mm

2 Aspergillus fumigates 3 4 Aspergillus oryzae Aspergillus terreus

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. 1 2 3 4 Fungus A Aspergillus niger 35 36 10 30 4 B Growth in mm C D 35 11 29 3 35 10 29 3

Average 35.25 mm 10.25 mm 29.25 mm 3.25 mm

Aspergillus fumigates 10 Aspergillus oryzae Aspergillus terreus 29 3

From the above observation table it was observed that plant extract of Lawsonia inermis shown the highest antifungal activity
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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. 1 2 3 4

Fungus A Aspergillus niger Aspergillus fumigates Aspergillus oryzae Aspergillus terreus 20 12 14 10 B 20 12 14 10

Growth in mm C 21 13 14 11 D 20 12 13 10 Average 20.25 mm 12.25 mm 13.75 mm 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

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Observation table 9: Antifungal activity of Mixture of Mancozeb and Czapeck-Dox-Agar media on Aspergillus species Sr. no. 1 2 3 4 Fungus A Aspergillus niger Aspergillus fumigates Aspergillus oryzae Aspergillus terreus B Growth in mm C 19 18 26 30 D Average 18 mm 17.75 mm 25.25 mm 30.25 mm

18 18 17 17 17 19 25 24 26 30 31 30

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. Growth in mm Fungus A 18 6 16 11 B 18 5 19 11 C 16 4 17 13 D 17 6 17 12 Average 17.25 mm 5.25 mm 17.25mm 11.75mm

1 Aspergillus niger 2 Aspergillus fumigates 3 Aspergillus oryzae 4 Aspergillus terreus

From the above observation table it was observed that plant extract of Copper oxychloride shown the highest antifungal activity
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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.

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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.

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Table 11:

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

concentration against Aspergillus species.

Sr. no Plant extract Pathogen Aspergillus Aspergillus niger fumigatus 39.16% 62.08% 32.08% 32.5% 41.25% 66.25% 70% 71.25% 39.39% 84.09% 65.00% 38.63% 68.93% 62.87% 46.21% 84.09% Aspergillus oryzae 22.58% 54.03% 41.93% 0.80% 5.64% 54.64% 18.84% 44.35% Aspergillus terreus 56.4% 82.69% 82.69% 37.825 91.66% 72.43% 32.43% 69.87%

1 Acacia nilotica 2 Azadirachat a indica 3 Emblica officinalis 4 Eucalyptus globules 5 Lawsonia inermis 6 Ocimum sanctum 7 Mancozeb 8 Copper oxychloride

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.
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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.

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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.

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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%).

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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.

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PHOTO GALLARY

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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.
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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%) 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. and inhibited Aspergillus

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