MATERIALS (21). Partially purified extracts in different solvents were

MATERIALS
AND METHODSCollection
and extract preparation of test plantsLeaves of Polyalthia longifolia and
lawsonia inermis,
fruit pulp of Cassia fistula and seeds
of Pongamia pinnata collected from
University College of Science campus, Udaipur. Collected plant parts were disinfected
with 0.1% HgCl2 and subsequently washed with distilled water. All
plant parts were shade dried at room temperature and powdered mechanically. The ground material was passed through sieve
No. 240 so as to obtain powder of mesh size 60, which was used to prepare
extract. 100% alcoholic and 50% alcoholic crude extracts was prepared according
to modified method of (21). Partially purified extracts in different
solvents were prepared by hot extraction methods (22-23). Test crop and fungus: Alternaria solani was
isolated from infected fruit of tomato collected from the local market of
udaipur. Identification of fungus was done by Dr. T. Prameela Devi (Head,
Division of Plant Pathology, Indian Type Culture Collection, IARI, New Delhi).
Tomato (Raja-1) seeds were collected from local market of Udaipur. Inoculum
development: Potato dextrose agar (PDA) medium which was sterilized in
an autoclave at 15 psi for 20 minutes was used for culture of Alternaria
solani. The inoculated petriplates were incubated at 250 ºC for seven days.
Pure culture thus obtained was used for mass culture of Alternaria solani. Preparation of Herbal Formulation: Herbal formulations
were prepared by using all test plant part extracts elicitor and binder in
different ratio. 20 gm elicitors like neem, mustard binders like cow dung were
dissolved in 100 ml of autoclaved water for 24 h. The mixture was then filtered
and used for antifungal activity.100% of neem oil cake, 100% mustard oil cake
and 100% of cow dung solutions were mix with crude extract and partially
purified extract. All ingredients of herbal formulation were used in following
combinations: Crude extracts: Neem oil cake: cow dungCrude extracts: Mustard oil cake: cow
dungPartially purified extract: Neem oil
cake: cow dungPartially purified extract: Mustard oil cake: cow
dungAntifungal
activity of herbal formulations: Antifungal activities of these formulations
were done by poison food technique (24) against Alternaria solani. 18 ml of molten sterile potato dextrose media
were mixed with 2 ml of formulation (Plant extract: oilcake: cowdung).This
mixture was poured into pre-sterilized petri-plates and allowed to solidify at
room temperature. Thus prepared petri plates were inoculated aseptically with 6
mm disc of test cultures. The plates were then incubated at 28±2°C for 3 days.
Antifungal activity was measured as a function of increase in growth of 6 mm
disc of inoculums of 7 days old culture of the fungus was aseptically
inoculated upside down in the centre of the Petriplate and incubated at 25±2ºC.
On the 7th day of incubation average diameter of the fungal colonies was
measured and percentage of mycelia growth inhibition was calculated by the
following formula given below.% Mycelial
growth inhibition = gc-gt / gc× 100Where,gc = Growth of mycelial colony after incubation period in control set
subtracting the diameter of inoculumsdisc;gt = Growth of mycelial colony after incubation period in treatment set
subtracting the diameter ofinoculum disc.      RESULTS AND DISCUSSIONAntifungal
activity of plant extracts, oilcake and cow dung were observed in the presented
case. The combination of plant extract, oilcake and cow dung in the form of
Bioformulation showed great inhibitory activity against A.solani. A.solani is a
causal agent of blight disease in tomato and potato crop. Plants and plant
based natural compounds and organic materials have thousands of constituents
and are important sources of new ecofriendly and biologically active molecules
show antimicrobial property. (25) The use of natural products for the control
of fungal diseases in plants is considered as an interesting alternative to
synthetic fungicides due to their less negative impacts on the environment (26).
In the present study leaf extract of
Lawsonia inermis, Polyalthia longifolia fruit pulp extract of Cassia fistula and seed extract of Pongamia pinnata were prepared and used
for develop the Bioformulation with mustard oilcake, neem oilcake extract and
cowdung extract.10 mg/ml concentration of all test plant extracts were mix with
100% of neem oilcake, mustard oilcake and cow dung solution. In this series 28 formulation
ratios were prepared and assayed for antifungal activity against A.solani. Among 28 formulations,
formulation no 2, 16, 20 and 28 showed best inhibitory activity against test
pathogen and percent inhibition of these formulations was 82.85%, 82%, 81.42
and 80% respectively. Extracts from higher plants that contain antifungal
substances in form of secondary metabolites, which help in resisting the
pathogens. The extraction of any plant material with solvent will yield a
mixture of compounds. The extract may contain a wide variety of compounds like
alkaloids, phenols, tannins, flavonoids, Volatile oils, Saponins and
Carbohydrates etc. neem cake and mustard cake proved superior and inhibited the
growth of pathogens also.(27) However,
this is the first report on comparative efficacy of different plant exracts in
combination with neem products, mustard products and cow dung used to control
the in vitro growth of A.solani.
Studies of various researchs suggested that neem products like neem leaf
powder, neem oil, neem seed powder completely suppress the plant pathogenic
fungi .(28) but neem oilcake in combination with plant extract and cow dung is
newly reported in this study. Plant preparations have been used for centuries
in medicine and pest control. Farmers in India use neem leaves to protect their
stored grain from insects. Herbs and spices, such as basil and clove, have been
used by many workers to protect food from spoilage, as both have antimicrobial property.
The antimicrobial activities of Lawsonia
inermis, Polyalthia longifolia, Cassia
fistula and Pongamia pinnata plant
parts have been studied earlier by many scientists (29-30) Leaf extract of Lawsonia inermis, Polyalthia longifolia, seed extract of Pongamia pinnata and Cassia fistula fruit pulp extract showed
antifungal activity against Aspergillus niger, Aspergillus
clavatus, Candida albicans. Solarizing soils plus use of
appropriate organic materials have also been observed to actuate a chain
reaction of chemical and microbial degradation, which increase toxicity against
soil flora and fauna, especially soil borne plant pathogens. These possibly
contributed to the high nutrient contents, which could be available with
organic manure amendment (31).

In present
study, we examined the In vitro
activity of  herbal formulation which
contains crude and partially purified extract of Lawsonia inermis, Polylathia
longifolia leaves  seed extract of Pongamia pinnata and  fruit pulp extract of Cassia fistula neem
oil cake, mustard oilcake and cow dung against Alternaria solani.

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