View of Effect of Some Plant Extracts: In Vitro Growth of Alternaria Sps

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Effect of Some Plant Extracts: In Vitro Growth of Alternaria Sps

Naina Srivastava

Department of Botany D.A.V. (P.G.) College, Dehradun, Uttar Pradesh, India

Abstract:

Alternaria usually attacks aerial parts of its host and symptoms appear as small, circular, dark spots. Control of this pathogen has become an obligatory requirement. In the present study, specie of Alternaria (Alternaria alternata) were selected on the basis of the ability to cause economically important plant diseases.Bioassays revealed that among aqueous and methanol extracts; methanol extract caused maximum inhibition of both the Alternaria species at high concentration of 4% after 5 and 10 days of incubation period. A. alternata displayed maximum inhibition of 100% at higher concentration of 4% after 5 days and 94% after 10 days of incubation.

Alternaria species, ubiquitous post harvest pathogens, contribute to the spoilage of 55% of the agricultural output. The study was to evaluate the antifungal activity of extracts some plant species against Alternaria spp.Methanolic extracts from different parts of Polygonum perfoliatum Cymbopogon citratus ,Lantana camara and Mimosa pudica were evaluated for potential antimicrobial activity against Alternaria strains. These plants can be used to discover bioactive natural products that may serve as leads in the development of new pharmaceuticals that address not fulfilled therapeutic needs.

Key words: Alternaria alternata, Methanolic extract, Agar well diffusion technique.

Introduction

Minimal inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of the extracts were determined medicinal plants contained substances inhibitory to conidial germination of Alternaria The present study aimed at evaluating the in vitro antimicrobial activity of methanolic extracts of medicinal plant against Alternaria alternata, solani using agar well diffusion technique. These extracts showed maximum activity, even at very low concentrations, and the same fungicide effects as chemical fungicide.This study concludes that aqueous and methanol extracts of aloe vera,Polygonum perfoliatum Cymbopogon citratus ,Lantana camara and Mimosa pudica possess the ability for substantial inhibition in growth of A. alternata and A. tenuissima and these extracts exhibit amazing fungicidal properties that support their traditional use as antiseptics formulations of plant extracts could have important roles in biologically based management strategies for control of diseases caused by Alternaria.

The plants used, as drugs are fairly innocuous and relatively free from toxic effects or were so toxic that lethal effects were well known. The nature has provided the storehouse of remedies to cure all ailments of mankind. There is no doubt that plants are a reservoir of potentially useful chemical compounds which serve as drugs, are provided newer leads and clues for modern drug design by synthesis.

Alternaria genus is cosmopolitan in occurrence. The members of this species like A.alternata, A. solani, A. porri, A. dauci, A. helianthi, A. carthami and A. macrosporecausing different diseases in their respective hosts (Rotem, 1998).

synthesis. There are several reports of antibiotic resistance of human pathogens to available antibiotics J., Mitsuyama et al, L .Gutmann et al, A. J Mathias., et al, R. Gangulyet al, Biomolecules of plant origin appear to be one of the alternatives for the control of theseantibiotic resistant human and plant pathogens besides small molecules from medicinal chemistry, natural

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products are still major sources.

Many researchers have tried to find safe and economical control of plant diseases by using extracts of different plant parts (Bajwa et al., 2004; Bajwa and Iftikhar, 2005; Shafique et al., 2011).

Among them, A. solani causing early blight of potato (Solanum tuberosum) and tomato (Lycopersiconesculentum) is the most destructive (Reni & Roeland, 2006) of field crops. It causes diseases on foliage (blight), basal stems of seedlings (collar rot and damping off), stems of adult plants (stem lesions), fruits (fruit rot) of tomato and may also infect egg plant and pepper. This disease can be very destructive if left uncontrolled, often resulting in complete defoliation of plants (Dater & Mayee, 1985). Nature has bestowed upon us a very rich botanical wealth and a large number of diverse types of plants grow wild in different parts of our country Indian medicinal plants are regularly used in various system of medicine because of minimal side effect and cost effectiveness. In this study, we evaluated the antimicrobial activity of methanolic extracts of Lantana camara and Mimosa pudica against several pathogenic microorganisms.

Although, chemical control suggests being the best method to control Alternaria diseases however in recent years, use of chemicals has increased consumer concern and their use is becoming more restricted due to carcinogenic effects, residual toxicity problems, environmental pollution, occurrence of microbial resistance, high inputs etc. (El-Rokiek et al., 2006).

Biological screening of plant extracts is carried out throughout the world for the determination of their antifungal activity. Synthetic chemicals used to control plant diseases not only pollute the environment, but are also harmful to human health. Because of environmental and economic considerations, plant scientists are involved to find the cheaper and more environmental friendly bio-compounds for the control of plant diseases using diffusates from different plants (Gerresten & Haagsma, 1951; Kumar et al., 1979; Naidu & John, 1981).

Many studies have shown that aromatic and medicinal plants are sources of diverse nutrient and non nutrient molecules, many of which showed antioxidant and antimicrobial properties which can protect the human body against both cellular oxidation reactions and pathogens. Thus it is important to characterize different types of medicinal plants for their antimicrobial potential (Mothana & Lindequist, 2005; Bajpai et al., 2005; Wojdylo et al., 2007).

The most common method for controlling these pathogens is the use of fungicides but the development of resistance in pathogenic fungi to common fungicides and increasing residual hazardous effects on human health and environmental pollution has given a thrust to search for new plant derivatives that can obstruct the fungal pathogencity. Use of natural products for the management of fungal diseases in the plants is considered as a good alternate to synthetic fungicides, due to their less negative impact on the environment. Many higher plants and their constituents have been successful in plant disease control and proved to be safe and nonphytotoxic;

unlike chemical fungicides. Three weedy plants namely Lantana camara and Capparis decidua has been used for this purpose (Sharma & Kumar, 2009).

Plant diffusates are not only easy to prepare but are also non-polluting and low priced as compare to commercial fungicides. Keeping in view, some plant diffusates were evaluated for their antifungal activity against Alternaria solani.

Materials and Methods

This work was conducted in Department of Botany, D.G.(P.G.) COLLEGE KANPUR determine the antifungal activities of Aloe vera,Polygonum perfoliatum Cymbopogon citratus ,Lantana camara and Mimosa pudica selected fungal pathogen viz., Alternaria alternata in methanol by employing food poisoning technique (Naz et al., 2006).

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Isolation of pathogens: pathogens viz., Alternaria alternata was selected for this experimental work. Pure culture of Diseased samples were surface sterilized with 5% Chlorox for one minute and washed three times with sterilized distilled water.

Preparation of pure culture: Pathogen were isolated with the help of sterilized forceps and plated on sterilized potato dextrose agar (PDA) medium (potato starch: 20 g, dextrose: 20 g, agar: 20 g and distilled water to make the volume 1 liter, which was sterilized in a gas operated autoclave at 15 pounds pressure per square inch (PSI) for 20 minutes. Plates were incubated at 25ºC and observed daily for emergence of colonies.Sub-culturing was done from single spore to obtain pure culture.

Collection and preservation of plants samples: Fresh leaves of Aloe vera,Polygonum perfoliatum Cymbopogon citratus ,Lantana camara and Mimosa pudica were collected from company garden kanpur. These were washed with tap water and air dried for one day to eliminate surface moisture.

Then leaves were packed into envelop and kept in oven at 60°C temperature until dried. Dried leaves were grinded separately in an eclectic grinder to obtain powder which was than kept in plastic bags for further use.

Preparation of extracts: The crude extracts of the different plant parts of different species were subjected to antimicrobial assay using the agar well diffusion method of Murray et al., 1995 modified by Olurinola 1996 Hundred gram of the dried powdered plant were soaked separately in 500ml of 98% ethanol. These mixtures were refluxed followed by agitation at 200 rpm (revolution per minute) for 1 hour. The ethanolic extracts were squeezed and then filtered by muslin cloth. The extracts were placed into a wide tray to evaporate ethanol and added with water to make plant extracts.

Food poison technique: Diffusates were added in Potato dextrose agar (PDA) @ 10, 50, 100 and 200 g L-1 and poured into Petri dishes. PDA medium added only with ethanol and water served as control. Each Petri dish was inoculated with 5 mm plug of pure isolate taken from margins of actively growing culture of pathogen. Then Petri plates were incubated at 25° ± 2oC.

Mycelial growth was recorded when the growth of three selected pathogens were completed in the control treatment. Each treatment was repeated five times. Mean radial mycelial growth of each plant diffusates was recorded and data were subjected to statistical analysis. Radial mycelial growths on different diffusates were transformed into inhibition percentage by using the following formula (Naz et al., 2006):

Mycelial growth on diffusates Inhibition percentage =100 - Mycelial growth control X 100

The agar plate diffusion plate method (Nene & Thaplliyal, 1979) was used to test antifungal activity of plants against plant pathogen.

Name of plants %Inhibition Inhibition in diameter

Aloe vera (77.85%) 4nm

Polygonum perfoliatum (66.96%) 3.2nm

Cymbopogon citratus (55.59%) 2.4nm

Lantana camara (52.90%) 1.5nm

Mimosa pudica (37.87%) .88nm

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Results and Discussion:

Inhibition in radial mycelial growth (rmg) of Alternaria solani induced by five medicinal plants:

Highest Inhibition in radial mycelial growth % induced by Alternaria solani was exhibited by Aloe vera (67.85%) Polygonum perfoliatum(56.96%) followed by Cymbopogon citratus (45.59%),,Lantana camara (42.90%) and Mimosa pudica (27.87%) difference found in inhibition of radial mycelial growth between Cymbopogon citratus (45.59%) and ,Lantana camara (42.90%), whereas significant difference in inhibition of radial mycelial growth was found between Polygonum perfoliatum (56.96%) and Cymbopogon citratus (45.59%). Lemongrass extract decreased Fusarium verticillioides growth in PDA by 90 and 100% at 500 and 1000 ppm, respectively, being in accordance with the present study (Mishra and Dubey, 1994). In the same way, Baratta et al. (1998) reported 91% inhibition of the growth of A. niger in liquid culture media when treated with 1000 ppm lemongrass extract.

0.00%

10.00%

20.00%

30.00%

40.00%

50.00%

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%Inhibition

Aloe vera

Polygonum perfoliatum Cymbopogon citratus Lantana camara Mimosa pudica

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

Inhibition in diameter

Aloe vera

Polygonum perfoliatum Cymbopogon citratus Lantana camara Mimosa pudica

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Results were found cognizant with previous findings of Hassanein et al., (2008) that the extract obtained from Lantana camara completely suppressed the growth of Alternaria solani on PDA In vitro.Statistically, significant increase in radial mycelial growth of Alternaria solani was observed with an increase in concentration of the diffusates (Fig. 2). Among 5 concentrations, the highest inhibition in radial mycelial growth was 55.72% at 200g/l followed by 48.60% at 100g/l, 44.51 at 50g/l and lowest inhibition was recorded 24.49 at 10g/l as compared to control. Similar results were recorded from preliminary investigations by Hassanein et al., (2008) reporting antifungal activity of Azadirachta indica leaf extract against Alternaria solani.

There are many examples in literature which support these findings. Similarly, Bajwa et al.

(2007) carried out the study on antifungal activity of aqueous and n-hexane shoot extracts of Aloe vera against A. alternata, Alternaria citri and A. tenuissima. They reported that inhibitory effect was variable with applied concentrations and caused a significant inhibition in biomass production of the three test fungi. Alternaria alternata had strong antifungal properties towards Aloe vera gel at same concentration . The result inclose conformity with the finding of Yolanta & Galon (1995) who tested antifungal activity of natural Aloe vera gel on plant pathogenic fungi Alternaria alternata.

The current results demonstrate that selected medicinal plant diffusates effectively suppressed the radial mycelial growth of Alternaria. The highest inhibition (77.85%) was recorded at 200g/l concentration in radial mycelial growth of pathogen, followed by (66.96%) followed by Cymbopogon citratus (45.59%),,Lantana camara (52.90%) and Mimosa pudica (37.87%) 100g/l (80.20%), 50g/l (62.11%), whereas the least inhibition of radial mycelial growth (42.90%) was observed at 10g/l. Statistically, the rate of increase in radial mycelial growth inhibition was regressed against five different concentrations of plant diffusates.

Biocontrol is the safest and economical method of controlling plant pathogens by using extracts of different plant parts . Presently, the study was conducted to assess the in vitro efficacy of aqueous and methanol extracts of C. citratus against two pathogenic test species of Alternaria.

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