Blight disease is caused by Alternaria solani belongs to the sub-division Deuteromycotina, class Hyphomycetes, family Dematiaceae. The genus is characterized by the formation of polymorphous conidia either singly or in short or longer chains and provided with cross, longitudinal as well as oblique septa and having longer or short beaks. The conidia are porospores produced from simple, dark, septate conidiophores in simple and borne singly. (1)
Among the different diseases caused by the genus Alternaria, blight disease is one of the most dominant one that causes average yield loss in the range of 32-57%(2).Symptoms of this disease include presence of irregular, often circular brown to dark brown colour leaf spots on the leaves with concentric lines inside the spots. Often the circular spots coalesce to form large patches resulting in the leaf blight. In several cases, small dark coloured spots are also formed on pods and tender twigs. (3).
For Alternaria blight management, early sowing (4) of properly stored clean certified seeds after deep ploughing along with clean cultivation, timely weeding and maintenance of optimum plant population , avoidance of irrigation at flowering and pod formation stages are some of the steps to be followed for an efficient management of the disease.Since a number of Alternaria species infect crops of economic importance, there is a strong need to effectively control for this pathogen. There are different methods which are there for needed for its control
The life cycle starts with the fungus over wintering in crop residues or wild members of the Solanaceae family. In the spring, conidia are produced. Multicellular conidia are splashed by water or by wind onto an uninfected plant. The conidia infect the plant by entering through small wounds, stomata, or direct penetration. Infections usually start on older leaves close to the ground. The fungus takes time to grow and eventually forms a lesion. From this lesion, more conidia are created and released. These conidia infect other plants or other parts of the same plant within the same growing season. Every part of the plant can be infected and form lesions. (5).
Alternaria solani infects stems, leaves and fruits of potato (S. tuberosum), tomato (Solanum lycopersicum L.), eggplant (S. melongena L.), bell pepper and hot pepper (Capsicum spp.), and other members of the Solanum family. Distinguishing symptoms of A. solani include leaf spot and defoliation, which are most pronounced in the lower canopy.
There are numerous fungicides on the market for controlling early blight. Some of the fungicides on the market are azoxystrobin, pyraclostrobin, chlorothalonil, copper products, hydrogen dioxide, mancozeb, potassium bicarbonate, and ziram. The most common and effective method for the control of early blight is through the application of foliar fungicides. Protectant fungicides recommended for late blight control (e.g. maneb, mancozeb, chlorothalonil, and triphenyl tin hydroxide) are also effective against early blight. (6)
On the other hand the pesticide treatment is not protected as chemicals pollute environment, effect health vulnerability in humans and when these harmful chemicals enter into the food chain become hazardous to all living entities (7). Botanical derivatives like phenols, tannins, and flavonoids are environmentally safe and may be used as an alternative to commercial fungicides for controlling pathogenic fungi (8).
Plants are rich in a wide variety of secondary metabolites such as tannin, terpenoids, alkaloids, flavonoids, phenols, steroids glycosides and volatile oils etc. These secondary metabolites posses antimicrobial property as well as play an important role in defense against attack by insects and herbivores (9) Investigation of antimicrobial properties of plants identifies them as but for developing a medicinal formulation from plants
The world is gradually turning to bioformulations which are known to be effective against a large repertoire of diseases and ailments. More importantly, they are not known to cause any notable derogatory effects and are readily available at affordable prices (10). However, add a note of caution stating that plant remedies are effective and without side-effects, provided they are selected properly and taken under proper medical supervision. Various organic substances such as cow dung, neem oil cake, mustard oil cake, coconut oil cake etc. are used by farmers as organic manure (11). Some workers also reported the significant antifungal properties of the organic substances (12). The present work proposes to develop a formulation by combining plant extracts with these traditionally used organic substances for control of early blight of tomato caused by Alternaria solani. This type of formulation will provide a cheap and environmentally safe herbal fungicide that can be further used as a commercial herbal bio-control agent. In the recent times, agricultural scenario is witnessing a trend towards organic farming. It is seen that agricultural products produced through organic farming, without using any inorganic fertilisers and pesticides, fetch high demand and price in the international market. Organic farming has its root in Nature and it makes use of only organic materials. Organic farming is not for a single crop but it envisages the entire farm. The main objective of organic farming includes mulching, crop rotation, cover cropping green manuring, animal waste, composting, bio-gas slurry, biofertilisers and organic recycling.
The world is gradually turning to bioformulations which are known to be effective against a large repertoire of diseases and ailments. More importantly, they are not known to cause any notable derogatory effects and are readily availabile at affordable prices. However, add a note of caution stating that plant remedies are effective and without side-effects, provided they are selected properly and taken under proper medical supervision. The active component, most often a secondary metabolite, varies in quality and quantity for a given plant species growing in different locations. The market value of such plants depends on their active content rather than merely their luxuriant growth. Reckon that close to 70,000 species of the plant kingdom have been used as herbal medicine at one time or other (13) discriminate use of fungicides may lead to toxic residues, development of fungicide resistance, environmental contaminations and carcinogenic, teratogenic and mutagenic effects in humans, animals and plants (14). In this context, biocontrol approaches may help to develop an eco-friendly control stratagey for management of serious plant diseases. Recent studies have shown the importance of biocontrol agents as possible non- phytotoxic systemaic and easily biodegradable alternative fungicides. (15).
Folk medicines of almost all civilizations of the world abound in herbal remedies. Majority of the traditional medicines used in healthcare are obtained from plants (16). In spite of several advancements in the field of synthetic drug chemistry and antibiotics, plants continue to be one of the major raw materials for drugs treating various ailments of humans. Clinical and pharmaceutical investigations have in fact elevated the status of medicinal plants by identifying the role of active principles present in them and elaborating on their mode of action in human and animal systems (17). Some workers noticed that the neem seed oil has more effective than the powder formulation in reducing egg-laying and adult emergence of the bruchid (18).The excessive misuse of a wide range of fungicides has led to it being harmful to the environment and increases the resistant pathogen populations (19). For this reason, alternative methods to control the disease had been studied with emphasis on biological control using fungi or bacteria to reduce fungicide application and decrease cost of production. Biological control has the potential to manage plant disease which occurred through different mechanisms such as antibiosis, competition, suppression, direct parasitism, induced resistance, hypovirulence and predation.
Hence, there would be no chance of residual toxicity of the oil on taste of the commodities. Similarly there are several reports which showed that herbal formulations or plant extracts do not leave any harmful residue (20), thus the chance of residual toxicity in case of extracts is also very less
MATERIALS AND METHODS
Collection and extract preparation of test plants
Leaves 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 dung
Crude extracts: Mustard oil cake: cow dung
Partially purified extract: Neem oil cake: cow dung
Partially purified extract: Mustard oil cake: cow dung
Antifungal 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× 100
gc = Growth of mycelial colony after incubation period in control set subtracting the diameter of inoculums
gt = Growth of mycelial colony after incubation period in treatment set subtracting the diameter of
RESULTS AND DISCUSSION
Antifungal 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.