ABSTRACT

Tomato (Lycopersicon esculentum Mill.) is one of most popular vegetables grown all over the world, particularly in developing countries. Tomato is vulnerable to many pathological problems which are most serious and significant in reducing yield and cause economic losses. Among these, Early blight of tomato caused by Alternaria solani has been reported to be most prevalent and destructive through out the tomato growing areas causing loss of million of dollars annually worldwide including Pakistan and is one of the major limiting factors in tomato production in the country. For the management of disease the use of Chemical treatments are prohibitively expensive besides the problems related to environmental pollution, chemical toxicity to humans and animals and pathogen resistance so in view of the high cost of chemical pesticides and their hazardous consequence, controlling the plant diseases by pollution free biocontrol methods are desirable now a days and are probably one of the most used approaches , which also provide an environmentally and economically appropriate means of the management of Early blight disease in Tomato. This research work has been proposed to assess the efficacy of bio-control by use of biodegradable material like fresh plant extracts and botanicals against early blight of tomato.

INTRODUCTION
Tomato (Lycopersicon esculentum Mill.) belongs to the family solanaceae and is one of most popular vegetable and a major horticultural crop grown all over the world and occupies a prominent postion in world vegetable economy (Chapagain and Wiesman, 2004). It is second most 1

consumed vegetable after potato, ranks first among the processing crops and unquestionably most popular garden crop (FAOSTAT, 2007). About 152956115 tonnes of tomatoes were produced in the world in 2009, (FAOSTAT, 2009). China is the largest producer of tomato in world accounted for about one quarter of the global production followed by USA and Turkey, where united state is leading importer received 25 % of the global value and volume.

Tomato is also an important vegetables crop of Pakistan. The popularity of tomato and its products, continue to rise as it contains a significant amount of the vitamin A and C. Among vegetables, tomato is the second major vegetable, produced in Pakistan (Mirza, 2007). Its area under cultivation, during 2008 tomatoes were grown over about 53.1 thousand hectares with an average yield of about 10.1 tons ha-1 (Anon, 2008) and in 2009-10 was 63 thousand/ha, with a total production of 562.9 thousand tones, and a yield 10522 Kg/ha (Agricultural Statistics of Pakistan, 2010). This yield is very low as compared to that of the developed countries, However, much higher tomato yield has been reported in other countries of the world e.g., 73.87 t ha-1 in USA, 63.55 t ha-1 in Spain, 88.91 t ha-1 in California and 146 t ha-1 in the Netherlands.Of the various factors, responsible for its low yield, in Pakistan, the diseases are important among which early blight is of most important disease. Tomatoes has largest number of varieties sold worldwide as compared to any other vegetable crop.Some of the important varieties of tomato in Pakistan are Roma, Money maker, Nagina, Pakit, Feston, Peelo, Cherry, Nemadina, Eva T-89, T-10, Red Top, Marglobe, Cun Dayri (Burney, 1996).Tomatoes are an important part of a diverse and balanced diet (Willcox et al., 2003). It ranks first among all fruits and vegetables as a source of vitamins, minerals (Rick, 1980) and phenolic antioxidants (Vinson et al., 1998). Fresh and processed tomatoes are the richest sources of the antioxidant lycopene (Nguyen and Schwartz, 1999), which arguably protects cells

from oxidants that have been linked to cancer (Giovannucci, 1999). Tomatoes are either consumed directly as raw vegetable or added to other food items. Variety of processed products such as tomato paste, whole peeled tomatoes, diced products, various forms of juices, sauces and soups have been marketed as well. One medium fresh tomato (135 g) provides 47% RDA of vitamin C, 22% RDA vitamin A, and 25 calories. Tomatoes also contain significant amounts of -carotene, niacin, riboflavin, thiamine, magnesium, iron, phosphorus, potassium and sodium (Meredith and Purcell, 1966; Davies and Hobson, 1981; Madhavi and Salunkhe, 1998; Fraser et al., 2001; Ranieri et al., 2004; Kaur et al., 2004). 52% of the total antioxidants (48% lycopene, 43% ascorbic acid, 53% phenolics) are located in the epidermis of the fruit, which in consequence should not be discarded during consumption (Toor and Savage, 2005). Tomato is susceptible to over 200 diseases caused by pathogenic fungi, bacteria, viruses and nematodes (Lukyanenko, 1991). Fungal diseases included Septoria leaf spot, early blight, Verticillium wilt, late blight, gray mold, leaf mold, powdery mildew, Fusarium wilt, southern blight, Buckeye rot, Phytophthora root and crown rot (Satour and Butler, 1967; Bolkan, 1985).The causal agent of early blight of tomato, Alternaria solani (Ell. and Mart) Jones and Grout, occurs on tomato wherever it is grown, but is more severe in tropical and sub-tropical areas. Although A. solani has been reported to be the most common pathogen causing early blight and fruit rot, A. alternata and A. alternata f.sp.lycopersici also cause leaf spots and stem canker in tomato (Grogan et al., 1975, Malathrakis, 1983, Sahi and Shayam, 1993). It is easy to recognize Alternaria sp. by the morphology of their large conidia typically ovoid to obclavate, often beaked pale brown to brown, multi celled and muriform (Ellis, 1976). Botanicals are derived from plants. Many plant products are said to have fungicidal properties. They are natural products and most of them break down quickly on the leaves or in the soil. However, there is very little information on their effective dose rates, their impact on beneficial 3

organisms or their toxicity to humans. Spraying of broad spectrum fungicides like mancozeb and captan has been recommended for the control of early blight of tomato by several workers (Ramakrishnan et al., 1971 and Stevenson, 1977). While the number of applications of these chemicals are more, they are less persistent on foliage (Thind and Jyothi, 1982). Thus, the control achieved by these chemicals is inadequate. One of the reasons attributed for the low sensitivity of A. solani to fungicides mentioned above is the production of dark brown to black pigment called melanin by the fungus, which enhanced survival and competitive abilities of the pathogen, under certain environmental conditions (Bell and Wheeler, 1986). Therefore, in the present study it is thought worthwhile to test the efficacy of more promising chemicals like iprodione, propiconazole, difenconazole, pyraclostrobin, benlate, ridomil against early blight fungus. Not much light has been shed on the biological control, botanicals which are effective against A. solani. Hence, an attempt has been made to test some of common botanicals against the pathogen. The objectives of the investigations are as below: 1. Isolation, identification and Purification of fungus causing Early blight in tomato. 2. Preparation of plant extracts in water and Ethanol. 3. Evaluation of antifungal activity of different Concentration of plant extracts on Alternaria solani using Poision food technique.

REVIEW OF LITERATURE
Tomato (Lycopersicon esculentum Mill.) is a key food and cash crop for many low income farmers in the tropics (Prior et al., 1994). Inspite of being a tropical plant, it is grown in almost every corner of the world from the tropics to the Arctic Circle. There are several diseases on tomato caused by fungi, bacteria, viruses, nematodes and abiotic factors (Balanchard, 1992). Among the fungal diseases, important foliar fungal disease of tomato, early blight also known as target spot disease incited by Alternaria solani is one of the worlds most catastrophic disease. The causal 4

organism is air borne and soil inhabiting and is responsible for early blight, collar rot and fruit rot of tomato (Datar and Mayee, 1981). This is the most common disease of the cultivated tomato in areas with heavy dew, frequent rainfall, and high humidity (Agrios, 2005). The necrotrophic nature of the pathogen can lead to complete defoliation of tomato plants and subsequent yield reductions. Almost all member species of the Solanaceae can serve as alternate host for overwintering of the pathogen. Early blight of tomato caused by A. solani was first recorded in 1882 in New Jersey, USA (Bose and Som, 1986). The mycelium consisted of septate, branched, light brown hyphae, which turned darker with age. The conidiophores were short, 50 to 90 m and dark coloured. Conidia were 120296 x 12-20 m in size, beaked, muriform dark coloured and borne singly. However in culture they formed short chains. According to Singh (1987) the conidia contained 5-10 transverse septa and 1-5 longitudinal septa.The Alternaria fungus can cause diseases on all parts of the plant (leaf blight, stem collar rot and fruit lesions) and results in severe damage during all stages of plant development (Abada et al., 2008).

The survey of field and post harvest diseases of hybrid and desi cultivars of tomatoes in West Bengal, India, revealed that among fungal diseases, blight caused by Alternaria sp. was the most predominant with the crop loss in the field ranging from 70-100% (Kanjilal et al., 2000).Early blight is a three-phase disease, which produce leaf spots, stem canker and fruit rot, but the foliar phase is the most common and destructive part of the disease (Maiero and Barksdale, 1989). Responsible for significant economic losses sustained by tomato producer each year. A.solani can cause extensive defoliation leading to a reduction of economic fruit yield (Spletzer and Enyedi, 1999).Control of early blight disease has been accomplished mostly by the application of chemical fungicides, long crop rotations, pasteurizing seedbeds with steam or fumigants (Spletzer and Enyedi, 1999). It is necessary to search for control measures that are cheap, ecologically sound and environmentally safe to eliminate or reduce the incidence of economic important pathogens and to increase both seed germination and yield of plant crops. In recent years much attention has been 5

given to non-chemical systems for seed treatment to protect them against many plant pathogens (Nwachukwu and Umechurub, 2001).Glance of earlier literatures indicates that attention has not been given for utilization of plant extracts in controlling F. oxysporum and other plant pathogens, even if their effectiveness has been reported in reducing many diseases of various plants (Bansal and Rajesh 2000).

This disease is controlled mainly with agro-chmicals however the world wide trend towards environmentally-safe methods of plant disease control in suitable agri demands for reducing the use of these synthetic chemical fungicides. In an attemp to modify this condition some alternative methods of control have been adopted. Recent efforts have focused on developing environmentally safe, long lasting and effective biocontrol methods for the management of plant diseses.natural plant products are imp sources of new agrocjemicals for thr control of plant diseases (Kagale et al., 2004). Furthermore, biocides of plant origin are non-phytotoxic, systemic and easily biodegradeable (Qasem and Aau-Blan, 1996). It is know that various natural plant products can reduce population of foliar pathogens and control disease development and then these plant extracts have potential as environmentally safe alternatives and as components in IPM programs (Bowers and Locke, 2004).

Many plant species have been reported to have natural substances that are toxic to several plant pathogenic fungi (Goussous et al., 2010). In a study the effect of 11 different plant extracts on mycelial growth of A.solani was checked and found that leaf extracts of some plants i.e Tamarix aphylla and Salsola baryosma totally inhibit the growth of the pathogen in-vivo (Dushyent and bohra, 1997). It was reported that garlic extract significantly reduces the early blight disease on tomato (Wszelaki and Miller, 2005). Addionionally, several plant extracts have shown

antimicrobial activity agaisnt fungal pathogens under in-vitro and in-vivo conditions (Kagale et al ., 2004).

Spraying tomato plants with 20 % aqueous neem leaf extract slightly decreased early blight incidence and disease severity while spray and irrigation with the same extract caused a marked decrease in both disease incidence and severity after two and four weeks of inoculation. Results were in conformity with those by Chattopadhyay (1999) who found that foliar spray of A. indica leaf extract and azadirachtin reduced mycelia growth of A.alternata (causing loss of sunflower and tomato), decreased disease severity and increased yield over control. Babu et al., (2000) mentioned that spraying with 3 % of neem oil in tomato pot cultures resulted in 53 % reduction in disease incidence over the control while Patil et al., (2001), found that incidence of tomato early blight caused by A.solani was affected by a botanical like neem seed extract with increased fruit yield between 156.43 and 168.56q / ha.Singh et al., (1980) observed inhibitory effects of essential oils from C. martinii, C. oliveri, and Trachysperumm ammi on Helminthosporium oryzae, as well as inhibitory effects of the essential oils from rhizomes and leaves of Zingiber chrysanthum on plant pathogens such as Alternaria sp. and Fusarium sp.

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).The trend towards the environmental friendly pesticides with alarming levels of pest resistance to commonly used pesticides has led to search new antimicrobial agents from various sources including medicinal plants. The major characteristics of such biopesticides are that they should have minimal toxic effects to human and other organisms, rapid degradation and often a narrow spectrum of the activity (Loper et al., 1991).Babu et al., (2000a) reported the effect of plant extracts, oils and Neem products (Neem leaf, neem seed kernel and neem cake) on tomato early blight in the field. Among 7

the plant products, Acacia concinna pod extract resulted in the lowest percent disease index in the field (23.1%) followed by neem oil (30.9%). The efficacy of fungicides, namely carbendazim (0.05%) and mancozeb (0.25%), botanicals i.e. neem seed and leaf extract (each at 5%) concentration and tobacco decoction (2%) was evaluated in a field experiment for the management of early blight of tomato. The lowest per cent disease incidence (PDI) was observed in carbendazim (13.93) and mancozeb (15.46) treatments. Similarly the highest yield of tomato fruits was recorded with carbendazim (200.86 q/ha) followed by mancozeb (179.10 q/ha). The plant products, namely neem seed extract (19.75 PDI), neem leaf extract (20.36 PDI) and tobacco decoction (23.87 PDI) were also effective in reducing disease incidence and increasing fruit yield by 168.56, 156.43 and 147.66q/ha, respectively (Patil et al., 2003).Whole flowering Marigold plant, soap and water. Fill-in a drum with 1/2-3/4 full of flowering plants. Leave to stand for 5-10 days. Stir occasionally. Strain before use. Dilute the filtrate with water at a ratio of 1:2. Add 1 teaspoon of soap in every litre of the extract (Stoll, 2000). 50 g of bulb onion and 1 litre distilled water. Finely chop the onion. Add to water. Mix well. Strain. Spray thoroughly on the infected plant, preferably early in the morning or late afternoon (Stoll, 2000).Patil et al., (2001) showed that neem leaf extract was effective in reducing early blight incidence with increased yield of tomato infected by Alt. solani.

MATERIALS AND METHODS

This research work will be conducted in Department of Plant Pathology, PMAS Arid Agriculture University, Rawalpindi during 2011 2012 to determine the antifungal activity of Neem (Azadirachta Indica), Eucalyptus (Eucalyptus spp.), Mint (Mentha Arvensis ), China

berry (Melia azedarach), Turmeric (Curcuma longa) Bhang, (Cannabis sativa) leaves Against Alternaria solani in water and ethanol by employing food poisoning technique. Survey of farmer fields will be conducted in Tomato growing areas of District Rawalpindi for diseased plant samples. Collection of diseased samples 8

Diseased samples of Tomato plants infected with early blight will be collected from tomato fields and Tunnels in the surrounding areas of Rawalpindi.After the collection of diseased plants, the samples will be collected in polythene bags and will be brought to the laboratory of Plant Pathology department, P.M.A.S Arid Agriculture University, Rawalpindi.The diseased plant samples will be stored at 4 oC in a refrigerator until processed. Isolation of pathogen The pathogen viz, Alternaria solani will be isolated from infected tomato plants with visible symptoms early blight of tomato. Diseased samples will be cut into small pieces up to 1.5 cm length and surface sterilized with 5% Chlorox for one minute and washed three times with sterilized distilled water. These surface sterilized samples will be placed onto 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) in Petri plates and will be incubated at 25 2C.for 3-5days. Preparation of pure culture After 3-5 days the fungal growth appeared on the infected pieces will be identified by microscopy and Sub-culturing will be done by using single spore technique to obtain pure

culture.Plates will be incubated at 25 2C.and observed daily for emergence of colonies. Collection and preservation of plants samples Fresh leaves of Neem (Azadirachta Indica), Eucalyptus (Eucalyptus spp.), Mint (Mentha Arvensis ), China berry (Melia azedarach), Turmeric (Curcuma longa) Bhang, (Cannabis sativa), will be collected from different locations of Rawalpindi/Islamabad. These will be washed with tap water and air dried for one day to eliminate surface moisture. Then leaves will be packed into envelop and kept inoven at 60C temperature until dried. Dried leaves will be grinded separately in an eclectic grinder to obtain powder which will be than kept in plastic bags for further use. Preparation of Plant extracts 9

Hundred gram of the dried powdered plant leaves of Neem (Azadirachta Indica), Eucalyptus (Eucalyptus spp.), Mint (Mentha Arvensis ), China berry (Melia azedarach), Turmeric (Curcuma longa) Bhang, (Cannabis sativa) will be soaked separately in 500ml of 98% ethanol. These mixtures will be refluxed followed by agitation at 200 rpm (revolution per minute) for 1 hour. The ethanolic extracts will be squeezed and then filtered by muslin cloth. The extracts will be placed into a wide tray to evaporate ethanol and added with water to make plant extracts (Rahber, 1986). Food poison technique Diffusates will be 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 will be inoculated with 5 mm plug of pure isolate taken from margins of actively growing culture of Alternaria solani. Then Petri plates will be incubated at 25 2C.Mycelial growth will be recorded when the growth of three selected pathogens will be completed in the control treatment. Each treatment will be repeated five times. Mean radial mycelial growth of each plant diffusates will record and data will be subjected to statistical analysis. Radial mycelial growths on different diffusates will be transformed into inhibition percentage by using the following formula (Naz et al., 2006).

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

Statistical analysis

X 100

Data regarding two parameters (concentration, pathogen and plants) will be taken following the procedure and analyzed statistically using MSTATC program with completely randomized design (CRD). Inhibition of radial mycelial growth will be examined using analysis of variance (ANOVA) and means will be separated by the test of least significant difference (LSD 0.05). Results will be helpful in finding out the best treatment for disease management (Naz et al., 2006). 10

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