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Owing to scarcity of new fungicides in the market and environmental problems, the researchers are now emphasizing other alternatives such as genetic potential of plants to resistance against pathogens and the use of biotic as well as abiotic agents for the development of induce or acquired resistance We investigate the role of resistance inducing substances (chemicals and plant extracts) in three chickpea cultivars C-44, Pb-91, Bitter-98 in a field experiments against Ascochyta blight disease. These cultivars were selected on the basis of better yield potential shown in the experiments. Aqueous solution of Salicylic acid @ 0.5, 1.0 and 1.5mM, Bion® @ 0.4, 0.8 and 1.2 mM while KOH @ 25, 50 and 75mM were applied whereas the plant extracts of Azadirachta indica, Datura metel and Allium sativum were applied @ 5, 10 and 15% were used. The data regarding the reduction in disease was recorded with different intervals from 4 day to 14 days after the induction and inoculation with the pathogen. The overall results revealed that significant disease reduction (79%) was provided by Bion® in the cultivar C-44 at 1.2mM dose rate as compared to salicylic acid however the least was showed by KOH. Among the plant extracts the maximum disease reduction (26%) against the disease was observed by the application of Azadirachta indica leaf extract and the extracts of Datura metel and Allium sativum did not proved effective in reducing the disease. The present findings suggest that enhancement of resistance before infection of chickpea plants could be an innovative control method for ascochyta blight of chickpea.
Key words: Induce resistance, Chemicals, Plant extracts, Reduction.
Chickpea blight caused by Ascochyta rabiei (Pass.) Labrousse, is the most devastating foliar disease of chickpea (Cicer arietinum L.) in many countries of the world (Pande et al. 2005). This disease caused considerable losses that may ranges from 5 to 100% around the world (Haware 1998). In Pakistan, number of epiphytotics has been reported with complete failure of the crop (Aslam, 1984). Chickpea breeders in Pakistan are focusing their attention to develop blight resistant cultivars (Ilyas et al, 2007: Iqbal et al, 1993). Different aspect of this disease like severity, lifecycle, pathogenicity, epidemiology, breeding for resistance and control measures cultural, chemical have been explored in different part of the world with great success but still this disease posses a great threat to chickpea crop (Mohamed et al, 2009: Trapero-Cases and Kaiser, 2009). This disease can be managed by the use of different fungicides through out the world (Gan et al. 2006), but the most economical management strategy is the use of resistance cultivars (Turkkan and Dolar, 2009). However, breeding of resistance chickpea cultivars is more difficult task due to the fact that great variation exists in the pathogenicity of A. rabiei isolates (Ali et al, 2009).
Since the pathogen is constantly changing in nature (Chaudhry et al, 2001), the already resistant cultivars become susceptible. Therefore it is need to restrain or alleviate infection by using recent management methods such as induce resistance a new technology for plant disease control is based on the activation of the plant's own defense system with the aid of low molecular weight synthetic molecules (Cohen et al, 1999). Induced resistance is defined as an increase in the level of resistance without any changes in the basic genetic constitution (Baysal, 2001) which is associated with an enhanced ability of the plant to resist infection by an intense activation of defence responses. This method is alternative to fungicides in plant protection as there is more awareness of deleterious effect of fungicides on the natural ecosystem (Vimala and Suriachandraselvan, 2009). At the same time development of resistance in pathogen to pesticides, health and environmental concern, real and perceived and increasing popularity of organic crop and sustainable agriculture (KuÄ‡, 2001). Induce resistance can appear locally (i.e in the organ where the resistance is applied) or it may be systemically in the plant part which are spatially separated from the site of inducing agents (Walter, 2009) and there are at least three types of induce resistance, systemic acquired resistance (SAR) developed by localized necrosis which resulted in hypersensitive reaction (HR), infection by virulent pathogen or by treatment with certain chemicals Induce systemic resistance (ISR) that developed due to colonization of plant growth promoting rhizobacteria (PGPR) and wound induce resistance is usually elicited by injury caused by feeding of insects. In systemic acquired resistance an invading pathogen is not needed and various defence pathway are stimulated in the induced plant which resulted in the production of diverse defence products that are included lignin, pathogenesis-related proteins some of which show chitinase or β-1,3-glucanase activity (van Loon 1997; Neuhaus, 1999), phytoalexins, thionins and defensins (Kessmann et al., 1994; Sticher et al., 1997; Kombrink and Schmelzer, 2001: Ziadi et al., 2001).
Induce resistance is age old phenomena for the management of plant diseases that was first reported by (Ray, 1901) in rust diseases and numerous studies have showed the appearance of defense responses against several important plant diseases (Abo-Elyousr and El-Hendawy, 2008: Jiang et al, 2008) . Up till now various chemicals salicylic acid, isonicotinic acid (INA), benzothiadiazole (BTH), Rigel (salicylic acid analog), β-aminobutyric acid (BABA), NaClO3, HgCl2, Oryzemate® (Probenzole), paraquat, polyacrylic acid, SiO2, Messenger, (Harpin protein), Phoenix (potassium phosphate) etc have been used as inducer of resistance against fungi, bacteria and viruses (Schneider et al., 1996; Kuc, 2001; Percival et al., 2009). The extracts of various plants have also been explored as natural resistance inducers like Azadirachta indica against Alternaria leaf spot of sesame (Guleria and Kumar, 2006), Datura metel against Rhizoctonia solani, Xanthomonas oryzae pv oryzae, Alternaria solani (Kagale et al., 2004; Latha et al., 2009).
Very less work has been carried out on the induction of resistance in ascochyta blight of chickpea except from (Chaudhry et al, 2001), studied the induction mechanism in susceptible chickpea cultivar C-727 by the application of aqueous solution of salicylic acid dipotassium hydrogen phosphate and cuprous chloride all the treatment showed significant results.
The present studies have been conducted to evaluate the reduction in ascochyta blight disease due to induce resistance by the application of simple chemicals and plant extracts which are safe, cheap and easy to apply in the field.
5.2 Material and Methods:
5.2.1 Plant material
Chickpea cultivars viz C-44, Pb-91 and Bitter-98 which were susceptible but have shown good yield character in the pervious experiment were taken and the seed were cultivated in the small plots of 20 sq ft in the experimental area of the Department of Plant Pathology, University of Agriculture, Faisalabad during the year 2007-08 (November-April). The experiment was laid out under factorial arrangement of randomized complete block design with three replications with eighteen plots per repeat. Each plot was consists of seven rows of 10 plants each with three rows were induced and inoculated and other three were induced but with out the inoculation and seventh row act as untreated control.
5.2.2 Preparation of plant extracts
The extracts of Neem (Azadirachta indica Juss.), Dautra (Datura metel) and Garlic (Allium sativum L.) were prepared in the phyto-bacteriology laboratory of the Department of Plant Pathology, University of Agriculture, Faisalabad. Leaves of Neem and Datura were collected from research area of Agronomy and square No.32, University of the Agriculture, Faisalabad while the cloves of the Gralic were purchased from local market. Leaves of Neem and Datura after thorough washing under tap water, the materials were surface sterilized with 1% sodium hypochlorite solution followed by thorough washing with sterilized water. Leaves were homogenized (???homogenizer name) in sterile distilled water at 1:1 (w/v) in a pestle and mortar, and filtered through a muslin cloth to produce a 100% crude plant extract. For preparing extract of Gralic from fresh samples, the outer, dry peel of cloves was first removed, surface-sterilized for 2 min in 70% ethanol, and washed in three changes of sterile distilled water. Cloves were crushed into a pulp in sterile porcelain mortar using a pestle and the pulp was suspended 100 ml water in 250 ml Erlenmeyer flask and filtered through a muslin cloth. The plant extracts were heated to 40°C for 10 min to avoid contamination (Jaganathan and Narasimhan, 1988) and diluted to the required concentration (5%, 10% and 15%) with sterile distilled water (v/v).
5.2.3 Induction treatment and chemical
Chemicals like Salicylic acid (Sigma Aldrich, Germany), Bion® (acibenzolar-S-methyl), provided by Syngenta Crop Sciences, Germany, and KOH (Sigma Aldrich, Germany) were used for the induction of resistance. Aqueous solution of Salicylic acid @ 0.5, 1.0 and 1.5mM, Bion® @ 0.4, 0.8 and 1.2 mM while KOH @ 25, 50 and 75mM were applied however all the plant extracts were applied @ of 5%, 10% and 15% respectively. At early flowering stage all the resistance inducing agents (chemicals and plant extracts) were sprayed on the plants until the runoff while the control plants were sprayed with distilled water only.
5.2.4 Challenge treatment
The mass preparation of already isolated and preserved inoculum of A. rabiei was carried by the method of Ilyas and Khan (1986). The conidial counts were adjusted with the help of haemocytometer. Normal agronomic practices were followed throughout the experiment. Four days after the induction of the resistance the plants were challenged with spore suspension of A. rabiei (1x105 spores L-1) with the help of lady hand sprayer until run off in the evening with an idea that temperature gets lower in the night and it help better germination of conidia and continued for three days to ensure maximum infection to plants.
5.2.5 Reduction percentage
Data regarding the disease reduction percentage was calculated at 4, 6, 7, 11, and 14 days interval by using the following formula (Altamiranda et al., 2008) with some modification
Disease reduction (%) =100(1-x/y), where x= disease rating in the resistance induce plants and y= disease rating of the control plants.
5.2.6 Statistical analysis
Data were statistically analyzed using SAS software by analysis of variance and the significance of the treatments was determined using Duncan's multiple range test (P = 0.05).
5.3 Results and Discussion:
5.3.1 Disease reduction % by the application of chemicals
There were highly significant differences (P <0.05)
5.3.1 Disease reduction % by the application of plant extracts
There were highly significant differences (P <0.05)
The researcher is highly obliged to Dr. Shakeel Ahmad, Head Research and Development Syngenta, Pakistan for providing me Bion and all assistance for its import from Germany.