The quantities of methionine, isolucine, tyrosine and phenylalanine content increased after induction and inoculation in three cultivars of chickpea and comparatively higher than induced/un-inoculated plants. Increased in lysine content more in C-44 upon inoculation in case of Bion and salicylic acid treatment but it decrease in Bitter-98. Arginine, and aspartic acid contents also increased in all the cultivars expect in Bitter-98 however aspartic acid content increased in case of garlic application. Invariable response was shown by threonine content by the application of chemicals and inoculation with pathogen in all the cultivars. Serine contents decrease in KOH, neem and datura extracts application in Bitter-98. Gultamic acid show invariable response while proline was decreased in C-44 and Bitter-98 after application of garlic extract. Glycine content decrease in Bitter-98 otherwise it increase in all the cultivar Gralic application showed decrease in alanine content inPb-91 and Bitter-98.
Key words: chickpea (Cicer arietinum L.), Ascochyta rabiei, (Pass) Labr, resistant vs susceptible cultivars, amino acid contents.
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Plant pathologist continued to faces immense challenges in the years ahead due to continuing problems of breakdown of host resistance, and fungicide insensitivity, furthermore there is a threat of global climate change, its impact on the pathogen spread and the ever-increasing human population (Walter et al., 2005). Plant Pathologists should be one step ahead of the pathogen, by understanding host pathogen interaction, providing the means to protect crops, due to fewer chemicals are available for disease control (Walters and Fountaine, 2009). In this context application of biotechnology would be a better choice to minimize the incidence of disease in agricultural crops (Lyon and Newton, 1999). One such approach might be through the induction and improvement of the plant's own mechanisms of defence rather than the application of toxic compounds. The induction of such a plant defence strategy seems logical as the induced disease resistance in plants is likely to offer protection against different pathogens; the attractive alternative which is natural, safe, effective and sustainable in controlling plant diseases (Van Wees et al., 1997) further it is preferred due to the reason that it activate the natural defense system of the host so chances of resistance development are less and its affectivity remain for longer period of time (Kuc, 2001).
This study can unravel many of the complicated biochemical interactions between the host and the pathogen. Plants can defend themselves against different pathogens through a wide array of mechanisms that may be local, systemic, inducible or constitutive. The induced is the stimulation of natural defense system in plants by the application of biological agent of chemicals (inducers) that are compatible with the environment (Altamiranda et al., 2008). They don't have the ability to produce the identical responses and it may vary according to host pathogen interaction (Gozzo, 2003). The induced resistance is not due to antimicrobial activity per se or their ability to be transformed into antimicrobial agents but different mechanisms may involve not only some pre-formed components but also a 'cascade' of induced responses (Ryals et al, 1996). The compounds like phytoalexins, reactive oxygen species/free radicals, calcium, silicon/silicates, polyphenoloxidases, peroxidases (has been implicated in the hypersensitive response), phenolic cross-linked cell wall polymers, hydroxyproline and glycine-rich glycoproteins, thionins, antimicrobial proteins and peptides, chitinases, Î²-1,3-glucanases, ribonucleases, proteases, callose, lignin, which help in the reinforcement of cell wall due to its deposition, polymer, lipoxygenases and phospholipases are synthesized and accumulated which may contribute to resistance after the infection by the pathogen (Hutcheson, 1998; Sticher et al., 1997; Kessman et al., 1994; van Loon et al., 1998). This cascade of resistance factors is induced only when a plant recognizes the presence of a potential pathogen, and the compounds capable of triggering such responses are termed elicitors. Application of salicylic acid, Bion, KOH, and plant extracts of neem, garlic, datura induce systemic resistance in different host pathogen interaction by enhancing their defence mechanism by releasing different enzymes, peroxidases, polyphenoloxidases pathogenesis related proteins (PR) chitinases, Î²-1,3-glucanases etc.(Boava et al., 2009: Ajay and Baby, 2009: Guleria and Kumar, 2006: Paul and Sharma, 2002). It has been reported that some PR proteins have chitinase (PR-2) group and Î²-1,3-glucanases (PR-3 group) activity in vitro (Mauch et al., 1988). These hydrolytic enzymes capable to degrading fungal cell wall polysaccharides, chitin and Î²-1,3-glucan, so inhibit the fungal growth (Roulin and Buchala, 1995).
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Chickpea blight disease caused by Ascochyta rabiei (Pass) Labr is devastating disease which upsets production statistics of this crop in the country (Ilyas, 2000; Bashir and Malik, 1998). Under environmental conditions conducive to the development of disease epidemics of disease may be encountered causing 50-70% crop losses (Malik and Bashir, 1984; Malik and Tufail, 1984). The disease can be managed by the use of disease free seed (Kaiser, 1984), destruction of plant debris (Chaube and Pandey, 1986), foliar and seed dressing fungicides (Reddy and Singh, 1984; Rauf et al., 1996) and host plant resistance (Iqbal et al., 2002; Ahmad et al., 2006; Malik et al., 2006). Since various bio-chemical factors like phytoalexins, phenolic compounds, amino acids and minerals contribute to resistance of host plants against their pathogens (Jamil et al., 1990; Vir and Grewal, 1994). Lot of information's regarding the role of different biochemical substance in resistance against A. rabiei are available like (Sarwar et al., 2001) reported maximum PAL activity was record 12 to 24 hours after inoculation in the resistant chickpea cultivar, while it took about five to nine day in the susceptible cultivar yet very few reports are available on the role of induce resistance in chickpea against A. rabiei and its effect on biochemical factors of induced plant. Chaudhary et al., (2001) reported that total phenolic contents and peroxidase activity was enhanced in the treated plants with salicylic acid, dipotssium hydrogen phosphate, cuprous chloride as compared to control. The objective of this study was to study the variation in amino acid contents of induced/un-inoculated and induced/inoculated chickpea cultivars after treatment with resistance inducer chemicals and plant extracts and inoculation with A. rabiei.
7.2 Materials and Methods:
The plant samples were collected from the induced/un-inoculated and induced/inoculated, with A. rabiei. The samples were dried in an oven at 55ï‚°C for 106 hours (to get constant weight) and ground. The determination of amino acid profile 100 mg of ground sample was placed in Pyrex glass test tubes (30 ml cap.), prepared by heat stretching in the middle in order to look like a damsel fly. Ten mL of 6 N HCl (reagent grade) was added to each of these test tubes. The oxygen from these test tubes was removed by a continuous flow of nitrogen gas. The test tubes were sealed with a pin point oxygen flame. The sealed tubes were placed in an upright position in an oven regulated at 110 ï‚± 1ï‚°C for 22 hours. The tubes were allowed to cool at room temperature. The hydrolyzate so formed was evaporated under vacuum at 60ï‚°C to dryness. The dry film of hydrolyzate was dissolved in 5 mL of 0.02 N HCl (pH = 2.2). The soluble material was centrifuged to remove the sediments and the samples were stored in glass stoppered bottles at 4ï‚°C until used for amino acid analysis. 20 ÂµL of the hydrolyzate was loaded on an ion exchange column of the automatic amino acid analyzer (Beckman Model 120C, U.S.A.) installed in the Institute of Animal Nutrition, University of Agriculture, Faisalabad. Acidic and neutral amino acids were eluted with sodium citrate buffer at pH 3.25 and 4.25 respectively. The elusion was done at flow rate of 15 and 30 mL/hour for ninhydrin and citrate buffer, respectively. The concentration of amino acids was calculated by the following formula:
Amino acid (%) = Peak area/constant
The study on determination of amino acids included the analysis of lysine, arginine, aspartic acid, threonine, serine glutamine, proline, glycine, alanine, valine, methionine, iso-leucine, leucine, tyrosine and phenylalanine. Various amino acids were determined by the Amino Acid Analyzer in the form of graphic peaks and the concentration of each amino acid was calculated by the above mentioned formula.
7.3 Results and Discussion:
Due to involvement of heavy cost for the purchase chemicals in amino acid analysis, only 14th day after induction and inoculation were subjected to studies regarding the role of amino acids. The whole plant samples from induced/un-inoculated and induced/inoculated of three chickpea. They were oven-dried at 55°C for 96 hours to get constant weight. These samples were processed for the estimation of amino acid profile. Content of different amino acids are expressed in terms of Âµg/100 mg dry weight of plant sample. The results are depicted as below.
The lysine content of induced/un-inoculated and an induced/inoculated plant of three chickpea cultivars with chemicals and plant extracts are given in Fig 1., the lysine content (p=0.05) of cultivar C-44 was highest (range 487.8 to 498.33) by the application of higher dose of Bion, but it was less (466.33) with out inoculation. The same trend was followed in the cultivar Pb-91 with maximum (449.36). Bion application and inoculation with pathogen decrease the lysine content in Bitter-98 (range 403.46 to 411.0) as compared to (414.83 to 418.96) Âµg/100 mg with out inoculation respectively at three dose rates.
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The response of all the three cultivars by the application of salicylic acid exhibited the increasing trend with maximum produce (range 382.46 to 395.06) in cultivar C-44 while in case of un-inoculated (range 328.93 to 339.6). The cultivar Bitter-98 exhibited decreasing trend upon inoculation with the fungus (range 211.93 to 225.86) Âµg/100 mg. In case of KOH application, the lysine content were more in C-44 and upon inoculation with the pathogen the lysine content showed increased level even in the cultivar Bitter-98, further cultivar Pb-91 at the higher dose rate showed increasing behavior (range 167.133 to 176.16) Âµg/100 mg with maximum in case of C-44. The plant extracts also showed significant (p=0.05) increasing trend after induction and inoculation with A. rabiei with maximum increased occur in case of neem leaf extract (161.9) at highest dose rate while on the lower dose rate the increase was (136.43 to 156.56). The cultivar Bitter-98 showed decreasing trend after inoculation with the pathogen (range 64.36 to 75.23) as compared to (range 79.4 to 85.56) with out inoculation. The extract of datura caused enhancement in the lysine content with maximum (81.0) Âµg/100 mg in case of C-44, the cultivar Bitter-98 showed reverse response as earlier treatment. The garlic extract induce the enhancement of lysine content but it was quite low as compared to other treatments with (2.8) was recorded in case of Bitter-98 upon induction and at the same time the increase in case of Pb-91 and C-44 was not significant at medium and high dose rate.
The quantity of arginine presented in the Fig 2 showed significant (p=0.05) production in both the induced/un-inoculated and inoculated with A.rabiei, salicylic acid induce increase in arginine contents in all the three cultivars but it was high in case of C-44 (range 544.06 to 557.96 Âµg/100 mg) after inoculation with fungus while in case of un inoculated it was (520.16) Âµg/100 mg. The highest amount of arginine was produced by in C-44 by the application of Bion at higher dose rate (range 616.43 to 624.73 Âµg/100 mg). Arginine content was increased by the application of KOH in the cultivar C-44 (range 300.53 to 315.63Âµg/100 mg) but it was reverse incase of Pb-91 and Bittet-98 exhibited declined trend by the application of KOH with least in Bitter-98 (range 91.5 to 109.2 Âµg/100 mg). The KOH application did not produced as much amount of arginine as in case of salicylic acid and Bion treatments.
The plant extracts also exhibited increase in the content of arginine significantly (p=0.05), maximum being produced by the application of neem leaf extract (range 153.46 to 164.83 Âµg/100 mg) in the cultivar C-44 while Bitter-98 displayed decrease (101.567 to 91.86 Âµg/100 mg) in the arginine content after inoculation with the pathogen, this decreasing trend was also followed in case of Bitter-98 (43.1 to 29.56) and (34.23 to 23.16 Âµg/100 mg) after treatment with extracts of datura and garlic and inoculation with pathogen but in case of C-44 and Pb-91 there is increasing tendency.
7.3.3 Aspartic acid
Fig 3 represents the aspartic acid content (p=0.05) of both induced/un-inoculated and inoculated plants of three chickpea cultivars. The data revealed that among chemicals applied for induction of resistance against A.rabiei, KOH was the least effective in the production of aspartic acid content with maximum in cultivar C-44 (range 375.5 to 423.46 Âµg/100 mg), but the declined tendency was followed in case of Bitter-98 (range 92.1 to 125.23 as compared to (114.96 to 139.5 Âµg/100 mg) in case of un-inoculated respectively. Application of Bion and salicylic acid increased the aspartic contents in all the cultivars with maximum (range 701.56 to 723.1) and (range 636.3 to 676.2 Âµg/100 mg) in the cultivar C-44. The amount of aspartic acid in cultivars Pb-91 and Bitter 98 increased by the application of Bion and salicylic acid with less (range 424.36 to 467.63 Âµg/100 mg) in case of Bion treated plants.
The aspartic acid content was decreased (73.43 to 63.5 Âµg/100 mg) by the application of datura extract in the cultivar Bitter-98, while increased trend was followed in case of neem leaf extract (range 219.4 to 241 Âµg/100 mg) in cultivar C-44. The least amount (range 37.36 to 58.26Âµg/100 mg) of aspartic acid content was shown in the garlic treated plants. Among all the resistance inducers (chemicals and extracts) the less was produce by the extracts as compared to chemical with Bion proved to be superior.
There was an increase in the threonine content of both the induced/un-inoculated and inoculated though it was more pronounced by the application of chemicals in cultivar C-44 (Fig 4). The application of neem leaf extract resulted increase (range 501.26 to 545.3 Âµg/100 mg) in C-44, moreover this tendency continued in other two cultivar Pb-91 and Bitter-98. The application of datura and garlic extract did not followed this pattern as there is decrease in threonine content in cultivar Bitter-98 with minimum (203.23 to 155.433 Âµg/100 mg) in case garlic extract.
In general, there was an increased in threonine content by the application of chemical with maximum (range 1406.38 to 1438.53 Âµg/100 mg) in the cultivar C-44 and higher content was recorded in Pb-91 but reverse was in case of Bitter-98 (1142. 2 to 1052.2 Âµg/100 mg). Although there was decreased but threonine content was almost near to the content in case of salicylic acid application in the cultivar Pb-91 with (1029.47 to 1055.67 Âµg/100 mg) yet the maximum was recorded in case of C-44 (range 1172.47 to 1194.33Âµg/100 mg) The application of KOH as resistance inducer resulted in an increase in the threonine content but it was decreased in case of Bitter-98 (from 659.27 to 539.27 Âµg/100 mg). More threonine content in case of chemical and less in case of plant extracts.
The quantity of serine was increased in all the treated and inoculated plants but it decreases in the cultivar Bitter-98 by the application of chemicals and plant extracts expect garlic extract Fig.5. The maximum increase (range 803.6 to 856.16 Âµg/100 mg) in the serine content was recorded in salicylic acid treatment with higher dose in the cultivar C-44 followed by Bion (range 781.53 to 821.83 Âµg/100 mg). The cultivar Pb-91 also showed increasing tendency (range 751.36 to 789.96 Âµg/100 mg) but with comparison to C-44 it was less in serine content in Bion treated plants. The KOH treated plants exhibited decrease (372.1 to 273.53 Âµg/100 mg) in the serine contents in the cultivar Bitter-98 but was high in case of C-44 and Pb-98.
Neem extract with maximum (range 416.96 to 462.33 Âµg/100 mg) serine content increase in cultivar C-44 was best as compared to datura and garlic but on over all bases it was less compare to chemical application. The serine content was decreased (170.7 to 74.66 Âµg/100 mg) in case of neem extract and (91.83 to 57.53Âµg/100 mg) datura extract but it showed increase (39.6 to 61.56 Âµg/100 mg) in the cultivar Bitter-98. The cultivar Pb-91 also exhibited less serine content upon inoculation with A.rabiei and application of datura extract (181.73 to 137.56 Âµg/100 mg) but more content in case neem (238.7 to 318.53 Âµg/100 mg) and garlic (137.46 to 198.56 Âµg/100 mg) extract.
7.3.6 Glutamic Acid
Glutamic acid content in Bion treated plant and inoculated with A. rabiei (range 846.73 to 934.96) in the cultivar C-44 (Fig 6), however it was (503.23 to 560.5 Âµg/100 mg) in the salicylic acid treated plants. The cultivar Bitter-98 showed decrease trend with (649.03 to 556.7 Âµg/100 mg) in case of Bion application (289.36 to 163.33 Âµg/100 mg) in case of salicylic acid treatment. On the other hand the same cultivar showed increasing (204.16 to 285.26 Âµg/100 mg) trend by the application of KOH. Cultivar Pb-91 with exhibited highest (855.33Âµg/100 mg) gultamic acid content in case of Bion application and lowest (405 Âµg/100 mg) with KOH.
Garlic extract with (181.8 to 134.3 Âµg/100 mg) reduction in the gultamic acid content in the cultivar C-44 but it was increased in the cultivar Pb-91 and Bitter-98. The increased tendency was followed in case of neem and datura extract in the three cultivars maximum being (375. 2 Âµg/100 mg) in cultivar C-44 while minimum (182.2 Âµg/100 mg) was in case of Bitter-98.
The increased trend was exhibited in the proline content by the application of chemical inducers and upon inoculation with the pathogen (Fig 7), maximum (112.3 to 161.56 Âµg/100 mg) was in case Pb-91 by the application of Bion followed (140.23 Âµg/100 mg) in case of C-44. Upon inoculation with the pathogen, in salicylic treated plant of cultivar C-44, proline content was (102.63 Âµg/100 mg) while it was quite less in case of KOH application in the same cultivar (52.2 Âµg/100 mg). Cultivar Bitter-98 exhibited (83.2, 170.26 and 36.2 Âµg/100 mg) praline content in salicylic acid, Bion and KOH treated plants.
Proline content in case of garlic extract was reduced in C-44 (20.6 to 17.46Âµg/100 mg) and Bitter-98 (14.43 to 12.16 Âµg/100 mg), however the opposite was shown by the extracts of neem and datura with maximum increase (range 22-41 to 41.83 Âµg/100 mg) occur in C-44 while the increase in case of Pb-91 and Bitter-98 was also significant.
The glycine content of both the groups increased (Fig 8) but it was more distinct in case of chemicals than the plant extracts. The maximum glycine content (from 390.8 to 418.2 Âµg/100 mg) was produced in the Bitter-98 in Bion treatment followed by Pb-91 with (399.43Âµg/100 mg) glycine content. There was less increase in the glycine content in C-44 in all the treatment salicylic acid, Bion and KOH (273.33, 385.23 and 189.23 Âµg/100 mg) and consequence of inoculation with the pathogen. Cultivar Pb-91 exhibited enhanced content of glycine but it was less than Bitter-98 and more in comparison with C-44.
Extract of datura increases the glycine content (125.23 to 130.66 Âµg/100 mg) in the cultivar C-44 but higher (154.56 Âµg/100 mg) glycine content was shown in case of Bitter-98. The highest among the plant extract was demonstrated by neem (171.26 Âµg/100 mg) in the cultivar Pb-91. On overall basis invariable response in the increae of glycine content was shown by the cultivars after treatment with plant extract and inoculation with pathogen.
Alanine content was influenced by the application of plant extracts but not greatly as in case of chemicals. There was declining trend in the alanine content by the application of garlic extract in Pb-91 and Bitter-98. The maximum (from 36.06 to 58.13 Âµg/100 mg) alanine content was produced in the neem treated and inoculated plant in C-44.
The alanine content of was maximum (493.26 Âµg/100 mg) in the cultivar C-44 as a result of inoculation with A. rabiei, however the increase was less (129.46 Âµg/100 mg) in case of KOH treated plant, salicylic acid application to the same variety resulted in 193.26 Âµg/100 mg) alanine content (Fig 9). Alanine content was low (68.33 Âµg/100 mg) in case of Bitter-98 after the application of KOH and inoculation with the pathogen. Cultivar Pb-91 displayed higher alanine content (235.2 Âµg/100 mg) in case of Bion treated and inoculated plants.
The valine content show increased trend due to stimuli with the chemicals and upon inoculation with the pathogen (Fig 10). The maximum (from 245.2 to 271.467 Âµg/100 mg) valine content was achieved by the application of Bion application in the cultivar C-44 followed by (211.53 to 229.26 Âµg/100 mg). KOH application also increases (108.23Âµg/100 mg) the valine content but not as much as in case of Bion and salicylic acid. There is also enhanced in the valine content after treatment with salicylic acid and inoculation with the pathogen in all the cultivars but most significant (207.33 to 232.53 Âµg/100 mg) in case of C-44.
Plant extracts of neem, datura and garlic contents increase invariably, neem extract increase the valine content (from 145.2 to 166.23Âµg/100 mg) in inoculated plants while in Bitter-98 the valine decrease (from 97.2 to 91.93Âµg/100 mg). In case of datura the increase was only in case of C-44 but in Pb-91 and Bitter-98, the decreasing trend was displayed upon inoculation. Similar trend was followed in case of garlic extract with decline in the valine content with minimum (from 10.16 to 25.2 Âµg/100 mg) in Bitter-98.
As regards the methionine content of induced/un-inoculated and inoculated plants of chickpea cultivar, it increased in all the treatment upon inoculation with A. rabiei (Fig 11). The more methionine content was more (from 156.3 to 178.76 Âµg/100 mg) in the cultivar C-44 by the application of Bion, this was followed by more content in Pb-91 and Bitter-98. Salicylic acid also increases the methionine content more (85.3 to 106.3 Âµg/100 mg) and least was in case of KOH application (26.43 to 49.43 Âµg/100 mg) in cultivar Bitter-98 after inoculation and induction. Plant extracts also increased the methionine content but less quantity as compared with chemicals. The maximum (64.3 to 88.3Âµg/100 mg) was produced in case of neem leaf extract, followed by Pb-91 nad Bitter-98. Methionine content was very (low 7.5 to 22.6 Âµg/100 mg) after treatment with garlic extract in the cultivar Bitter-98.
Isoleucine content of induced/un-inoculated and inoculated plants of is presented in Fig 12. In general, there was a tendency of increase in isoleucine content in all the three cultivars of chickpea as a result of inoculation with the pathogen. Thus, there is an increase (from 176.33 to 197.36 Âµg/ 100 mg) in C-44 followed by Pb-91 and Bitter-98 after treatment with Bion. The application of salicylic acid increases the isoleucine content with maximum in (132.53 Âµg/ 100 mg) in cultivar C-44 followed by Pb-91 (105.2 Âµg/ 100 mg) and Bitter-98 (81.93Âµg/ 100 mg). Application of KOH also enhances the isoleucine content but was not too high as compared to Bion and salicylic treatment and inoculated pathogen.
Gralic extract with highest (24.23 to 40.3 Âµg/ 100 mg) isoleucine content in C-44 was quite low as in case of neem and datura extract. The lowest content was recorded in Bitter-98 with (50.4, 44.66 and 27.23 Âµg/ 100 mg) after application of neem, datura and garlic extracts. Over all the trend of increased isoleucine content was observed in chemicals as compared to plant extracts.
The leucine content of the chickpea cultivar belonging to the two groups is given in Fig 13. In cultivar C-44 after application of garlic extract, the increase ranges from (86.83 to 106.0 Âµg/100 mg) followed by (76.36 Âµg/100 mg) in Pb-91 and (53.3 Âµg/100 mg) in Bitter-98. Neem extract with (161.4 Âµg/100 mg) was considered as best among all the extract in increasing the leucine content. The extract of datura was also proved effectual but less to neem.
The application of chemical and than inoculation with the pathogen increases the leucine content but it was more significant in Bion treated plants in all the varieties (501.4, 473.16 and 463.36 Âµg/100 mg) in C-44, Pb-91 and Bitter-98. This tendency was also followed by the application of salicylic acid with (305.23Âµg/100 mg) content of leucine in cultivar C-44 followed (284.26, 241.43 Âµg/100 mg) by Pb-91 and Bitter 98. Leucine content of (246.2 Âµg/100 mg) was produced by the application of KOH in the cultivar C-44 while (204.26 and 168.2 Âµg/100 mg) was produced in cultivar Pb-91 and Bitter-98.
The quantity of tyrosine in the induced/un-inoculated and inoculated plants of three chickpea cultivar demonstrated increase trend in case of application resistance inducers and plant extracts (Fig 14). Salicylic acid application in all the cultivars under study, gave increase (41.4 to 64.16 Âµg/100 mg) as compared to un-inoculated plants (36.33 to 54.23 Âµg/100 mg). Tyrosine content was also on higher side in Pb-91 (51.23Âµg/100 mg) and Bitter-98 (45.2 Âµg/100 mg). The maximum increase (61.7 to 83.23 Âµg/100 mg) was recorded in case of Bion application in cultivar C-44 which was different in case of Pb-91 (67.6 Âµg/100 mg) and Bitter-98 (50.33 Âµg/100 mg). The application of KOH in all the three cultivars increased (48.26, 36.53 and 28.53 Âµg/100 mg) the tyrosine content but the increase was less as compared to Bion application. Among the plant extract only the neem extract (67.26 Âµg/100 mg) tyrosine content as compared to (54.32 and 40.5 Âµg/100 mg) in the cultivar Pb-91 and Bitter-98. Gralic and dutara extract resulted in increasing the content of tyrosine with maximum (42.3 Âµg/100 mg) in case of C-44. It is interesting to note that Bitter-98 exhibited slight increase (30.3 Âµg/100 mg) tyrosine content in induced and inoculated as compared to Pb-91 (27.36).
The phenylalanine content of all the three cultivars showed increase trend (298.3, 268.2 and 241.2 Âµg/100 mg) in C-44, Pb-91 and Bitter-98 (Fig 15). KOH application in inoculated plants showed enhance tendency but it was less (63.43 Âµg/100 mg) as compared to Bion and salicylic acid application There was (298.3, 268 and 241.2 Âµg/100 mg) phenylalanine content by the application of salicylic acid in three cultivar. The plant extracts also effectual in boost the phenylalanine content (146.03, 127.4 and 105.33 Âµg/100 mg) by the application of neem extract in the cultivars C-44, Pb-91 and Bitter-98. Gralic extract increase the phenylalanine contents (40.76 Âµg/100 mg) but it was less as compared to neem and datura extracts. Over all the phenylalanine contents increased in all the cultivars after inoculation of the induced plant although the increase was also observed in un-inoculated plants but on comparison basis, it was less after inoculation.
A novel technology in the management of plant diseases is the activation of the plant's own defence system with the aid of biotic and abiotic inducers (Satya et al., 2007). Several chemicals like, salicylic acids and its derivatives (Schreiber and Desveaux, 2008), Bion and Di-potassium Phosphate (Kamal, 2008) and plant extracts can induce resistance which is often associated with increased activity of PO, lignin deposition, increase in phenolic content and induction of PR-proteins (Hammerschmidt & Kuc 1982). There are 17 families of pathogenesis related proteins in host pathogen interaction produced in various plants (Tuzan and Somanchi, 2006). These are produced in response of different stresses or stress related plant hormones including osmotic stress, ethylene, wounding, drought, etc. Pathogenesis related proteins (PR) chitinases, Î²-1,3-glucanases break down the Î²-1,3-glucans and chitin in the cell wall of fungi but the protection provided by them may vary (Manandhar et al., 1999) and it may depends upon the genotype, physiological condition of the plant and nature of inducing agent used (Ton et al., 1999). PAL is the considered as key enzyme of phenylpropanoid metabolism in higher plants which catalyzes the conversion of phenylalanine to trans-cinnamic acid which supplies the precursors for flavonoid pigments, lignins and phytoalexins (Hahlbrock and Scheel, 1989). Several studies indicated that disease resistance is associated with the activation of PAL and subsequent increase in phenolic content in plants.
Amino acids are responsible for the synthesis and build-up of metabolites that collectively form a defence mechanism for an individual. Although first proof of the involvement of amino acid/proteins in resistance was observed by Kuc et al, (1957) in susceptible apple leave and found that petiole injection of phenylalanine made the leaves resistant to Venturia inaequalis, the cause of apple scab disease, yet different hypothesis still exist about the association of amino acid in resistance/susceptibility in host pathogen interaction. van Andel, (1966) discussed the amino acids role in two way i) amino acids have direct effect on the pathogen, it kill the pathogen directly possess fungicidal ability or decrease the pathogenicity. ii) Affect the metabolism of the host due to formation of any compound that is toxic to pathogen. Amino acids glycine, DL-asparagine, and DL-glutamine found inactive in all tests when they were applied (van Andel, 1958: Samborski and Forsyth, 1960: Papavizas and Davey, 1960), while DL-Iysine, DL-proline, hydroxyproline, cause serious injury to the plants (Samborski and Forsyth, 1960: van Andel, 1958).
The chickpea cultivar exhibited increased in lysine content more in C-44 upon inoculation in case of Bion and salicylic acid treatment but it decrease in Bitter-98. Arginine, and aspartic content also increased in all the cultivars expect in Bitter-98 however aspartic acid content increased in case of garlic application. Invariable response was shown by threonine content by the application of chemicals and inoculation with pathogen in all the cultivars. Serine contents decrease in KOH, neem and datura extracts application in Bitter-98. Gultamic acid show invariable response while praline was decreased in C-44 and Bitter-98 after application of garlic extract. Glycine content decrease in Bitter-98 otherwise it increases in all the cultivar. Gralic application showed decrease in alanine content inPb-91 and Bitter-98. Methionine, isolucine, tyrosine and phenylalanine content increased after induction and inoculation. As many as 15 individual amino acids were obtained from chickpea plants during the present study whereas only two amino acids namely, methionine and cystine could be noted during the earlier studies (Bhatti et al., 1987). They did not indicate the amount of two amino acids recovered from the resistant cultivar (CM-72) and the susceptible cultivar (6153). Only one study regarding the induction of resistance in chickpea against A.rabiei reported an increase in phenolic and peroxidase content after treatment with salicylic acid (Chaudhar et al, 2001). Hence, the present results could not be compared to the aforementioned study. These results were in partially in line with (Sahi, 1999) but contrary to those of Randhawa (1994) who observed marked decrease in the content of total amino acids in both the chickpea groups, resistant as well as susceptible to Ascochyta rabiei. Similarly (Gokulakumar and Narayanaswamy, 2009) found that Serine, Threonine, Arginnine, Phenylalanine and Leucine was higher in resistant roots and lower in disease roots in fifteen sesame cultivars after the attack of Macrophomina phaseolina. Present study clearly indicate that upon inoculation in the three susceptible cultivars of chickpea, the appreciable increase was recorded by the application of chemicals than by the extracts, it might be due to more production of PRs proteins, neem leaf extract enhance the amino acid content more as compared to datura, garlic extract. These results are in line with that of Singh and Prithiviraj (1997) reported that Neemazal, a product of neem induced resistance in pea against Erysiphe pisi with concomitant increase in PAL activity. Paul and Sharma (2002) reported that aqueous extract of leaves of neem provided control of leaf stripe pathogen (Drechslera graminea) on barley and the treated leaves exhibited significantly high activity of PAL along with rapid and distinct accumulation of fungitoxic phenolic compounds.
The results of the present study are also similar to Lewis and MC Clure, (1975) reported that upon infection, in the root-knot susceptible cultivar, M8, there is greater percentage increase in certain individual free amino acids than the resistant cultivar, Clevewilt, but over all the sum total of free amino acids was greatest in the resistant cultivar further free amino acid content of tissues has been related to the susceptibility or resistance of plants to various other pathogenic organisms, as in our case there in increase in the amino acid content after inoculation with A.rabiei likewise as in case of C-44 the highest amount of alanine was recorded similar to results of (Lakshminarayanan, 1955) who reported that Cystine and alanine play a role in resistance to diseases caused by Fusarium sp. (Hashem and Rehim, 1967). A relationship also exists between the level of alanine and susceptibility to Verticillium by cotton (Booth, 1969: Singh et al., 1971). Chiang and Nip (1973) observed higher levels of alanine, asparagine, and tyrosine in tissues of cabbages resistant to clubroot. Similarly more recently Matsubara et al., (2010.) reported the influence of arbuscular mycorrhizal fungi (AMF) on the total amino acid content production in strawberry plants, after inoculation with Glomus mosseae yielded more amino acids among thm Serine, glutamic acid, glycine, alanine, leucine and GABA were higher in both mycorrhizal plants after inoculation as compared to non-inoculated plants with/out the influence of phosphorus. Amino acids and carbohydrates in nine clone of cacao were analyzed revealed that seven amino acids (asparagine, cysteine, glycine, isoleucine, proline, serine and tyrosine) were identified but total amino acid content was 74.5% higher in the less susceptible as compared to highly susceptible clone and the occurrence of each in the pods varied with the genotype and with the treatment (Omokolo et al., 2002). Aspartic acid, tyrosine and valine showed a gradual increase with advancing age in case of infection with Fusarium solani to turmeric plants (Reddy et al., 2005).