Antioxidative Enzyme Responses against Fusarium wilt ( Fusarium oxysporum f. sp. ciceris ) in Chickpea Genotypes

Three wilt resistant chickpea genotypes activity profile of in wilt antioxidative enzymes pre and post three wilt susceptible checks. of antioxidative enzyme profile normal and wilt activity of three antioxidative ., APX guaiacol superoxide Vikas at post infection resistant susceptible group of genotypes from 2.07 to 2.38 µmoles ascorbate oxidized mg -1 protein min -1 with mean percent increase of 97.94%. Though APX activity increased at post infection stage in wilt sick soil in the wilt resistant genotypes percent increase was from 33.03 to 57.64 with a mean percent increase of 40.4%. Same trend was recorded in GPX and SOD activity.


INTRODUCTION
Chickpea (Cicer arietinum L.) is a self pollinated leguminous crop, diploid (2n=16) grown since 7000 BC in different area of the world but its cultivation is mainly concentrated in semiarid environments [1]. Several diseases are known to limit worldwide production of chickpeas, of which Fusarium oxysporum f. sp. ciceris (Fusarium wilt) is one of the most important. Management of Fusarium wilt has been primarily through development of resistant cultivars as part of an integrated management approach. However, the high pathogenic variability in populations of F. oxysporum f. sp. ciceris presents problems for sustainability of resistant cultivars. Two pathotypes and eight races of the pathogen have been identified. The reliance on resistant cultivars for disease management of Fusarium wilt therefore places significant importance on the confident and efficient identification of pathogenic races of F. oxysporum f. sp. ciceris.
Amongst these wilt of chickpea caused by F. oxysporum f. sp. ciceris is a major limiting factor in the Indian subcontinent and causes 10 to 15% annual yield losses [2], but the disease can completely destroy the crop under specific conditions. Previous studies have demonstrated that, in Fusarium wilt of chickpea, roots are most susceptible to inoculation. The most common site of penetration of the fungus is at or near the root tip. In initially attacked cell(s), rapid responses may ultimately lead to cell death within few hours of pathogen contact [3]. This host cell death has been classically termed as hypersensitive response (HR). The HR has been proposed to play a causal role in disease resistance against bacterial and fungal pathogens [4].
One of the earliest observable responses of plant cells in many incompatible interactions is oxidative burst. Oxidative burst is generally defined as rapidly stimulated production of reactive O 2 species (ROS) including superoxide anion (O 2 ), hydroxyl radical (OH) and hydrogen peroxide (H 2 O 2 ). Doke and colleagues [5] were the first to report that superoxide anions were produced in incompatible interactions, initially between potato and Phytophthora infestans and then between tobacco and tobacco mosaic virus. Later it was recognized that oxidative burst is employed in many plant microbe interactions [6]. The production of reactive oxygen species (ROS) is the first response detected within minutes of an attack by virulent or avirulent pathogen [7]. Weak and transient ROS generation is due to a biologically non-specific reaction. After some hours, a massive and prolonged ROS production, called oxidative burst, occurs in cells attacked by avirulent pathogens. This two-phase kinetics of ROS production is typical of incompatible plant-pathogen interactions that are characterized by HR [8]. The present research work has therefore been undertaken to study the interaction between antioxidative enzymes in relation to wilt disease in chickpea.

Growth Conditions and Plant Material for Analysis
Ten chickpea genotypes viz., wilt resistant [7] namely GJG 0919, BCP-2010-1, JG 24,Vijay, Digvijay, WR 315, ICC 4958 and wilt susceptible [3] namely JG 62, SAKI 9516 and Vikas were grown in normal and wilt sick soil contains inoculum load of 2x10 7 cfu /g soil in pots in triplicate. After proper and uniform germination the root tissues of these genotypes were evaluated for activity of antioxidative enzymes both at preinfection and post infection stages of growth.

Enzyme Extraction and Activity Assays
Antioxidative enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and guaiacol peroxidase (GPX) were extracted from leaf tissue by using the method of Costa et al. [9]. For assays of SOD, CAT, APX and GPX, 200 mg cleaned root samples were homogenized in a chilled mortar and pestle with 2 ml of an ice-cold 0.1 mM potassium phosphate buffer (pH 7.5) containing 1 mM EDTA, 1 mM PMSF and 5% (w/v) PVP.
The homogenates were filtered through four layers of cheesecloth and then centrifuged at 4°C for 20 min at 15,000xg. The supernatant fraction was used as crude extract for enzyme activity assays.

Superoxide dismutase
Superoxide dismutase activity was determined by measuring its ability to inhibit the photochemical reduction of nitroblue tetrazolium using the method described by Dhindsa et al. [10]. Three ml enzyme reaction mixture contained: 50 mM phosphate buffer (pH 7. A complete reaction mixture without enzyme extract, which gave the maximal colour, served as irradiated control. After 15 min, the reaction was terminated by switching off light and covering the tubes with black cloth. A complete reaction mixture without enzyme extract kept in dark served as non-irradiated blank. The absorbance of the reaction mixture was red at 560 nm. One unit of SOD was defined as the amount of enzyme required to cause 50 per cent inhibition of NBT reduction per min at 560 nm.

Catalase
Catalase activity was measured immediately in fresh extract as described by Aebi [11]. Three ml enzyme reaction mixture contained: 50 mM potassium phosphate buffer (pH 7.0) (1.5 ml of 100 mM), 200 µl enzyme extract, 800 µl of distilled water and 12.5 mM hydrogen peroxide (0.5 ml of 75 mM).The reaction was initiated with addition of 0. ). The enzyme activity was expressed as µmoles of H 2 O 2 decomposed mg -1 protein min -1 .

Ascorbate peroxidase
Ascorbate peroxidase activity was measured immediately in fresh extract which was assayed as per the method described by Nakano and Asada [12]. Three milliliter of enzyme reaction mixture contained: 50 mM potassium phosphate buffer (pH 7.0) (1.5 ml of 100 mM), 0.5 mM ascorbic acid (0.5 ml of 3 mM), 0.1 mM EDTA (0.1 ml of 3 mM), 100 µl enzyme extract, 0.6 ml of distilled water and 0.1 mM hydrogen peroxide (0.1 ml of 3 mM). The reaction was initiated by the addition of 0.1 ml of 3 mM H 2 O 2 . The hydrogen peroxide dependent oxidation of ascorbic acid was followed by a decrease in the absorbance measured at 290 nm for three min at the interval of 30 sec. The amount of ascorbate oxidized was determined from molar extinction coefficient (ε 2.8 mM

Guaiacol peroxidase
For GPX, the rate of decomposition of hydrogen peroxide by peroxidase, with guaiacol as a hydrogen donor was measured by the increase in absorbance at 436 nm per min as per the method described by Castillo et al. [13]. Three ml of enzyme reaction mixture contained: 50 mM phosphate buffer (pH 7.0) (1.5 ml of 100 mM), 16 mM guaiacol (0.5 ml of 96 mM) 100 µl enzyme extract, 0.4 ml of distilled water and 2 mM hydrogen peroxide (0.5 ml of 12 mM).
The above mixture was mixed properly by using a spinner for 3-5 seconds. The reaction was initiated with adding 0.5 ml of 12 mM H 2 O 2 . An increase in absorbance due to the formation of tetra-guaiacol was measured at 470 nm for three min at an interval of 30 sec. The enzyme activity was calculated as per molar extinction coefficient of its oxidization product, tetra-guaiacol ε = 26.6 mM -1 cm -1 . The enzyme activity was expressed as nmoles of tetra-guaiacol formed mg -1 protein min -1 .

Lipid peroxidation rate
The level of lipid peroxidation product was measured in terms of malondialdehyde as thiobutaric acid reactive substance [14]. Root samples 0.2 g was homogenized in 2 ml of 0.1% TCA. The homogenate was centrifuged at 15000 g for 15 min and the supernatant was used for the estimation of MDA content. Leaf extract, 0.2 ml was thoroughly mixed with 0.4 ml of TBA reagent. The mixtures were heated for fifteen min at 100°C, cooled and cleared by centrifugation at 1000 g for 10 min. The absorbance was taken at 535 nm on spectrophotometer. Results were expressed as A 535 per gram of plant fresh weight.

Data Analysis
The data on biochemical constituents was statisticaly analyzed by using completely randomized block design [15].

RESULTS AND DISCUSSION
The transient production of AOS, in an oxidative burst, is frequently an early plant response in pathogen attack. Under wilt sick soil wilt susceptible chickpea genotypes have more pathogen attack in root tissues than wilt tolerant genotypes.
At preinfection stage mean APX activity did not vary significantly from normal soil to wilt sick soil, however significant increase was observed at post infection stage with 1.08 µmoles ascorbate oxidized mg with mean percent increase of 97.94%. Though APX activity increased at post infection stage in wilt sick soil in the wilt resistant genotypes percent increase was from 33.03 to 57.64 with a mean percent increase of 40.4%. Garcia et al. [16] reported remarkably increased root APX activity in wilt susceptible genotype JG 62 at post infection stage. APX activity was also increased but not significant in wilt immune genotype WR 315 at post infection stage. Maximal increase in root APX activity was recorded in JG 62 at post infection stage as compared to wilt resistant genotype JCP 27 by Joshi et al. [17].
At preinfection stage of growth the mean root GPX activity of chickpea genotypes did not vary significantly and increased from 0.44 to 0.45 µmoles of tetra guaiacol formed mg -1 protein min -1 in normal and wilt sick soil respectively, while the GPX activity increased significantly in root tissue at post infection stage from 0.83 to 1.01 µmoles of tetra guaiacol formed mg -1 protein min -1 in wilt sick soil. Wilt susceptible genotype JG 62 recorded maximum increase in root GPX activity at both pre and post infection stages. The GPX activity increased from 0.79 to 1.23 µmoles of tetra guaiacol formed mg -1 protein min -1 in JG 62 with 55.51 percent increase at post infection stage in wilt sick soil ( Table 2). All the wilt resistant chickpea genotypes recorded minimum increase in GPX activity at both growth stages from normal to wilt sick soil. Levels of GPX activity increased in wilt susceptible genotypes at post infection stage in wilt sick soil to counter the oxidative burst due to more pathogen attack in the roots of susceptible genotypes.    At post infection stage wilt susceptible genotypes JG 62, SAKI 9516 and Vikas recorded maximum increase with 16.14, 15.87 and 11.47, respectively confirms that pathogen attack is more in roots of wilt susceptible genotypes ( Table 4). The SOD activity increased in roots of chickpea genotypes 5 infected plants as compared to control plants at post infection stage lants SOD, APX and CAT activities were increased remarkably when f. sp. Melonis Race 1.2 as compared to uninoculated plants [18].   [20]. In flax, powdery mildew resistant varieties recorded .5 fold increases in MDA content as [21].

CONCLUSIONS
The evaluation of antioxidative enzyme profile in normal and wilt sick soil of the three wilt resistant genotypes along with four wilt resistant and three wilt susceptible checks exhibited a differential response. The activity of three antioxidative enzymes viz., APX, GPX and SOD increased significantly in wilt susceptible checks at post infection stage. The non compatible interaction between wilt resistant genotype and pathogen demonstrated lesser increase.

COMPETING INTERESTS
Authors have declared that no competing interests exist.