Timing and intensity of upregulated defensive enzymes is a key factor determining resistance in chickpea to Ascochyta rabiei

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Highlights

  • Desi, kabuli and wild chickpea genotypes were characterised on basis of Ascochyta blight resistance.

  • Upregulation of SOD, APX-GR, CAT and POD at the time of initiation of disease provide resistance in chickpea genotypes.

  • Higher H2O2 content in resistant and MDA in susceptible genotypes led to differential disease resistance behaviour.

  • Up regulation of defensive enzymes in susceptible genotypes at later stages of infection failed to mediate disease resistance.

  • The time and intensity of induced defensive mechanism is an essential contributing factor towards disease resistance.

Abstract

Ten genotypes comprising five resistant genotypes namely PBG 7, GLK 08–104 and GL 12021 (desi), ILWC 292 and C. judaicum 182 (wild); one moderately resistant genotype HC 5 (desi); three susceptible genotypes GLK 14313 and L552 (kabuli) and C. pinnatifidum 212 (wild) and one highly susceptible wild genotype ILWC 115 characterized on the basis of disease severity scale to Ascochyta blight caused by infection of Ascochyta rabiei were investigated for elicited biochemical responses. These genotypes were sown separately in Ascochyta screening plot and in control plot under normal conditions to study the response of enzymatic antioxidants and signalling molecules in leaves at four different stages of infection i.e. S0 (pre-inoculatory) stage, S1 stage (7 days after infection), S2 stage (20 days after infection), S3 stage (30 days after infection). The specific activity of superoxide dismutase increased during S0 and S1 and catalase activity was enhanced at later stages of infection. The specific activities of peroxidase, ascorbate peroxidase and glutathione reductase increased with the incidence of disease in resistant genotypes as compared to susceptible genotypes. The timely upregulation and more intensity of defensive enzymes such as SOD, APX-GR followed by CAT and POD in the resistant genotypes as compared to susceptible genotypes might be responsible for resistance to Ascochyta blight. The higher accumulation of H2O2 in resistant genotypes and higher MDA content in susceptible genotypes also contribute to their differential tolerance behaviour towards infection. The upregulation of defensive enzymes at later stages of infection in GLK14313, L552, C. pinnatifidum 212 and their lower activities in ILWC115 might be responsible for their susceptible behaviour indicating that the time and intensity of induced defensive mechanism is an important contributing factor towards disease tolerance.

Introduction

Chickpea (Cicer arietinum L.) is an essential second largest pulse crop, planted and utilized throughout the world, mostly in the Afro-Asian countries. Its global production is about 17.19 mt from 17.81 mha area with an average yield of 965 kg per ha [1]. It is an excellent source of energy and nutrients having high quality protein, with a wide range of essential amino acids) [2].. Chickpea used as human food as well as animal feed, coupled with its ability to fix atmospheric nitrogen makes it a very important crop. There are two distinct types of chickpea genotypes; desi and kabuli, primarily based on seed size, shape and colour. The desi type is mostly grown in Asia and Africa while the kabuli type is commonly found in Mediterranean region and also widely grown in North America, particularly in Mexico and US [3]. Wild Cicer species of chickpea have purple flowers, coloured and thin seed coat and plants are shorter in height and have smaller leaflets. Wild Cicer species have reported to be source of resistance to multiple stresses as compared to cultivated species. Wild chickpea species can enhance the genetic variation by contributing in a broader range of ecotypes that have sources for resistance against pest and other diseases [4].

Chickpea yield are less than the estimated yield due to various abiotic (drought, salinity, temperature, nutrient deficiency, etc.) and biotic stresses that are prevalent during crop growth and affecting its yield. Among the biotic stresses, fungal diseases are the most important limiting factor for grain production in chickpea. Ascochyta blight is the most important fungal disease worldwide affecting the chickpea production [5].. Aschochyta blight is seed- and residue-borne disease [6]. The fungi Ascochyta rabiei (Pass.) Labr. (syn. Didymella rabiei) is a highly virulent necrotrophic pathogen which causes ascochyta blight in chickpea. Ascochyta blight can infect crops at all developmental stages and cause over 40–50% yield reduction under conditions suitable for disease development [7].). However, the crop is more susceptible at flowering as well as podding stages causing substantial economic damage to the crop [8]. Cool and wet conditions such as low temperature 15-25 °C and 65–100% relative humidity favours disease development and spread. It has been observed that under heavy infection, the seed quality is reduced and there is complete yield loss [9]. Symptoms of aschochyta blight disease can develop on foliar and stem parts which leads to stem breakage and also cause seed rot. Lesions may appear as bended or lengthened darker brown-red lines on leaflets. Pycnidia in concentric circles on leaves, stem and pods are changed into black lines, 3–4 cm length of brown lesions with black spots on stem and petioles [10].

Plants develop a set of defense mechanisms like induction of the antioxidant system, activation of defense signaling pathways by production of reactive oxygen species (ROS), reinforcement of the cell wall by deposition of lignin and suberin at the site of infection as well as induction of pathogenesis related genes (PR genes) under pathogen attack [11]. Normally there is equivalence between generation and scavenging of free radicals but under stressed conditions this order is disturbed. This disorder in balance stimulates sudden increment in intracellular levels of ROS which can bring about critical harm to cell structure [12]. Many environmental stresses causes enhanced production of ROS that leads to advanced oxidative burst and ultimately cell death. In plants, ROS are also part of the reactions which are activated when plants undergo priming for defences [13]. ROS are considered to play a dual role; as they have destructive activity but also act as secondary messengers in a various cellular processes and provide tolerance to stresses [14]. Plants have enzymatic and non-enzymatic antioxidant mechanisms to cope up with the deleterious effects of ROS.

The respiration rate increases in plants after infection caused by the pathogen and it continued to rise during the multiplication and sporulation of the pathogen. The large amount of energy is needed for a rapid generation or mobilization of defence mechanisms in the cells of infected plants due to increased respiration rates as compared to resistant plants the activity of several enzymes of the respiratory pathways also rises. Plants which possess sufficient energy for mobilization of defence mechanism under infection might have tolerance to the pathogen attack [15].

The achievement of resistance in chickpea towards ascochyta blight is very difficult due to the absence of cultivars with a desirable level of resistance to blight and the low efficiency of the available fungicides. However, cultivars with enhanced resistance have been developed using moderately resistant genotypes. Along with fungicide application, these cultivars were used to manage the disease. This strategy, however, is often ineffective when the conditions for Ascochyta rabiei infection are highly conducive [8]. Management of ascochyta blight requires an integrated approach including the use of certified disease-free seeds, deep seeding depth, crop rotations of at least 3 years, tillage to bury plant debris, fungicide seed treatment to reduce seed transmission, the use of resistant cultivars and foliar fungicides for prevention or treatment of disease symptoms [16]. Fungicide treatments are prohibitively expensive and leads to ecological contamination such as environmental pollution, chemical toxicity to humans and animals. Therefore the use of resistant cultivars is likely the most ideal approach to deal with these disease [17]. Even the resistant resistant chickpea sources available are not sufficient and new sources needs to be identified from time to time [18].It was found that the production of rapid oxidative burst accompanied by the host resistance occur in the chickpea-ascochyta interaction in tolerant varieties [19]. Gharbi et al.) [20] revealed that higher H2O2 burst due to early upregulation of superoxide dismutase (SOD) and repressed activity of catalase (CAT) in the olive resistant cultivar as compared to the susceptible cultivars under Verticillium dahlia attack. The aim of present study was to compare the biochemical responses like activities of antioxidative enzymes such as superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX) and glutathione reductase (GR) elicited in chickpea genotypes along with content of H2O2, MDA after ascochyta blight disease at four stages of infection i.e. S0 (pre-inoculatory stage), S1(7 days after infection), S2 (20 days after infection), S3 (30 days after infection), corresponding to 0%, 25%, 50% and 75% of disease incidence, respectively.

Section snippets

Plant material and pathogen

Ten chickpea (Cicer arietinum L.) genotypes namely PBG 7, HC5, GL 12021 (Desi) and GLK 14313, GLK-08-104, L 552 (Kabuli) and ILWC 115, C. judaicum 182, ILWC 292, C. pinnatifidum 212 (Wild) were sown at the Research Farms of Pulses Section, Department of Plant Breeding and Genetics, Punjab Agricultural University, Ludhiana. The chickpea plants were sown separately in Ascochyta screening plot and in control plot under normal conditions. Each cultivar was sown in two rows of 2 m length spaced at

Results

In the present study, ten chickpea genotypes were grown separately under control and infected conditions. These genotypes were categorized on the basis of disease incidence; PBG 7, GLK 08–104, GL 12021, ILWC 292 and C. judaicum 182 as resistant genotypes, HC 5 as moderately resistant genotype, GLK 14313, L552 and C. pinnatifidum 212 as susceptible genotypes and ILWC 115 as highly susceptible (Table 2). The biochemical parameters were estimated in leaves of genotypes at four stages at different

Discussion

Ascochyta blight is the major disease of chickpea, especially in regions where cool, cloudy, and humid weather persists during the crop season. It have been reported that ascochyta blight causes the complete yield loss under favourable epidemic conditions [31]. It was observed that ascochyta blight symptoms appeared in sick plot after 10–15 days of inoculation. Disease intensity increased day by day and was at extreme after 20–25 days after inoculation on all aerial parts of the plants.

Conclusion

We conclude from our results that timely upregulation and intensity of antioxidative enzymes such as superoxide dismutase, catalase, peroxidase, ascorbate peroxidase and glutathione peroxidase in response to Ascochyta blight disease in resistant chickpea played an important role for the suppression of oxidative stress caused by invading fungal pathogen in them as compared to susceptible cultivars. The selection of resistant chickpea cultivars is a promising alternative to fight against Ascochyta

Author statement

Kaur K : Analysis, investigation Writing and editing. Grewal SK: Conceptualization, supervision, Writing and editing. Singh S: Conceptualization, Resources, Writing and editing. Rani U: Field trials, field management Bhardwaj RD: Methodology, Writing and editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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