Elucidating oxidative burst during interaction of rice-elicitor/pathogen (Xanthomonas oryzae pv. Oryzae)

Elucidating oxidative burst during interaction of rice-elicitor/pathogen (Xanthomonas oryzae pv. Oryzae) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Elucidating oxidative burst during interaction of rice-elicitor/pathogen (Xanthomonas oryzae pv. Oryzae) Tusar Mondal, Sudhamoy Mandal, Binod Saradar This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9124704/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The oxidative burst is one of the earliest and most prominent defence responses of plants to microbial infection and is characterized by a rapid and transient accumulation of reactive oxygen species (ROS), including hydrogen peroxide (H 2 O 2 ). The present study investigated the timing and magnitude of ROS production and associated antioxidant enzyme responses in five rice cultivars (TN-1, Satabdi, Naveen, Annapurna, and Tapaswini) following treatment with two defence elicitors, salicylic acid (SA) and chitosan (CH), and infection with the bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo). Xoo-inoculated plants exhibited a biphasic oxidative burst, marked by an early increase in H 2 O 2 accumulation at 24 h post-inoculation (hpi), followed by a second, more sustained phase after 72 hpi. Enhanced lipid peroxidation accompanied ROS generation, indicating oxidative stress during pathogen interaction. Superoxide anion (O 2 •⁻) and hydroxyl radical (•OH) levels increased predominantly between 72 and 120 hpi, depending on the cultivar. Antioxidant capacity, assessed by DPPH radical scavenging activity, increased following pathogen inoculation. Enzymatic antioxidants showed dynamic responses, with superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPX), and ascorbate peroxidase (APX) activities being differentially regulated across cultivars and time points, particularly during the later stages of infection. The early oxidative burst is consistent with PAMP-triggered immunity (PTI), while the sustained ROS accumulation at later stages may reflect prolonged defence signalling or pathogen proliferation during a compatible interaction. Both SA and CH acted as PTI mimics, inducing ROS production and activating antioxidant defence mechanisms. Collectively, these findings highlight the role of oxidative burst dynamics and antioxidant regulation in rice defence responses against bacterial blight. Antioxidative mechanism Oxidative burst Reactive oxygen species Rice–pathogen interaction Xanthomonas oryzae pv. Oryzae Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Plants are continuously exposed to numerous environmental challenges throughout their life cycle. Because plants are sessile, they have evolved a wide range of defence or stress-response mechanisms to cope with both biotic and abiotic stresses. Among the most serious biotic threats faced by plants are pathogenic fungi, bacteria, and viruses ( 1 ). Reactive oxygen species (ROS), including hydrogen peroxide (H 2 O 2 ), hydroxyl radicals (•OH), and superoxide anions (O 2 •⁻), are naturally produced in plant cells as by-products of photosynthetic and respiratory metabolism in chloroplasts and mitochondria (2;3). In addition, ROS are generated through redox reactions mediated by membrane-bound, cytoplasmic, and extracellular enzymes ( 4 ). Under non-stress conditions, ROS production and scavenging are tightly regulated, maintaining cellular redox homeostasis. However, exposure to biotic and abiotic stressors such as pathogen invasion, dehydration, and low temperature can disrupt this balance, leading to excessive ROS accumulation and cellular damage, including enzyme inactivation and programmed cell death ( 5 ). To mitigate ROS-induced damage, plants possess an elaborate antioxidant defence system comprising both enzymatic and non-enzymatic components. Key ROS-scavenging enzymes include superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPX), and ascorbate peroxidase (APX), which play essential roles in detoxifying excess ROS during stress conditions ( 6 ). In addition, plants utilize a range of non-enzymatic antioxidants such as ascorbic acid, glutathione, phenolic compounds, flavonoids, carotenoids, and tocopherols to maintain redox balance (7; 8; 9). The controlled production and scavenging of ROS are crucial for normal cellular processes, including growth, development, and signalling (10; 11). Conversely, excessive ROS accumulation under stress conditions disrupts cellular homeostasis, resulting in oxidative damage and yield loss ( 12 ). An oxidative burst is a hallmark early defence response in plants, characterized by the rapid and transient overproduction of ROS following pathogen or elicitor perception. Numerous studies have demonstrated that ROS generation and the activation of ROS-metabolizing enzymes are closely associated with defence responses in plant–pathogen interactions. Due to their rapid production and cytotoxic nature, ROS function as an effective first line of defence by directly restricting pathogen growth or delaying infection. In addition, ROS contribute to cell wall strengthening through oxidative cross-linking of cell wall proteins and act as signalling molecules that activate downstream defence pathways. Recent evidence suggests that oxidative bursts also facilitate the propagation of systemic defence signals in hypersensitive tissues ( 13 ). Bacterial blight, caused by Xanthomonas oryzae pv. oryzae is one of the most destructive diseases affecting rice. The pathogen enters plants through wounds in leaves and roots and colonizes the xylem vessels, leading to characteristic symptoms such as yellowish-white lesions, leaf desiccation, curling, wilting, and eventual plant death. During interactions with phytopathogenic bacteria, rice plants exhibit oxidative bursts similar to those observed in plant–fungal interactions, characterized by increased ROS production ( 14 ). In rice leaves infected with Xanthomonas oryzae pv. oryzae, elevated levels of hydrogen peroxide have been reported, correlating with pathogen proliferation. In this context, the present study investigates the dynamics of the oxidative burst, antioxidant defence mechanisms, and associated biochemical responses in five rice cultivars—TN-1, Satabdi, Naveen, Annapurna, and Tapaswini—following infection with the bacterial blight pathogen Xanthomonas oryzae pv. oryzae and treatment with defence elicitors. Understanding these early defence responses provides insight into cultivar-specific resistance mechanisms against bacterial blight in rice. 2. Materials and methods 2.1. Chemicals All solvents used in this study, including ethyl acetate, formic acid, HPLC-grade methanol, ethanol, acetone, and cyclohexane, were procured from Merck (India Pvt. Ltd.), HiMedia Laboratories (India), SRL (India), and Sigma-Aldrich (India). The chemicals used included salicylic acid (SA), chitosan, trichloroacetic acid (TCA), nitro blue tetrazolium (NBT), 2-deoxyribose (DOR), 2,2-diphenyl-1-picrylhydrazyl (DPPH), L-methionine, potassium iodide (KI), thiobarbituric acid (TBA), hydrogen peroxide (H 2 O 2 ), guaiacol, ascorbic acid, riboflavin, and ethylenediaminetetraacetic acid (EDTA). All chemicals were of analytical grade. 2.2. Plant materials Five rice (Oryza sativa L.) cultivars—TN-1, Satabdi, Naveen, Annapurna, and Tapaswini—were used in this study. Seeds of these cultivars were obtained from the previous season’s harvest. Details of plant growth conditions and experimental treatments are described in the respective sections corresponding to individual experiments conducted to achieve the objectives of the study. 2.3. Preparation of chitosan and salicylic acid Chitosan (CH) was prepared according to the method described by Villegas and Brodelius ( 15 ), with minor modifications. Briefly, 1 g of chitosan was dissolved in 75 mL of distilled water by heating to 90°C, followed by gradual titration with hydrochloric acid to a pH of 5.0. The final volume was adjusted to 100 mL with distilled water. The stock solution was autoclaved at 121°C for 15 minutes under 15 psi pressure. Salicylic acid (SA) was prepared by dissolving it in deionized water containing 10% methanol to obtain a final concentration of 200 µM, as described by Bhalerao and Raje Harshal ( 16 ). 2.4. Media for pathogen culture Potato dextrose agar (PDA) medium was obtained in ready-to-use powdered form from HiMedia Laboratories (India Pvt. Ltd.). The medium contained potato infusion equivalent to 200 g L⁻¹. For preparation, 39 g of PDA powder was suspended in 1 L of deionized water, and the pH was adjusted to 6.0. The medium was sterilized by autoclaving at 121°C and 15 psi for 15 minutes before use. 2.5. Assay of hydrogen peroxide (H 2 O 2 ) during rice–elicitor/pathogen interaction Hydrogen peroxide (H 2 O 2 ) content was determined following the method of Velikova et al. ( 17 ). Rice leaf tissue was homogenized in 0.1% (w/v) trichloroacetic acid (TCA) at a ratio of 1:10 (w/v) and centrifuged at 4°C for 15 minutes using a refrigerated centrifuge (REMI CPR-24 PLUS). One millilitre of the supernatant was mixed with 1 mL of 1 M potassium iodide (KI) solution and incubated at room temperature for 5 minutes. Absorbance was measured at 390 nm using a UV–Vis spectrophotometer (EVOLUTION 300, Thermo Fisher Scientific). 2.6. Determination of lipid peroxidation during rice–elicitor/pathogen interaction Lipid peroxidation was estimated by measuring malondialdehyde (MDA), a thiobarbituric acid–reactive substance, following the method described by Heath and Packer (18; 19). Rice leaf samples were homogenized in 0.1% (w/v) TCA at a ratio of 1:5 (w/v) and centrifuged at 12,000 × g for 30 minutes at 4°C. One millilitre of the supernatant was incubated with 4 mL of 20% (w/v) TCA containing 0.5% (w/v) thiobarbituric acid at 95°C for 30 minutes. The reaction was terminated by placing the tubes on ice for 10 minutes, followed by centrifugation at 10,000 × g for 15 minutes. Absorbance of the supernatant was measured at 532 nm. MDA concentration was calculated using an extinction coefficient of 155 mM⁻¹ cm⁻¹ and expressed as µmol g⁻¹ fresh weight (FW). 2.7. Determination of superoxide anion (O 2 •⁻) concentration (nitro-blue tetrazolium-reducing activity) Superoxide anion (O 2 •⁻) production was estimated by measuring nitro-blue tetrazolium (NBT) reduction, as described by Doke ( 20 ). Five leaf discs (0.5 cm diameter) were incubated in 3 mL of 0.01 M potassium phosphate buffer (pH 7.8) containing 0.05% NBT and 10 mM sodium azide (NaN₃) for 1 hour. After incubation, leaf discs were removed, and the reaction mixture was heated to 85°C for 15 minutes, then cooled rapidly. The increase in absorbance was measured at 580 nm and expressed as NBT-reducing activity per hour per gram fresh weight (h⁻¹ g⁻¹ FW). 2.8. Determination of hydroxyl radical (•OH) concentration during rice–elicitor/pathogen interaction Hydroxyl radical (•OH) production was quantified following the method of Tiedemann ( 21 ) using 2-deoxyribose (DOR) as a molecular probe. Twenty leaf discs (0.5 cm diameter) were incubated in 2 mL of 1 mM DOR solution for 45 minutes at room temperature (22°C) in the dark. Following incubation, 0.5 mL of 1% (w/v) thiobarbituric acid prepared in 0.05 M NaOH and 0.5 mL of 2.8% (w/v) TCA were added. The mixture was heated for 10 minutes, then cooled on ice for 10 minutes. Absorbance was recorded at 540 nm, and results were expressed as absorbance units g⁻¹ FW. 2.9. DPPH radical scavenging activity during rice–elicitor/pathogen interaction DPPH radical scavenging activity was determined according to the method described by Burits and Bucar ( 22 ). One gram of leaf tissue was homogenized in 40 mL of 90% methanol. An aliquot of 10 µL of the extract was mixed with 4 mL of distilled water and 1 mL of 250 µM DPPH solution. The mixture was incubated in the dark for 30 minutes. Absorbance was measured at 517 nm using a UV–Vis spectrophotometer (EVOLUTION 300, Thermo Fisher Scientific). DPPH radical scavenging activity was expressed as percentage inhibition relative to the control. 2.10. Assay of antioxidant enzyme activities during oxidative burst Rice leaf tissue was homogenized in 10 mL of chilled 0.1 M phosphate buffer (pH 7.0). The homogenate was centrifuged at 15,000 × g for 30 minutes at 4°C. The resulting supernatant was used as the crude enzyme extract for the estimation of superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPX), and ascorbate peroxidase (APX) activities. Protein concentration in the enzyme extract was determined following the Bradford method ( 23 ). 2.10.1. Superoxide dismutase (SOD) activity assay Superoxide dismutase activity was assayed by measuring its ability to inhibit the photochemical reduction of nitro blue tetrazolium (NBT), as described by Beauchamp and Fridovich ( 24 ). The reaction mixture (4 mL) consisted of 63 µM NBT, 13 mM L-methionine, 0.1 mM EDTA, 13 µM riboflavin, 0.05 M sodium carbonate, and 0.5 mL of enzyme extract. For the control, distilled water was used instead of enzyme extract. The reaction mixture was incubated at 25°C for 15 minutes under two 15-W fluorescent lamps, then kept in the dark for 15 minutes. Absorbance was recorded at 560 nm. One unit of SOD activity was defined as the amount of enzyme required to inhibit NBT reduction by 50%. 2.10.2. Catalase (CAT) activity assay Catalase activity was determined by monitoring the rate of hydrogen peroxide (H 2 O 2 ) decomposition at 240 nm, as described by Cakmak and Marschner ( 25 ). The reaction mixture (2 mL) contained 25 mM phosphate buffer (pH 7.0), 10 mM H 2 O 2, and 0.2 mL of enzyme extract. A decrease in absorbance of 0.1 unit per minute was defined as one unit of catalase activity under the assay conditions. Enzyme activity was expressed as nkat mg⁻¹ protein. 2.10.3. Guaiacol peroxidase (GPX) activity assay Guaiacol peroxidase activity was measured by monitoring the increase in absorbance at 470 nm due to the oxidation of guaiacol to tetraguaiacol, following the method of Chance and Maehly ( 26 ). The reaction mixture (3 mL) consisted of 20 mM guaiacol (0.5 mL), 0.1 M acetate buffer (pH 5.0; 2.1 mL), 40 mM H 2 O 2 (0.2 mL), and 0.2 mL of enzyme extract. Enzyme activity was calculated from the linear portion of the reaction curve and expressed as nkat mg⁻¹ protein. One unit of GPX activity was defined as the amount of enzyme catalyzing the oxidation of 1 µmol of guaiacol per minute. 2.10.4. Ascorbate peroxidase (APX) activity assay Ascorbate peroxidase activity was determined by measuring the decrease in absorbance at 290 nm due to the oxidation of ascorbic acid, following the method of Nakano and Asada ( 27 ). The reaction mixture (2 mL) contained 25 mM phosphate buffer (pH 7.0), 0.1 mM EDTA, 0.25 mM ascorbic acid, 1.0 mM H 2 O 2 , and 0.2 mL of enzyme extract. The decrease in absorbance was recorded 60 seconds after the addition of enzyme extract. A change in absorbance of 0.1 unit was defined as one unit of APX activity, and enzyme activity was expressed as nkat mg⁻¹ protein. 2.11. Statistical analysis All experiments were conducted with three biological replicates. Data are presented as mean ± standard deviation (SD). Statistical significance among treatments and time points was assessed using analysis of variance (ANOVA), and differences were considered significant at p < 0.05. 3. Results 3.1. Hydrogen peroxide (H 2 O 2 ) accumulation during rice–elicitor/pathogen interaction 3.1.1. Interaction between chitosan and rice plants Chitosan treatment increased H 2 O 2 production in all rice cultivars, with a noticeable increase observed at 24 h post-inoculation (hpi). TN-1 and Tapaswini exhibited the highest H 2 O 2 accumulation, showing 2.3- and 3.2-fold increases, respectively, at 96 hpi compared with the control. Overall, a single-phase pattern of H 2 O 2 production was observed in chitosan-treated plants (Fig. 1 ). 3.1.2. Interaction between salicylic acid and rice plants Salicylic acid (SA) treatment showed a similar trend in H 2 O 2 accumulation across all cultivars, with an initial increase followed by a gradual decline at 48 hpi (Fig. 1 ). Satabdi showed a significant increase in H 2 O 2 content, reaching 1.8-fold that of the control at 72 hpi. A triphasic pattern of H 2 O 2 accumulation was observed in SA-treated plants. 3.1.3. Interaction between pathogen and rice plants Following Xanthomonas oryzae pv. oryzae (Xoo) inoculation, Naveen, Annapurna, and Tapaswini showed a significant increase in H₂O₂ production at 24 hpi, with levels 2.3-, 2.9-, and 2.0-fold higher than the control, respectively. This was followed by a sharp increase after 72 hpi, indicating a biphasic oxidative burst (Fig. 1 ). In contrast, Satabdi and TN-1 did not exhibit a pronounced second phase of H 2 O 2 accumulation. 3.2. Lipid peroxidation during rice–elicitor/pathogen interaction 3.2.1. Interaction between chitosan and rice plants Lipid peroxidation, assessed by malondialdehyde (MDA) content, varied among cultivars following chitosan treatment. Control plants exhibited relatively higher baseline MDA levels. Among treated plants, Satabdi, Naveen, and Annapurna showed greater increases in MDA content compared with TN-1 and Tapaswini. The highest MDA levels were recorded at 120 hpi in Satabdi (7.06 µmol g⁻¹ FW), Naveen (5.75 µmol g⁻¹ FW), and Annapurna (5.62 µmol g⁻¹ FW). 3.2.2. Interaction between salicylic acid and rice plants In SA-treated TN-1 and Tapaswini plants, lipid peroxidation decreased significantly at 96 hpi, by 2.0- and 1.7-fold, respectively, compared with the control. In contrast, Satabdi, Naveen, and Annapurna exhibited the lowest MDA levels at 48 hpi, which were 1.6-, 2.0-, and 2.2-fold lower than the control, respectively (Fig. 2 ). 3.2.3. Interaction between pathogen and rice plants Following pathogen inoculation, TN-1 and Tapaswini showed a slight decrease in lipid peroxidation up to 96 hpi. In contrast, Satabdi, Annapurna, and Naveen displayed a decline in MDA content up to 72 hpi, followed by an increase until 120 hpi (Fig. 2 ). 3.3. Superoxide anion (O 2 •⁻) accumulation 3.3.1. Interaction between chitosan and rice plants Chitosan treatment resulted in significantly increased O 2 •⁻ levels at 72 hpi in TN-1, Naveen, and Annapurna. In Satabdi and Tapaswini, the highest O 2 •⁻ accumulation was observed at 120 hpi, reaching 2.24 and 2.40 µmol g⁻¹ FW, respectively (Fig. 3 ). 3.3.2. Interaction between salicylic acid and rice plants SA-treated TN-1 and Tapaswini plants exhibited maximum O 2 •⁻ accumulation at 120 hpi, with values of 2.42 and 2.50 µmol g⁻¹ FW, respectively. 3.3.3. Interaction between pathogen and rice plants In pathogen-inoculated plants, O 2 •⁻ production was detected shortly after infection. The highest accumulation was recorded at 120 hpi in TN-1 among the five cultivars studied (Fig. 3 ). 3.4. Hydroxyl radical (•OH) accumulation 3.4.1. Interaction between chitosan and rice plants Hydroxyl radical production increased rapidly following chitosan treatment. In TN-1 and Tapaswini, •OH levels increased up to 72 hpi and declined slightly thereafter at 96 hpi (Fig. 4 ). Naveen exhibited a continuous increase up to 96 hpi, reaching 1.77 µmol g⁻¹ FW. 3.4.2. Interaction between salicylic acid and rice plants SA-treated TN-1, Annapurna, and Tapaswini plants showed peak •OH levels at 72 hpi, with values of 1.72, 1.90, and 1.91 µmol g⁻¹ FW, respectively. 3.4.3. Interaction between pathogen and rice plants In pathogen-infected plants, •OH accumulation increased from 24 hpi onwards. Maximum levels were observed at 120 hpi in TN-1, Satabdi, and Tapaswini (Fig. 4 ). 3.5. DPPH radical scavenging activity 3.5.1. Interaction between chitosan and rice plants Chitosan treatment induced a similar trend in DPPH radical scavenging activity across all cultivars, with a gradual decline observed at 72 hpi. Satabdi showed a significant increase, reaching 4.5-fold higher activity than the control at 96 hpi. A biphasic pattern of DPPH activity was observed in chitosan-treated plants (Fig. 5 ). 3.5.2. Interaction between salicylic acid and rice plants In SA-treated plants, DPPH scavenging activity increased from 24 hpi across all cultivars (Fig. 5 ). Naveen exhibited the highest activity at 96 hpi, reaching 1.70 µmol g⁻¹ FW. 3.5.3. Interaction between pathogen and rice plants Following pathogen inoculation, DPPH scavenging activity increased within a few hours. TN-1 (1.61 µmol g⁻¹ FW) and Tapaswini (1.56 µmol g⁻¹ FW) exhibited the highest activity among the five cultivars at 96 hpi. 3.6. Antioxidant enzyme activities during oxidative burst 3.6.1. Superoxide dismutase (SOD) activity 3.6.1.1. Interaction between chitosan and rice plants SOD activity increased within a few hours of chitosan treatment in all rice cultivars. The highest SOD activity was observed at 96 h post-inoculation (hpi) in TN-1 (23.06 nkat mg⁻¹ protein) and Tapaswini (25.59 nkat mg⁻¹ protein) compared with the other cultivars (Fig. 6 ). 3.6.1.2. Interaction between salicylic acid and rice plants SA treatment resulted in a marked increase in SOD activity in TN-1, Naveen, and Tapaswini. Maximum activity was recorded at 96 hpi, with values of 27.48, 20.32, and 21.42 nkat mg⁻¹ protein, respectively (Fig. 6 ). In TN-1, SOD activity was approximately 2.9-fold higher than the control at this time point. 3.6.1.3. Interaction between pathogen and rice plants Pathogen inoculation induced a progressive increase in SOD activity from 24 hpi onwards. TN-1 and Tapaswini exhibited the highest SOD activity at 120 hpi (Fig. 6 ). In contrast, Satabdi, Naveen, and Annapurna showed a slight decline in SOD activity after 96 hpi. 3.6.2. Catalase (CAT) activity 3.6.2.1. Interaction between chitosan and rice plants In chitosan-treated plants, CAT activity peaked at 24 hpi in Tapaswini, reaching 21.93 nkat mg⁻¹ protein, which was 1.7-fold higher than the control. Thereafter, CAT activity declined gradually. In the remaining cultivars, CAT activity exhibited a biphasic pattern, with control values often exceeding early post-inoculation levels (Fig. 7 ). 3.6.2.2. Interaction between salicylic acid and rice plants SA treatment increased CAT activity from 24 hpi onwards. In TN-1, CAT activity peaked at 72 hpi with a value of 15.9 nkat mg⁻¹ protein, representing a 1.4-fold increase over the control (Fig. 7 ). Satabdi, Naveen, and Tapaswini showed a significant reduction in CAT activity, whereas Annapurna exhibited an increase after 48 hpi, reaching 16.29 nkat mg⁻¹ protein. 3.6.2.3. Interaction between pathogen and rice plants Pathogen inoculation resulted in increased CAT activity from 24 hpi. In TN-1, CAT activity was 2.1-fold higher than the control. Annapurna exhibited a marked decrease at 48 hpi (10.21 nkat mg⁻¹ protein below control levels), followed by a peak increase at 120 hpi of approximately 4.2 nkat mg⁻¹ protein above the control (Fig. 7 ). 3.6.3. Guaiacol peroxidase (GPX) activity 3.6.3.1. Interaction between chitosan and rice plants GPX activity increased in all chitosan-treated cultivars. Satabdi showed a rapid increase at 24 hpi (165 nkat mg⁻¹ protein), followed by a slight decrease at 48 hpi and a subsequent peak at 72 hpi (177 nkat mg⁻¹ protein; Fig. 8 ). Naveen exhibited a moderate increase, reaching 113 nkat mg⁻¹ protein at 24 hpi. At this time point, GPX activity was 1.9-fold higher in Satabdi and 1.4-fold higher in Naveen compared with their respective controls. 3.6.3.2. Interaction between salicylic acid and rice plants SA treatment also enhanced GPX activity across cultivars. Annapurna exhibited an early increase at 24 hpi (75.6 nkat mg⁻¹ protein), which was approximately 2-fold higher than the control (Fig. 8 ). The highest GPX activity was observed at 72 hpi in TN-1 (219 nkat mg⁻¹ protein) and Tapaswini (198.6 nkat mg⁻¹ protein), both representing a 2.1-fold increase over control levels, followed by a gradual decline. 3.6.3.3. Interaction between pathogen and rice plants Pathogen-inoculated plants showed elevated GPX activity from 24 hpi onwards. The highest activity was recorded at 72 hpi in TN-1 (219 nkat mg⁻¹ protein) and Tapaswini (222 nkat mg⁻¹ protein), exceeding control values by more than 2-fold. Annapurna displayed a significant early increase at 24 hpi, followed by a gradual decline. 3.6.4. Ascorbate peroxidase (APX) activity 3.6.4.1. Interaction between chitosan and rice plants APX activity increased at 24 hpi in all chitosan-treated cultivars (Fig. 9 ). Tapaswini exhibited the highest activity at this time point, reaching 18.41 nkat mg⁻¹ protein. A biphasic pattern of APX activity was observed in most cultivars following chitosan treatment. 3.6.4.2. Interaction between salicylic acid and rice plants In SA-treated plants, TN-1, Naveen, and Tapaswini showed maximum APX activity at 72 hpi, with values of 7.16, 11.24, and 5.61 nkat mg⁻¹ protein, respectively. In Naveen, APX activity was approximately 1.4-fold higher than the control at this time point (Fig. 9 ). 3.6.4.3. Interaction between pathogen and rice plants Pathogen inoculation resulted in a sharp increase in APX activity from 24 hpi in all cultivars. Tapaswini showed the highest APX activity at 24 hpi, reaching 18.24 nkat mg⁻¹ protein (Fig. 9 ). 4. Discussion Local infection of plant tissues by obligate biotrophic or hemibiotrophic bacterial pathogens triggers a rapid, transient increase in reactive oxygen species (ROS) production, commonly referred to as the oxidative burst ( 28 ). In the present study, rice leaves infected with Xanthomonas oryzae pv. oryzae (Xoo) exhibited elevated levels of ROS and malondialdehyde (MDA), along with enhanced antioxidant enzyme activities. Increased ROS production during the oxidative burst is recognized as one of the earliest defence responses activated following pathogen perception (28; 29). Significant variation in hydrogen peroxide (H 2 O 2 ) accumulation was observed in rice leaves treated with Xoo, chitosan (CH), and salicylic acid (SA), with a pronounced increase at 24 h post-inoculation (hpi). This early oxidative burst likely corresponds to PAMP-triggered immunity (PTI), which represents the first layer of plant defence following pathogen recognition. Both SA and CH act as defence elicitors and PTI mimics by inducing basal defence signalling and ROS generation. In contrast, the sustained or later-phase accumulation of ROS observed at subsequent time points may reflect prolonged defence signalling, reduced antioxidant efficiency, or enhanced pathogen proliferation during a compatible host–pathogen interaction. The accumulation of H 2 O 2 in infected rice leaves likely contributes to defence against the hemibiotrophic bacterial pathogen. Lipid peroxidation, one of the most prominent indicators of oxidative stress in both plants and animals, reflects damage to unsaturated membrane lipids ( 30 ). In the present study, reduced MDA levels at 24 hpi suggest an initial protective response against membrane damage, whereas later increases indicate progressive oxidative stress. Additionally, the levels of superoxide anion (O 2 •⁻) and hydroxyl radical (•OH) increased up to 72 hpi, further supporting the occurrence of sustained oxidative pressure during pathogen interaction. To counteract ROS-mediated damage, plants activate an array of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPX), and ascorbate peroxidase (APX). The cellular concentration of free radicals is regulated by a dynamic balance between ROS production and scavenging. In this study, pathogen inoculation enhanced SOD activity from 24 hpi onwards, with the highest increases recorded in TN-1 and Tapaswini up to 120 hpi. A slight decline in SOD activity after 96 hpi in Satabdi, Naveen, and Annapurna may indicate reduced scavenging efficiency during later infection stages. Catalase plays a crucial role in regulating intracellular H 2 O 2 levels ( 31 ). In the present investigation, CAT activity displayed a biphasic pattern in most cultivars following CH treatment. SA-treated TN-1 plants showed a 1.4-fold increase in CAT activity at 72 hpi; however, CAT activity declined at later stages, suggesting reduced detoxification efficiency over time. This decline may contribute to the sustained accumulation of H₂O₂ observed during advanced stages of infection. Guaiacol peroxidase activity was markedly elevated in Xoo-, SA-, and CH-treated plants, particularly in TN-1, Tapaswini, Satabdi, and Naveen at 72 hpi. Increased GPX activity has been associated with peroxide detoxification and protection of cellular membranes and proteins from oxidative damage (32; 14). Annapurna exhibited an early increase in GPX activity at 24 hpi, indicating cultivar-specific differences in antioxidant responses. Ascorbate peroxidase, present as multiple isoenzymes, plays a central role in fine-tuning ROS signalling and H₂O₂ metabolism in higher plants ( 33 ). In agreement with previous studies, APX activity increased significantly in rice leaves treated with Xoo, CH, and SA. Elevated APX activity was associated with increased H₂O₂ and MDA levels, suggesting an adaptive response aimed at limiting oxidative damage. APX activity increased markedly at 24 hpi in all cultivars, declined slightly at 48 hpi, and peaked again at 72 hpi, indicating a tightly regulated antioxidant response during the oxidative burst. Overall, the oxidative burst induced by rice–elicitor and rice–pathogen interactions represent an important early defence mechanism against the hemibiotrophic bacterial pathogen. However, the apparent decline in antioxidant efficiency, particularly the reduction in APX activity at later stages following SA and CH treatments, may allow sustained ROS accumulation and increased lipid peroxidation. These biochemical responses suggest that, despite the activation of defence mechanisms, the host-pathogen interaction remains compatible. This interpretation is consistent with previous reports indicating that ROS production during Xoo infection in rice may ultimately favour pathogen survival and establishment. To our knowledge, this is the first exhaustive study on rice-Xoo interaction in respect to oxidative burst. The precise origin of ROS during rice–elicitor–pathogen interactions, and the relative contributions of host- and pathogen-derived ROS, remain unresolved. Further investigations are required to elucidate these mechanisms and to clarify the role of oxidative signalling in determining resistance or susceptibility in rice. Abbreviations ROS: Reactive oxygen species H₂O₂: Hydrogen peroxide O₂•⁻: Superoxide anion •OH: Hydroxyl radical SOD: Superoxide dismutase CAT: Catalase GPX: Guaiacol peroxidase APX: Ascorbate peroxidase SA: Salicylic acid CH: Chitosan Xoo: Xanthomonas oryzae pv. oryzae Declarations 7. Acknowledgements The authors acknowledge the excellent technical assistance provided by Mr Manindra Nath Das (Senior Technician) and Mr Tripata Ojha throughout this work. The authors are also grateful to the Central Rice Research Institute, Cuttack, India, for providing the laboratory facilities required to carry out this study. 8. Conflict of Interest The authors declare that there are no conflicts of interest regarding the publication of this manuscript. 9. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. 10. CRediT authorship contribution statement Tusar Mondal: Writing – original manuscript, Data curation, Methodology, Formal analysis, Visualization; Sudhamoy Mandal : Conceptualization, Supervision, Drafting & Final review; Binod Saradar : Data analysis and Manuscript editing. All authors have read and approved the final version of the manuscript. References Przemysław W. Oxidative burst: an early plant response to pathogen infection. Biochem J. 1997; 322:681–692. Al-Dalaen SM. Oxidative stress versus antioxidants. Am J Biosci Bioeng. 2014; 2:60. https://doi.org/10.11648/j.bio.20140205.11 Lamb C, Dixon RA. The oxidative burst in plant disease resistance. Annu Rev Plant Biol. 1997; 48:251–275. Foyer CH, Lelandais M, Kunert KJ. Photooxidative stress in plants. Physiol Plant. 1994;92(4):696–717. https://doi.org/10.1111/j.1399-3054.1994.tb03042.x Vitiello G, Serpe L, Blázquez-Castro A. The role of reactive oxygen species in chemical and biochemical processes. Front Chem. 2021; 9:33. Mandal S, Das RK, Mishra S. Differential occurrence of oxidative burst and antioxidative mechanism in compatible and incompatible interactions of Solanum lycopersicum and Ralstonia solanacearum. Plant Physiol Biochem. 2011; 49:117–123. Gill SS, Tuteja N. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol Biochem. 2010; 48:909–930. https://doi.org/10.1016/j.plaphy.2010.08.016. Hasanuzzaman M, Hossain MA, da Silva JAT, Fujita M. Plant response and tolerance to abiotic oxidative stress: antioxidant defence is a key factor. In: Crop Stress and Its Management: Perspectives and Strategies. Springer; 2012. p. 261–315. Kaur N, Kaur J, Grewal SK, Singh I. Effect of heat stress on antioxidative defence system and its amelioration by heat acclimation and salicylic acid pretreatments in three pigeonpea genotypes. Indian J Agric Biochem. 2019; 32:106–110. Mittler R. ROS are good. Trends Plant Sci. 2017; 22:11–19. https://doi.org/10.1016/j.tplants.2016.08.002 Hasanuzzaman M, Bhuyan MHMB, Anee TI, Parvin K, Nahar K, Mahmud J, Fujita M. Regulation of ascorbate–glutathione pathway in mitigating oxidative damage in plants under abiotic stress. Antioxidants. 2019; 8:384. Raja V, Majeed U, Kang H, Andrabi KI, John R. Abiotic stress: interplay between ROS, hormones and MAPKs. Environ Exp Bot. 2017; 137:142–157. Noctor G, Reichheld JP, Foyer CH. ROS-related redox regulation and signalling in plants. Semin Cell Dev Biol. 2018; 80:3–12. Mandal S, Mitra A, Mallick N. Biochemical characterization of oxidative burst during interaction between Solanum lycopersicum and Fusarium oxysporum f. sp. lycopersici. Physiol Mol Plant Pathol. 2008; 72:56–61. Villegas M, Brodelius PE, Kylin A. Elicitor-induced hydroxycinnamoyl-CoA: tyramine hydroxycinnamoyl transferase in plant cell suspension cultures. Physiol Plant. 1990; 78:414–420. Bhalerao SS, Raje Harshal A. Preparation, optimization, characterization and stability studies of salicylic acid liposomes. Drug Dev Ind Pharm. 2003; 29:451–467. Velikova V, Yordanov I, Edreva A. Oxidative stress and some antioxidant systems in acid rain-treated bean plants: protective role of exogenous polyamines. Plant Sci. 2000; 151:59–66. Heath RL, Packer L. Photoperoxidation in isolated chloroplasts: I. Kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys. 1968; 125:189–198. Heath RL, Packer L. Photoperoxidation in isolated chloroplasts: II. Role of electron transfer. Arch Biochem Biophys. 1968; 125:850–857. Doke N. Involvement of superoxide anion generation in the hypersensitive response of potato tuber tissues to infection with an incompatible race of Phytophthora infestans. Physiol Plant Pathol. 1983; 23:345–357. Tiedemann AV. Evidence for a primary role of active oxygen species in induction of host cell death during infection of bean leaves with Botrytis cinerea. Physiol Mol Plant Pathol. 1997; 50:151–166. Burits M, Bucar F. Antioxidant activity of Nigella sativa essential oil. Phytother Res. 2000; 14:323–328. He F. Bradford protein assay. Bio-Protocol. 2011;1: e45. Beauchamp C, Fridovich I. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem. 1971; 44:276–287. Cakmak I, Marschner H. Magnesium deficiency and high light intensity enhance activities of superoxide dismutase, ascorbate peroxidase and glutathione reductase in bean leaves. Plant Physiol. 1992; 98:1222–1227. Chance B, Maehly AC. Assay of catalases and peroxidases. Methods Enzymol. 1955; 2:764–775. https://doi.org/10.1016/S0076-6879(55)02300-8 Nakano Y, Asada K. Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol. 1981; 22:867–880. Mehdy MC. Active oxygen species in plant defence against pathogens. Plant Physiol. 1994; 105:467–472. Levine A, Tenhaken R, Dixon R, Lamb C. H₂O₂ from the oxidative burst orchestrates the plant hypersensitive disease resistance response. Cell. 1994; 79:583–593. Catalá Á. Lipid peroxidation modifies the assembly of biological membranes: the lipid whisker model. Front Physiol. 2015; 5:520. Bolwell GP, Bindschedler LV, Blee KA, Butt VS, Davies DR, Gardner SL, Gerrish C, Minibayeva F. The apoplastic oxidative burst in response to biotic stress in plants: a three-component system. J Exp Bot. 2002; 53:1367–1376. Loggini B, Scartazza A, Brugnoli E, Navari-Izzo F. Antioxidative defence system, pigment composition and photosynthetic efficiency in two wheat cultivars subjected to drought. Plant Physiol. 1999; 119:1091–1100. Grant JJ, Loake GJ. Role of reactive oxygen intermediates and redox signalling in disease resistance. Plant Physiol. 2000; 124:21–29. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9124704","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":618157072,"identity":"c130161d-faaa-4cfb-b88c-95a7f9bff985","order_by":0,"name":"Tusar Mondal","email":"","orcid":"","institution":"Orissa University of Agriculture \u0026 Technology","correspondingAuthor":false,"prefix":"","firstName":"Tusar","middleName":"","lastName":"Mondal","suffix":""},{"id":618157074,"identity":"16e52b66-c7f1-4ea5-98cb-32d42f4b878b","order_by":1,"name":"Sudhamoy Mandal","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+ElEQVRIiWNgGAWjYHACMwglwdxwgIHBBshibDxApBbGhgMHGNJAWhqI18JwgOEwmI1Xi/yM5G0PfubYJDZINzYe/vDnvN3a9sNAW2psonFpMbiRVm7Yuy0tsUHmINBhPLeTt51JBGo5lpbbgEuLRI6ZBO+2w4kNEkCVByRuJ5sdADIYGw7j1CI/I8dM8i9ci8G5ZLPzD/FrYbiRYyaNsCXhgJ3ZDQK2GJx5ViYtuy3NuA3klzMHkhPMbgBtScDjF/n25G2Sb7fZyPZLNx/+UPHHzt7sfPrDBx9qbHA7DAoc26CMRLDKBALKQcAegzEKRsEoGAWjAAYABL1sg/WIA9wAAAAASUVORK5CYII=","orcid":"","institution":"ICAR-Central Rice Research Institute","correspondingAuthor":true,"prefix":"","firstName":"Sudhamoy","middleName":"","lastName":"Mandal","suffix":""},{"id":618157077,"identity":"631c4666-3f9d-42d8-9cf1-9a54289f2911","order_by":2,"name":"Binod Saradar","email":"","orcid":"","institution":"University of Calcutta","correspondingAuthor":false,"prefix":"","firstName":"Binod","middleName":"","lastName":"Saradar","suffix":""}],"badges":[],"createdAt":"2026-03-14 19:38:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9124704/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9124704/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106317612,"identity":"6cd8a018-30af-4bfd-9335-de14d2b066a6","added_by":"auto","created_at":"2026-04-07 11:42:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":110957,"visible":true,"origin":"","legend":"\u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e generation (expressed as µmol g\u003csup\u003e-1\u003c/sup\u003e FW) in rice plants on a time course after inoculation of the plants with Xoo, CH, SA and in the control. Data presented are the means ± SD of three biological replicates.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9124704/v1/3ddf8708c39b16b85eaca396.png"},{"id":106317812,"identity":"89bcde66-a9d3-44a0-a744-c418cdc8af0b","added_by":"auto","created_at":"2026-04-07 11:43:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":117609,"visible":true,"origin":"","legend":"\u003cp\u003eLipid peroxidation (measured in terms of malondialdehyde (MDA) content and expressed as µmol g\u003csup\u003e-1\u003c/sup\u003e FW in the rice plants on a time course after inoculation of the plants with Xoo, CH, SA and in the control. Data presented in graphs are the means ± SD of three biological replicates.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9124704/v1/24b5fd1fbbc710ab34d84435.png"},{"id":106317662,"identity":"0a52d23a-319a-404b-b2f8-f3b6014826c0","added_by":"auto","created_at":"2026-04-07 11:42:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":112918,"visible":true,"origin":"","legend":"\u003cp\u003eNitro-blue tetrazolium (NBT)-reducing activity for determination of superoxide anion (expressed as A/g/h FW) in the rice plants on a time course after inoculation of the plants with Xoo, CH, SA and in the control. Data presented in graphs are the means ± SD of three biological replicates.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9124704/v1/285ee60417406601d23c1e8d.png"},{"id":106317675,"identity":"148528d1-3ba9-4a34-bb14-dee4b5ad977c","added_by":"auto","created_at":"2026-04-07 11:42:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":110638,"visible":true,"origin":"","legend":"\u003cp\u003eHydroxyl radical concentration\u003cstrong\u003e \u003c/strong\u003e(expressed as A/g/h FW) in the rice plants on a time course after inoculation of the plants with Xoo, CH, SA and in the control. Data presented in graphs are the means ± SD of three biological replicates \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9124704/v1/3e62479bc4a3d7560e17b06c.png"},{"id":106317610,"identity":"e9172aec-f079-4a6e-8927-bb6157478b59","added_by":"auto","created_at":"2026-04-07 11:42:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":107558,"visible":true,"origin":"","legend":"\u003cp\u003eDPPH radical scavenging activity (expressed as µmol g⁻¹ FW) in the rice plants on a time course after inoculation of the plants with Xoo, CH, SA and in the control. Data presented in graphs are the means ± SD of three biological replicates \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9124704/v1/8cc56cafa056cdcc8cf21303.png"},{"id":106317609,"identity":"2ad6a1b7-4e08-409f-a8c0-6770e7436f51","added_by":"auto","created_at":"2026-04-07 11:42:51","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":103784,"visible":true,"origin":"","legend":"\u003cp\u003eSuperoxide dismutase (expressed as nkat.mg\u003csup\u003e-1\u003c/sup\u003e protein) in leaves of rice plants on a time course after inoculation of the plants with Xoo, CH, SA and in the control. Data presented in graphs are the means ± SD of three biological replicates.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9124704/v1/54f15e60a28493edbdbebec3.png"},{"id":106317611,"identity":"276dfc4e-2fa9-4d69-b57b-90125b59cf91","added_by":"auto","created_at":"2026-04-07 11:42:51","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":112479,"visible":true,"origin":"","legend":"\u003cp\u003eCatalase activity (expressed as nkat.mg\u003csup\u003e-1\u003c/sup\u003e protein) in leaves of rice plants on a time course after inoculation of the plants with Xoo, CH, SA and in the control. Data presented in graphs are the means ± SD of three biological replicates.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-9124704/v1/84cd3fc79f7079da256a902e.png"},{"id":106317613,"identity":"63893865-ef69-4d05-97b2-c5ae8adca02b","added_by":"auto","created_at":"2026-04-07 11:42:51","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":107395,"visible":true,"origin":"","legend":"\u003cp\u003eGuaiacol peroxidase activity (expressed as nkat.mg\u003csup\u003e-1\u003c/sup\u003e protein) in leaves of rice plants on a time course after inoculation of the plants with Xoo, CH, SA and in the control. Data presented in graphs are the means ± SD of three biological replicates.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-9124704/v1/62c9680e53e069f413958bf3.png"},{"id":106317710,"identity":"d5ccb3de-605d-48d3-8fc9-19077279f198","added_by":"auto","created_at":"2026-04-07 11:42:59","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":113722,"visible":true,"origin":"","legend":"\u003cp\u003eAscorbate peroxidase activity (expressed as nkat.mg-\u003csup\u003e1\u003c/sup\u003e protein) in leaves of rice plants on a time course after inoculation of the plants with Xoo, CH, SA and in the control. Data presented in graphs are the means ± SD of three biological replicates.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-9124704/v1/41cc467fb22195941a73c959.png"},{"id":107706533,"identity":"617fdaf7-5725-4ac2-8a7d-7fd1c4a2edbe","added_by":"auto","created_at":"2026-04-24 09:18:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1065090,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9124704/v1/d031ac4c-35fc-4681-9a5e-f41b4d2b8dc3.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eElucidating oxidative burst during interaction of rice-elicitor/pathogen (Xanthomonas oryzae pv. Oryzae)\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePlants are continuously exposed to numerous environmental challenges throughout their life cycle. Because plants are sessile, they have evolved a wide range of defence or stress-response mechanisms to cope with both biotic and abiotic stresses. Among the most serious biotic threats faced by plants are pathogenic fungi, bacteria, and viruses (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eReactive oxygen species (ROS), including hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), hydroxyl radicals (\u0026bull;OH), and superoxide anions (O\u003csub\u003e2\u003c/sub\u003e\u0026bull;⁻), are naturally produced in plant cells as by-products of photosynthetic and respiratory metabolism in chloroplasts and mitochondria (2;3). In addition, ROS are generated through redox reactions mediated by membrane-bound, cytoplasmic, and extracellular enzymes (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Under non-stress conditions, ROS production and scavenging are tightly regulated, maintaining cellular redox homeostasis. However, exposure to biotic and abiotic stressors such as pathogen invasion, dehydration, and low temperature can disrupt this balance, leading to excessive ROS accumulation and cellular damage, including enzyme inactivation and programmed cell death (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo mitigate ROS-induced damage, plants possess an elaborate antioxidant defence system comprising both enzymatic and non-enzymatic components. Key ROS-scavenging enzymes include superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPX), and ascorbate peroxidase (APX), which play essential roles in detoxifying excess ROS during stress conditions (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). In addition, plants utilize a range of non-enzymatic antioxidants such as ascorbic acid, glutathione, phenolic compounds, flavonoids, carotenoids, and tocopherols to maintain redox balance (7; 8; 9). The controlled production and scavenging of ROS are crucial for normal cellular processes, including growth, development, and signalling (10; 11). Conversely, excessive ROS accumulation under stress conditions disrupts cellular homeostasis, resulting in oxidative damage and yield loss (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAn oxidative burst is a hallmark early defence response in plants, characterized by the rapid and transient overproduction of ROS following pathogen or elicitor perception. Numerous studies have demonstrated that ROS generation and the activation of ROS-metabolizing enzymes are closely associated with defence responses in plant\u0026ndash;pathogen interactions. Due to their rapid production and cytotoxic nature, ROS function as an effective first line of defence by directly restricting pathogen growth or delaying infection. In addition, ROS contribute to cell wall strengthening through oxidative cross-linking of cell wall proteins and act as signalling molecules that activate downstream defence pathways. Recent evidence suggests that oxidative bursts also facilitate the propagation of systemic defence signals in hypersensitive tissues (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBacterial blight, caused by Xanthomonas oryzae pv. oryzae is one of the most destructive diseases affecting rice. The pathogen enters plants through wounds in leaves and roots and colonizes the xylem vessels, leading to characteristic symptoms such as yellowish-white lesions, leaf desiccation, curling, wilting, and eventual plant death. During interactions with phytopathogenic bacteria, rice plants exhibit oxidative bursts similar to those observed in plant\u0026ndash;fungal interactions, characterized by increased ROS production (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). In rice leaves infected with Xanthomonas oryzae pv. oryzae, elevated levels of hydrogen peroxide have been reported, correlating with pathogen proliferation.\u003c/p\u003e \u003cp\u003eIn this context, the present study investigates the dynamics of the oxidative burst, antioxidant defence mechanisms, and associated biochemical responses in five rice cultivars\u0026mdash;TN-1, Satabdi, Naveen, Annapurna, and Tapaswini\u0026mdash;following infection with the bacterial blight pathogen Xanthomonas oryzae pv. oryzae and treatment with defence elicitors. Understanding these early defence responses provides insight into cultivar-specific resistance mechanisms against bacterial blight in rice.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Chemicals\u003c/h2\u003e \u003cp\u003eAll solvents used in this study, including ethyl acetate, formic acid, HPLC-grade methanol, ethanol, acetone, and cyclohexane, were procured from Merck (India Pvt. Ltd.), HiMedia Laboratories (India), SRL (India), and Sigma-Aldrich (India). The chemicals used included salicylic acid (SA), chitosan, trichloroacetic acid (TCA), nitro blue tetrazolium (NBT), 2-deoxyribose (DOR), 2,2-diphenyl-1-picrylhydrazyl (DPPH), L-methionine, potassium iodide (KI), thiobarbituric acid (TBA), hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), guaiacol, ascorbic acid, riboflavin, and ethylenediaminetetraacetic acid (EDTA). All chemicals were of analytical grade.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Plant materials\u003c/h2\u003e \u003cp\u003eFive rice (Oryza sativa L.) cultivars\u0026mdash;TN-1, Satabdi, Naveen, Annapurna, and Tapaswini\u0026mdash;were used in this study. Seeds of these cultivars were obtained from the previous season\u0026rsquo;s harvest. Details of plant growth conditions and experimental treatments are described in the respective sections corresponding to individual experiments conducted to achieve the objectives of the study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Preparation of chitosan and salicylic acid\u003c/h2\u003e \u003cp\u003eChitosan (CH) was prepared according to the method described by Villegas and Brodelius (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e), with minor modifications. Briefly, 1 g of chitosan was dissolved in 75 mL of distilled water by heating to 90\u0026deg;C, followed by gradual titration with hydrochloric acid to a pH of 5.0. The final volume was adjusted to 100 mL with distilled water. The stock solution was autoclaved at 121\u0026deg;C for 15 minutes under 15 psi pressure.\u003c/p\u003e \u003cp\u003eSalicylic acid (SA) was prepared by dissolving it in deionized water containing 10% methanol to obtain a final concentration of 200 \u0026micro;M, as described by Bhalerao and Raje Harshal (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Media for pathogen culture\u003c/h2\u003e \u003cp\u003ePotato dextrose agar (PDA) medium was obtained in ready-to-use powdered form from HiMedia Laboratories (India Pvt. Ltd.). The medium contained potato infusion equivalent to 200 g L⁻\u0026sup1;. For preparation, 39 g of PDA powder was suspended in 1 L of deionized water, and the pH was adjusted to 6.0. The medium was sterilized by autoclaving at 121\u0026deg;C and 15 psi for 15 minutes before use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Assay of hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) during rice\u0026ndash;elicitor/pathogen interaction\u003c/h2\u003e \u003cp\u003eHydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) content was determined following the method of Velikova et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Rice leaf tissue was homogenized in 0.1% (w/v) trichloroacetic acid (TCA) at a ratio of 1:10 (w/v) and centrifuged at 4\u0026deg;C for 15 minutes using a refrigerated centrifuge (REMI CPR-24 PLUS). One millilitre of the supernatant was mixed with 1 mL of 1 M potassium iodide (KI) solution and incubated at room temperature for 5 minutes. Absorbance was measured at 390 nm using a UV\u0026ndash;Vis spectrophotometer (EVOLUTION 300, Thermo Fisher Scientific).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Determination of lipid peroxidation during rice\u0026ndash;elicitor/pathogen interaction\u003c/h2\u003e \u003cp\u003eLipid peroxidation was estimated by measuring malondialdehyde (MDA), a thiobarbituric acid\u0026ndash;reactive substance, following the method described by Heath and Packer (18; 19). Rice leaf samples were homogenized in 0.1% (w/v) TCA at a ratio of 1:5 (w/v) and centrifuged at 12,000 \u0026times; g for 30 minutes at 4\u0026deg;C. One millilitre of the supernatant was incubated with 4 mL of 20% (w/v) TCA containing 0.5% (w/v) thiobarbituric acid at 95\u0026deg;C for 30 minutes. The reaction was terminated by placing the tubes on ice for 10 minutes, followed by centrifugation at 10,000 \u0026times; g for 15 minutes. Absorbance of the supernatant was measured at 532 nm. MDA concentration was calculated using an extinction coefficient of 155 mM⁻\u0026sup1; cm⁻\u0026sup1; and expressed as \u0026micro;mol g⁻\u0026sup1; fresh weight (FW).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Determination of superoxide anion (O\u003csub\u003e2\u003c/sub\u003e\u0026bull;⁻) concentration (nitro-blue tetrazolium-reducing activity)\u003c/h2\u003e \u003cp\u003eSuperoxide anion (O\u003csub\u003e2\u003c/sub\u003e\u0026bull;⁻) production was estimated by measuring nitro-blue tetrazolium (NBT) reduction, as described by Doke (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Five leaf discs (0.5 cm diameter) were incubated in 3 mL of 0.01 M potassium phosphate buffer (pH 7.8) containing 0.05% NBT and 10 mM sodium azide (NaN₃) for 1 hour. After incubation, leaf discs were removed, and the reaction mixture was heated to 85\u0026deg;C for 15 minutes, then cooled rapidly. The increase in absorbance was measured at 580 nm and expressed as NBT-reducing activity per hour per gram fresh weight (h⁻\u0026sup1; g⁻\u0026sup1; FW).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Determination of hydroxyl radical (\u0026bull;OH) concentration during rice\u0026ndash;elicitor/pathogen interaction\u003c/h2\u003e \u003cp\u003eHydroxyl radical (\u0026bull;OH) production was quantified following the method of Tiedemann (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e) using 2-deoxyribose (DOR) as a molecular probe. Twenty leaf discs (0.5 cm diameter) were incubated in 2 mL of 1 mM DOR solution for 45 minutes at room temperature (22\u0026deg;C) in the dark. Following incubation, 0.5 mL of 1% (w/v) thiobarbituric acid prepared in 0.05 M NaOH and 0.5 mL of 2.8% (w/v) TCA were added. The mixture was heated for 10 minutes, then cooled on ice for 10 minutes. Absorbance was recorded at 540 nm, and results were expressed as absorbance units g⁻\u0026sup1; FW.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. DPPH radical scavenging activity during rice\u0026ndash;elicitor/pathogen interaction\u003c/h2\u003e \u003cp\u003eDPPH radical scavenging activity was determined according to the method described by Burits and Bucar (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). One gram of leaf tissue was homogenized in 40 mL of 90% methanol. An aliquot of 10 \u0026micro;L of the extract was mixed with 4 mL of distilled water and 1 mL of 250 \u0026micro;M DPPH solution. The mixture was incubated in the dark for 30 minutes. Absorbance was measured at 517 nm using a UV\u0026ndash;Vis spectrophotometer (EVOLUTION 300, Thermo Fisher Scientific). DPPH radical scavenging activity was expressed as percentage inhibition relative to the control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Assay of antioxidant enzyme activities during oxidative burst\u003c/h2\u003e \u003cp\u003eRice leaf tissue was homogenized in 10 mL of chilled 0.1 M phosphate buffer (pH 7.0). The homogenate was centrifuged at 15,000 \u0026times; g for 30 minutes at 4\u0026deg;C. The resulting supernatant was used as the crude enzyme extract for the estimation of superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPX), and ascorbate peroxidase (APX) activities. Protein concentration in the enzyme extract was determined following the Bradford method (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.10.1. Superoxide dismutase (SOD) activity assay\u003c/h2\u003e \u003cp\u003eSuperoxide dismutase activity was assayed by measuring its ability to inhibit the photochemical reduction of nitro blue tetrazolium (NBT), as described by Beauchamp and Fridovich (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). The reaction mixture (4 mL) consisted of 63 \u0026micro;M NBT, 13 mM L-methionine, 0.1 mM EDTA, 13 \u0026micro;M riboflavin, 0.05 M sodium carbonate, and 0.5 mL of enzyme extract. For the control, distilled water was used instead of enzyme extract. The reaction mixture was incubated at 25\u0026deg;C for 15 minutes under two 15-W fluorescent lamps, then kept in the dark for 15 minutes. Absorbance was recorded at 560 nm. One unit of SOD activity was defined as the amount of enzyme required to inhibit NBT reduction by 50%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.10.2. Catalase (CAT) activity assay\u003c/h2\u003e \u003cp\u003eCatalase activity was determined by monitoring the rate of hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) decomposition at 240 nm, as described by Cakmak and Marschner (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). The reaction mixture (2 mL) contained 25 mM phosphate buffer (pH 7.0), 10 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2,\u003c/sub\u003e and 0.2 mL of enzyme extract. A decrease in absorbance of 0.1 unit per minute was defined as one unit of catalase activity under the assay conditions. Enzyme activity was expressed as nkat mg⁻\u0026sup1; protein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.10.3. Guaiacol peroxidase (GPX) activity assay\u003c/h2\u003e \u003cp\u003eGuaiacol peroxidase activity was measured by monitoring the increase in absorbance at 470 nm due to the oxidation of guaiacol to tetraguaiacol, following the method of Chance and Maehly (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). The reaction mixture (3 mL) consisted of 20 mM guaiacol (0.5 mL), 0.1 M acetate buffer (pH 5.0; 2.1 mL), 40 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (0.2 mL), and 0.2 mL of enzyme extract. Enzyme activity was calculated from the linear portion of the reaction curve and expressed as nkat mg⁻\u0026sup1; protein. One unit of GPX activity was defined as the amount of enzyme catalyzing the oxidation of 1 \u0026micro;mol of guaiacol per minute.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e2.10.4. Ascorbate peroxidase (APX) activity assay\u003c/h2\u003e \u003cp\u003eAscorbate peroxidase activity was determined by measuring the decrease in absorbance at 290 nm due to the oxidation of ascorbic acid, following the method of Nakano and Asada (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). The reaction mixture (2 mL) contained 25 mM phosphate buffer (pH 7.0), 0.1 mM EDTA, 0.25 mM ascorbic acid, 1.0 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and 0.2 mL of enzyme extract. The decrease in absorbance was recorded 60 seconds after the addition of enzyme extract. A change in absorbance of 0.1 unit was defined as one unit of APX activity, and enzyme activity was expressed as nkat mg⁻\u0026sup1; protein.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.11. Statistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments were conducted with three biological replicates. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical significance among treatments and time points was assessed using analysis of variance (ANOVA), and differences were considered significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e3.1. Hydrogen peroxide (H\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eO\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003cb\u003e) accumulation during rice\u0026ndash;elicitor/pathogen interaction\u003c/b\u003e\u003c/h2\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e3.1.1. Interaction between chitosan and rice plants\u003c/h2\u003e \u003cp\u003eChitosan treatment increased H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e production in all rice cultivars, with a noticeable increase observed at 24 h post-inoculation (hpi). TN-1 and Tapaswini exhibited the highest H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e accumulation, showing 2.3- and 3.2-fold increases, respectively, at 96 hpi compared with the control. Overall, a single-phase pattern of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e production was observed in chitosan-treated plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003e3.1.2. Interaction between salicylic acid and rice plants\u003c/h2\u003e \u003cp\u003eSalicylic acid (SA) treatment showed a similar trend in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e accumulation across all cultivars, with an initial increase followed by a gradual decline at 48 hpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Satabdi showed a significant increase in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content, reaching 1.8-fold that of the control at 72 hpi. A triphasic pattern of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e accumulation was observed in SA-treated plants.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003e3.1.3. Interaction between pathogen and rice plants\u003c/h2\u003e \u003cp\u003eFollowing Xanthomonas oryzae pv. oryzae (Xoo) inoculation, Naveen, Annapurna, and Tapaswini showed a significant increase in H₂O₂ production at 24 hpi, with levels 2.3-, 2.9-, and 2.0-fold higher than the control, respectively. This was followed by a sharp increase after 72 hpi, indicating a biphasic oxidative burst (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In contrast, Satabdi and TN-1 did not exhibit a pronounced second phase of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e accumulation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Lipid peroxidation during rice\u0026ndash;elicitor/pathogen interaction\u003c/h2\u003e \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1. Interaction between chitosan and rice plants\u003c/h2\u003e \u003cp\u003eLipid peroxidation, assessed by malondialdehyde (MDA) content, varied among cultivars following chitosan treatment. Control plants exhibited relatively higher baseline MDA levels. Among treated plants, Satabdi, Naveen, and Annapurna showed greater increases in MDA content compared with TN-1 and Tapaswini. The highest MDA levels were recorded at 120 hpi in Satabdi (7.06 \u0026micro;mol g⁻\u0026sup1; FW), Naveen (5.75 \u0026micro;mol g⁻\u0026sup1; FW), and Annapurna (5.62 \u0026micro;mol g⁻\u0026sup1; FW).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003e3.2.2. Interaction between salicylic acid and rice plants\u003c/h2\u003e \u003cp\u003eIn SA-treated TN-1 and Tapaswini plants, lipid peroxidation decreased significantly at 96 hpi, by 2.0- and 1.7-fold, respectively, compared with the control. In contrast, Satabdi, Naveen, and Annapurna exhibited the lowest MDA levels at 48 hpi, which were 1.6-, 2.0-, and 2.2-fold lower than the control, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003e3.2.3. Interaction between pathogen and rice plants\u003c/h2\u003e \u003cp\u003eFollowing pathogen inoculation, TN-1 and Tapaswini showed a slight decrease in lipid peroxidation up to 96 hpi. In contrast, Satabdi, Annapurna, and Naveen displayed a decline in MDA content up to 72 hpi, followed by an increase until 120 hpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Superoxide anion (O\u003csub\u003e2\u003c/sub\u003e\u0026bull;⁻) accumulation\u003c/h2\u003e \u003cdiv id=\"Sec28\" class=\"Section3\"\u003e \u003ch2\u003e3.3.1. Interaction between chitosan and rice plants\u003c/h2\u003e \u003cp\u003eChitosan treatment resulted in significantly increased O\u003csub\u003e2\u003c/sub\u003e\u0026bull;⁻ levels at 72 hpi in TN-1, Naveen, and Annapurna. In Satabdi and Tapaswini, the highest O\u003csub\u003e2\u003c/sub\u003e\u0026bull;⁻ accumulation was observed at 120 hpi, reaching 2.24 and 2.40 \u0026micro;mol g⁻\u0026sup1; FW, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section3\"\u003e \u003ch2\u003e3.3.2. Interaction between salicylic acid and rice plants\u003c/h2\u003e \u003cp\u003eSA-treated TN-1 and Tapaswini plants exhibited maximum O\u003csub\u003e2\u003c/sub\u003e\u0026bull;⁻ accumulation at 120 hpi, with values of 2.42 and 2.50 \u0026micro;mol g⁻\u0026sup1; FW, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section3\"\u003e \u003ch2\u003e3.3.3. Interaction between pathogen and rice plants\u003c/h2\u003e \u003cp\u003eIn pathogen-inoculated plants, O\u003csub\u003e2\u003c/sub\u003e\u0026bull;⁻ production was detected shortly after infection. The highest accumulation was recorded at 120 hpi in TN-1 among the five cultivars studied (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Hydroxyl radical (\u0026bull;OH) accumulation\u003c/h2\u003e \u003cdiv id=\"Sec32\" class=\"Section3\"\u003e \u003ch2\u003e3.4.1. Interaction between chitosan and rice plants\u003c/h2\u003e \u003cp\u003eHydroxyl radical production increased rapidly following chitosan treatment. In TN-1 and Tapaswini, \u0026bull;OH levels increased up to 72 hpi and declined slightly thereafter at 96 hpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Naveen exhibited a continuous increase up to 96 hpi, reaching 1.77 \u0026micro;mol g⁻\u0026sup1; FW.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e \u003ch2\u003e3.4.2. Interaction between salicylic acid and rice plants\u003c/h2\u003e \u003cp\u003eSA-treated TN-1, Annapurna, and Tapaswini plants showed peak \u0026bull;OH levels at 72 hpi, with values of 1.72, 1.90, and 1.91 \u0026micro;mol g⁻\u0026sup1; FW, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section3\"\u003e \u003ch2\u003e3.4.3. Interaction between pathogen and rice plants\u003c/h2\u003e \u003cp\u003eIn pathogen-infected plants, \u0026bull;OH accumulation increased from 24 hpi onwards. Maximum levels were observed at 120 hpi in TN-1, Satabdi, and Tapaswini (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec35\" class=\"Section2\"\u003e \u003ch2\u003e3.5. DPPH radical scavenging activity\u003c/h2\u003e \u003cdiv id=\"Sec36\" class=\"Section3\"\u003e \u003ch2\u003e3.5.1. Interaction between chitosan and rice plants\u003c/h2\u003e \u003cp\u003eChitosan treatment induced a similar trend in DPPH radical scavenging activity across all cultivars, with a gradual decline observed at 72 hpi. Satabdi showed a significant increase, reaching 4.5-fold higher activity than the control at 96 hpi. A biphasic pattern of DPPH activity was observed in chitosan-treated plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec37\" class=\"Section3\"\u003e \u003ch2\u003e3.5.2. Interaction between salicylic acid and rice plants\u003c/h2\u003e \u003cp\u003eIn SA-treated plants, DPPH scavenging activity increased from 24 hpi across all cultivars (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Naveen exhibited the highest activity at 96 hpi, reaching 1.70 \u0026micro;mol g⁻\u0026sup1; FW.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec38\" class=\"Section3\"\u003e \u003ch2\u003e3.5.3. Interaction between pathogen and rice plants\u003c/h2\u003e \u003cp\u003eFollowing pathogen inoculation, DPPH scavenging activity increased within a few hours. TN-1 (1.61 \u0026micro;mol g⁻\u0026sup1; FW) and Tapaswini (1.56 \u0026micro;mol g⁻\u0026sup1; FW) exhibited the highest activity among the five cultivars at 96 hpi.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec39\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Antioxidant enzyme activities during oxidative burst\u003c/h2\u003e \u003cdiv id=\"Sec40\" class=\"Section3\"\u003e \u003ch2\u003e3.6.1. Superoxide dismutase (SOD) activity\u003c/h2\u003e \u003cdiv id=\"Sec41\" class=\"Section4\"\u003e \u003ch2\u003e3.6.1.1. Interaction between chitosan and rice plants\u003c/h2\u003e \u003cp\u003eSOD activity increased within a few hours of chitosan treatment in all rice cultivars. The highest SOD activity was observed at 96 h post-inoculation (hpi) in TN-1 (23.06 nkat mg⁻\u0026sup1; protein) and Tapaswini (25.59 nkat mg⁻\u0026sup1; protein) compared with the other cultivars (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec42\" class=\"Section4\"\u003e \u003ch2\u003e3.6.1.2. Interaction between salicylic acid and rice plants\u003c/h2\u003e \u003cp\u003eSA treatment resulted in a marked increase in SOD activity in TN-1, Naveen, and Tapaswini. Maximum activity was recorded at 96 hpi, with values of 27.48, 20.32, and 21.42 nkat mg⁻\u0026sup1; protein, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In TN-1, SOD activity was approximately 2.9-fold higher than the control at this time point.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec43\" class=\"Section4\"\u003e \u003ch2\u003e3.6.1.3. Interaction between pathogen and rice plants\u003c/h2\u003e \u003cp\u003ePathogen inoculation induced a progressive increase in SOD activity from 24 hpi onwards. TN-1 and Tapaswini exhibited the highest SOD activity at 120 hpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In contrast, Satabdi, Naveen, and Annapurna showed a slight decline in SOD activity after 96 hpi.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec44\" class=\"Section3\"\u003e \u003ch2\u003e3.6.2. Catalase (CAT) activity\u003c/h2\u003e \u003cdiv id=\"Sec45\" class=\"Section4\"\u003e \u003ch2\u003e3.6.2.1. Interaction between chitosan and rice plants\u003c/h2\u003e \u003cp\u003eIn chitosan-treated plants, CAT activity peaked at 24 hpi in Tapaswini, reaching 21.93 nkat mg⁻\u0026sup1; protein, which was 1.7-fold higher than the control. Thereafter, CAT activity declined gradually. In the remaining cultivars, CAT activity exhibited a biphasic pattern, with control values often exceeding early post-inoculation levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec46\" class=\"Section4\"\u003e \u003ch2\u003e3.6.2.2. Interaction between salicylic acid and rice plants\u003c/h2\u003e \u003cp\u003eSA treatment increased CAT activity from 24 hpi onwards. In TN-1, CAT activity peaked at 72 hpi with a value of 15.9 nkat mg⁻\u0026sup1; protein, representing a 1.4-fold increase over the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Satabdi, Naveen, and Tapaswini showed a significant reduction in CAT activity, whereas Annapurna exhibited an increase after 48 hpi, reaching 16.29 nkat mg⁻\u0026sup1; protein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec47\" class=\"Section4\"\u003e \u003ch2\u003e3.6.2.3. Interaction between pathogen and rice plants\u003c/h2\u003e \u003cp\u003ePathogen inoculation resulted in increased CAT activity from 24 hpi. In TN-1, CAT activity was 2.1-fold higher than the control. Annapurna exhibited a marked decrease at 48 hpi (10.21 nkat mg⁻\u0026sup1; protein below control levels), followed by a peak increase at 120 hpi of approximately 4.2 nkat mg⁻\u0026sup1; protein above the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec48\" class=\"Section3\"\u003e \u003ch2\u003e3.6.3. Guaiacol peroxidase (GPX) activity\u003c/h2\u003e \u003cdiv id=\"Sec49\" class=\"Section4\"\u003e \u003ch2\u003e3.6.3.1. Interaction between chitosan and rice plants\u003c/h2\u003e \u003cp\u003eGPX activity increased in all chitosan-treated cultivars. Satabdi showed a rapid increase at 24 hpi (165 nkat mg⁻\u0026sup1; protein), followed by a slight decrease at 48 hpi and a subsequent peak at 72 hpi (177 nkat mg⁻\u0026sup1; protein; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Naveen exhibited a moderate increase, reaching 113 nkat mg⁻\u0026sup1; protein at 24 hpi. At this time point, GPX activity was 1.9-fold higher in Satabdi and 1.4-fold higher in Naveen compared with their respective controls.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec50\" class=\"Section4\"\u003e \u003ch2\u003e3.6.3.2. Interaction between salicylic acid and rice plants\u003c/h2\u003e \u003cp\u003eSA treatment also enhanced GPX activity across cultivars. Annapurna exhibited an early increase at 24 hpi (75.6 nkat mg⁻\u0026sup1; protein), which was approximately 2-fold higher than the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). The highest GPX activity was observed at 72 hpi in TN-1 (219 nkat mg⁻\u0026sup1; protein) and Tapaswini (198.6 nkat mg⁻\u0026sup1; protein), both representing a 2.1-fold increase over control levels, followed by a gradual decline.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec51\" class=\"Section4\"\u003e \u003ch2\u003e3.6.3.3. Interaction between pathogen and rice plants\u003c/h2\u003e \u003cp\u003ePathogen-inoculated plants showed elevated GPX activity from 24 hpi onwards. The highest activity was recorded at 72 hpi in TN-1 (219 nkat mg⁻\u0026sup1; protein) and Tapaswini (222 nkat mg⁻\u0026sup1; protein), exceeding control values by more than 2-fold. Annapurna displayed a significant early increase at 24 hpi, followed by a gradual decline.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec52\" class=\"Section3\"\u003e \u003ch2\u003e3.6.4. Ascorbate peroxidase (APX) activity\u003c/h2\u003e \u003cdiv id=\"Sec53\" class=\"Section4\"\u003e \u003ch2\u003e3.6.4.1. Interaction between chitosan and rice plants\u003c/h2\u003e \u003cp\u003eAPX activity increased at 24 hpi in all chitosan-treated cultivars (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Tapaswini exhibited the highest activity at this time point, reaching 18.41 nkat mg⁻\u0026sup1; protein. A biphasic pattern of APX activity was observed in most cultivars following chitosan treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec54\" class=\"Section4\"\u003e \u003ch2\u003e3.6.4.2. Interaction between salicylic acid and rice plants\u003c/h2\u003e \u003cp\u003eIn SA-treated plants, TN-1, Naveen, and Tapaswini showed maximum APX activity at 72 hpi, with values of 7.16, 11.24, and 5.61 nkat mg⁻\u0026sup1; protein, respectively. In Naveen, APX activity was approximately 1.4-fold higher than the control at this time point (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec55\" class=\"Section4\"\u003e \u003ch2\u003e3.6.4.3. Interaction between pathogen and rice plants\u003c/h2\u003e \u003cp\u003ePathogen inoculation resulted in a sharp increase in APX activity from 24 hpi in all cultivars. Tapaswini showed the highest APX activity at 24 hpi, reaching 18.24 nkat mg⁻\u0026sup1; protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eLocal infection of plant tissues by obligate biotrophic or hemibiotrophic bacterial pathogens triggers a rapid, transient increase in reactive oxygen species (ROS) production, commonly referred to as the oxidative burst (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). In the present study, rice leaves infected with Xanthomonas oryzae pv. oryzae (Xoo) exhibited elevated levels of ROS and malondialdehyde (MDA), along with enhanced antioxidant enzyme activities. Increased ROS production during the oxidative burst is recognized as one of the earliest defence responses activated following pathogen perception (28; 29).\u003c/p\u003e \u003cp\u003eSignificant variation in hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) accumulation was observed in rice leaves treated with Xoo, chitosan (CH), and salicylic acid (SA), with a pronounced increase at 24 h post-inoculation (hpi). This early oxidative burst likely corresponds to PAMP-triggered immunity (PTI), which represents the first layer of plant defence following pathogen recognition. Both SA and CH act as defence elicitors and PTI mimics by inducing basal defence signalling and ROS generation. In contrast, the sustained or later-phase accumulation of ROS observed at subsequent time points may reflect prolonged defence signalling, reduced antioxidant efficiency, or enhanced pathogen proliferation during a compatible host\u0026ndash;pathogen interaction.\u003c/p\u003e \u003cp\u003eThe accumulation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in infected rice leaves likely contributes to defence against the hemibiotrophic bacterial pathogen. Lipid peroxidation, one of the most prominent indicators of oxidative stress in both plants and animals, reflects damage to unsaturated membrane lipids (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). In the present study, reduced MDA levels at 24 hpi suggest an initial protective response against membrane damage, whereas later increases indicate progressive oxidative stress. Additionally, the levels of superoxide anion (O\u003csub\u003e2\u003c/sub\u003e\u0026bull;⁻) and hydroxyl radical (\u0026bull;OH) increased up to 72 hpi, further supporting the occurrence of sustained oxidative pressure during pathogen interaction.\u003c/p\u003e \u003cp\u003eTo counteract ROS-mediated damage, plants activate an array of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPX), and ascorbate peroxidase (APX). The cellular concentration of free radicals is regulated by a dynamic balance between ROS production and scavenging. In this study, pathogen inoculation enhanced SOD activity from 24 hpi onwards, with the highest increases recorded in TN-1 and Tapaswini up to 120 hpi. A slight decline in SOD activity after 96 hpi in Satabdi, Naveen, and Annapurna may indicate reduced scavenging efficiency during later infection stages.\u003c/p\u003e \u003cp\u003eCatalase plays a crucial role in regulating intracellular H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). In the present investigation, CAT activity displayed a biphasic pattern in most cultivars following CH treatment. SA-treated TN-1 plants showed a 1.4-fold increase in CAT activity at 72 hpi; however, CAT activity declined at later stages, suggesting reduced detoxification efficiency over time. This decline may contribute to the sustained accumulation of H₂O₂ observed during advanced stages of infection.\u003c/p\u003e \u003cp\u003eGuaiacol peroxidase activity was markedly elevated in Xoo-, SA-, and CH-treated plants, particularly in TN-1, Tapaswini, Satabdi, and Naveen at 72 hpi. Increased GPX activity has been associated with peroxide detoxification and protection of cellular membranes and proteins from oxidative damage (32; 14). Annapurna exhibited an early increase in GPX activity at 24 hpi, indicating cultivar-specific differences in antioxidant responses.\u003c/p\u003e \u003cp\u003eAscorbate peroxidase, present as multiple isoenzymes, plays a central role in fine-tuning ROS signalling and H₂O₂ metabolism in higher plants (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). In agreement with previous studies, APX activity increased significantly in rice leaves treated with Xoo, CH, and SA. Elevated APX activity was associated with increased H₂O₂ and MDA levels, suggesting an adaptive response aimed at limiting oxidative damage. APX activity increased markedly at 24 hpi in all cultivars, declined slightly at 48 hpi, and peaked again at 72 hpi, indicating a tightly regulated antioxidant response during the oxidative burst.\u003c/p\u003e \u003cp\u003eOverall, the oxidative burst induced by rice\u0026ndash;elicitor and rice\u0026ndash;pathogen interactions represent an important early defence mechanism against the hemibiotrophic bacterial pathogen. However, the apparent decline in antioxidant efficiency, particularly the reduction in APX activity at later stages following SA and CH treatments, may allow sustained ROS accumulation and increased lipid peroxidation. These biochemical responses suggest that, despite the activation of defence mechanisms, the host-pathogen interaction remains compatible. This interpretation is consistent with previous reports indicating that ROS production during Xoo infection in rice may ultimately favour pathogen survival and establishment. To our knowledge, this is the first exhaustive study on rice-Xoo interaction in respect to oxidative burst.\u003c/p\u003e \u003cp\u003eThe precise origin of ROS during rice\u0026ndash;elicitor\u0026ndash;pathogen interactions, and the relative contributions of host- and pathogen-derived ROS, remain unresolved. Further investigations are required to elucidate these mechanisms and to clarify the role of oxidative signalling in determining resistance or susceptibility in rice.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eROS: Reactive oxygen species\u003c/p\u003e\n\u003cp\u003eH₂O₂: Hydrogen peroxide\u003c/p\u003e\n\u003cp\u003eO₂•⁻: Superoxide anion\u003c/p\u003e\n\u003cp\u003e•OH: Hydroxyl radical\u003c/p\u003e\n\u003cp\u003eSOD: Superoxide dismutase\u003c/p\u003e\n\u003cp\u003eCAT: Catalase\u003c/p\u003e\n\u003cp\u003eGPX: Guaiacol peroxidase\u003c/p\u003e\n\u003cp\u003eAPX: Ascorbate peroxidase\u003c/p\u003e\n\u003cp\u003eSA: Salicylic acid\u003c/p\u003e\n\u003cp\u003eCH: Chitosan\u003c/p\u003e\n\u003cp\u003eXoo: \u003cem\u003eXanthomonas oryzae\u003c/em\u003e pv. \u003cem\u003eoryzae\u003c/em\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e7. Acknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge the excellent technical assistance provided by Mr Manindra Nath Das (Senior Technician) and Mr Tripata Ojha throughout this work. The authors are also grateful to the Central Rice Research Institute, Cuttack, India, for providing the laboratory facilities required to carry out this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e8. Conflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no conflicts of interest regarding the publication of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e9. Funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e10. CRediT\u0026nbsp;authorship\u0026nbsp;contribution statement\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTusar Mondal:\u003c/strong\u003e Writing\u0026nbsp;\u0026ndash; original manuscript, Data curation, Methodology, Formal analysis,\u0026nbsp;Visualization; \u003cstrong\u003eSudhamoy Mandal\u003c/strong\u003e: Conceptualization, Supervision, Drafting \u0026amp; Final review; \u003cstrong\u003eBinod Saradar\u003c/strong\u003e: Data analysis and Manuscript editing. All authors have read and approved the final version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003ePrzemysław W. Oxidative burst: an early plant response to pathogen infection. Biochem J. 1997; 322:681\u0026ndash;692.\u003c/li\u003e\n \u003cli\u003eAl-Dalaen SM. Oxidative stress versus antioxidants. Am J Biosci Bioeng. 2014; 2:60. https://doi.org/10.11648/j.bio.20140205.11\u003c/li\u003e\n \u003cli\u003eLamb C, Dixon RA. The oxidative burst in plant disease resistance. 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Physiol Plant. 1990; 78:414\u0026ndash;420.\u003c/li\u003e\n \u003cli\u003eBhalerao SS, Raje Harshal A. Preparation, optimization, characterization and stability studies of salicylic acid liposomes. Drug Dev Ind Pharm. 2003; 29:451\u0026ndash;467.\u003c/li\u003e\n \u003cli\u003eVelikova V, Yordanov I, Edreva A. Oxidative stress and some antioxidant systems in acid rain-treated bean plants: protective role of exogenous polyamines. Plant Sci. 2000; 151:59\u0026ndash;66.\u003c/li\u003e\n \u003cli\u003eHeath RL, Packer L. Photoperoxidation in isolated chloroplasts: I. Kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys. 1968; 125:189\u0026ndash;198.\u003c/li\u003e\n \u003cli\u003eHeath RL, Packer L. Photoperoxidation in isolated chloroplasts: II. Role of electron transfer. Arch Biochem Biophys. 1968; 125:850\u0026ndash;857.\u003c/li\u003e\n \u003cli\u003eDoke N. 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Lipid peroxidation modifies the assembly of biological membranes: the lipid whisker model. Front Physiol. 2015; 5:520.\u003c/li\u003e\n \u003cli\u003eBolwell GP, Bindschedler LV, Blee KA, Butt VS, Davies DR, Gardner SL, Gerrish C, Minibayeva F. The apoplastic oxidative burst in response to biotic stress in plants: a three-component system. J Exp Bot. 2002; 53:1367\u0026ndash;1376.\u003c/li\u003e\n \u003cli\u003eLoggini B, Scartazza A, Brugnoli E, Navari-Izzo F. Antioxidative defence system, pigment composition and photosynthetic efficiency in two wheat cultivars subjected to drought. Plant Physiol. 1999; 119:1091\u0026ndash;1100.\u003c/li\u003e\n \u003cli\u003eGrant JJ, Loake GJ. Role of reactive oxygen intermediates and redox signalling in disease resistance. Plant Physiol. 2000; 124:21\u0026ndash;29.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Antioxidative mechanism, Oxidative burst, Reactive oxygen species, Rice–pathogen interaction, Xanthomonas oryzae pv. Oryzae","lastPublishedDoi":"10.21203/rs.3.rs-9124704/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9124704/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe oxidative burst is one of the earliest and most prominent defence responses of plants to microbial infection and is characterized by a rapid and transient accumulation of reactive oxygen species (ROS), including hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e). The present study investigated the timing and magnitude of ROS production and associated antioxidant enzyme responses in five rice cultivars (TN-1, Satabdi, Naveen, Annapurna, and Tapaswini) following treatment with two defence elicitors, salicylic acid (SA) and chitosan (CH), and infection with the bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo).\u003c/p\u003e \u003cp\u003eXoo-inoculated plants exhibited a biphasic oxidative burst, marked by an early increase in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e accumulation at 24 h post-inoculation (hpi), followed by a second, more sustained phase after 72 hpi. Enhanced lipid peroxidation accompanied ROS generation, indicating oxidative stress during pathogen interaction. Superoxide anion (O\u003csub\u003e2\u003c/sub\u003e\u0026bull;⁻) and hydroxyl radical (\u0026bull;OH) levels increased predominantly between 72 and 120 hpi, depending on the cultivar. Antioxidant capacity, assessed by DPPH radical scavenging activity, increased following pathogen inoculation. Enzymatic antioxidants showed dynamic responses, with superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPX), and ascorbate peroxidase (APX) activities being differentially regulated across cultivars and time points, particularly during the later stages of infection.\u003c/p\u003e \u003cp\u003eThe early oxidative burst is consistent with PAMP-triggered immunity (PTI), while the sustained ROS accumulation at later stages may reflect prolonged defence signalling or pathogen proliferation during a compatible interaction. Both SA and CH acted as PTI mimics, inducing ROS production and activating antioxidant defence mechanisms. Collectively, these findings highlight the role of oxidative burst dynamics and antioxidant regulation in rice defence responses against bacterial blight.\u003c/p\u003e","manuscriptTitle":"Elucidating oxidative burst during interaction of rice-elicitor/pathogen (Xanthomonas oryzae pv. 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