Effectiveness of ethylene diurea in ameliorating ozone stress in blackgram varieties (Vigna mungo L.)

ABSTRACT Tropospheric ozone, a phytotoxic air pollutant, has inadvertently increased since industrial era and its effect on agricultural crops has gained urgent scientific attention. The present study aims to evaluate the response of blackgram varieties (Vigna mungo L.) exposed to elevated ozone stress (100 ppb) with antiozonant (Ethylene diurea at 300 ppm). The study was conducted during two seasons and the effectiveness of ethylene diurea (EDU) was explored in terms of plant physiology, biochemistry, antioxidant enzymes, growth, yield and spectral reflectance of blackgram varieties. Ethylene diurea ameliorates the ozone stress by increasing photosynthetic rate by 40.78 and 37.21% in season I and 43.03 and 35.77% in season II for VBN3 and VBN8, respectively, and antioxidant enzyme superoxide dismutase by 24.31 and 19.42% in season I and 22.59 and 18.59% in season II for VBN3 and VBN8, respectively. The growth indices and yield traits were significantly increased with the application of EDU and the effect was more pronounced in sensitive cultivar (VBN3) compared to the tolerant (VBN8) one. Spectral reflectance, a non-destructive study, further validates the effect of tropospheric ozone on blackgram varieties. Newly identified spectral indices [R566, R573], and [R564, R586] demonstrated the greatest potential in detecting ozone sensitivity of blackgram varieties.


Introduction
Industrial revolution since 1850s until today increases the emission of anthropogenic sources of ozone-forming precursors like nitrogen oxides and volatile organic compounds, which accelerate the formation of ground level ozone (O 3 ) (Monks et al. 2015). For instance, globally the monthly average tropospheric ozone concentrations at daytime has mounted approximately from 10 ppb in the late 1800s to more than 40-50 ppb at present (Brauer et al. 2016). Further in India, the observations from 2008 to 2013 based on multi-model simulations from the Chemistry Climate Model Initiative (CCMI) and from 2009 to 2012 based on International Hemispheric Transport of Air Pollution Phase II (HTAPII) models estimated the tropospheric ozone concentration as 37.3 to 56.1 ppb (Hakim et al. 2019). Moreover, the maximum ozone concentration upto 56 ppb was recorded during summer in southern Tamil Nadu (Krishna and Nagaveena 2016). The seasonal variation in ozone concentrations was maximum during summer (62 ppb in march) at higher altitude of Western Ghats (Udayasoorian et al. 2013). The increase in global tropospheric ozone causes adverse impact on flora and fauna at regions (Osborne et al. 2019), proving that the O 3 concentration has exceeded the standard permissible limit (Singh et al. 2018a, b). The response of O 3 stress in plants was identified through reduction in photosynthetic activity, biomass production and yield; wherein, it varied from crop to crop and from cultivar to cultivar (Feng et al. 2011). Significant reduction in yield recorded in important food crops like wheat, rice, maize and soybean clearly indicates that global food security is threatened by the current concentration of tropospheric O 3 and is expected to intensify in future (Singh et al., 2009a;Ashrafuzzaman et al. 2017;Mills et al. 2018).
Pulses sharing major protein requirement (16.47 million tons) is one of the most sensitive crops against O 3 and the critical level Accumulated Ozone exposure over a Threshold of 40 ppb (AOT40) responsible for 5% reduction in yield was 3.03 ppm h (Mills et al. 2007;DAC&FW 2018). Further, a yield loss of about 10-65% in Asian region was reported, where mean ambient O 3 concentration varied between 35 to 75 ppb during the growing season (Emberson et al. 2009). With evidence to earlier reports, it was observed that, higher O 3 concentration in Indian region affects the pulse production (Singh and Agrawal 2011). Blackgram accounts for nearly 19% of total pulses acreage in India and contributes for about 23% of total pulse production. Furthermore, nearly 18 to 79% yield loss in Blackgram due to ozone pollution have been reported (Agrawal et al 2006). In this regard, application of ozone protectants would alleviate ozone induced stress on crops and reduces yield loss. Ethylenediurea (EDU), an antiozonant, has been used since early research stages to protect the crops against ozone stress including pulses (Singh et al. 2010a, b). It was found to improve the net assimilation rate, stomatal conductance, chlorophyll content, antioxidant enzymes, growth and yield of wheat (Fatima et al. 2019), maize (Chaudhary and Rathore 2020), mung bean (Agrawal et al. 2005) and chilli (Khan et al. 2021).
Furthermore, studying leaf optical properties through non-destructive method would pose a great potential in ozone stress monitoring on crop species (Gosselin et al. 2020). The mechanisms of the changes in leaf optical properties are related to the changes in the chlorophyll, carotenoid and xanthophyll pigments, and (or) chloroplast shrinkage which subsequently induced light scattering at red region (Sukhov et al. 2021). Monitoring leaf phenotypic characteristics like chlorophyll content and photosynthesis using spectral reflectance was found to be highly effective and precise (Gitelson et al. 2003). In addition, identification of the best correlated reflectance indices with plant parameters using heatmap has shown several advantages. In recent years, estimation of ozone induced damage has been studied using leaf spectral reflectance in various crops i.e. rice (Ashrafuzzaman et al. 2017) and soybean (Sagan et al. 2018). Though identification of ozone stress through leaf properties is cost effective and rapid method, studies on leaf spectral properties under ozone stress is still limited. Nevertheless, pulses contribute major share of protein in India, studies on the influence of O 3 stress in pulses is scarce and its sensitivity to ozone has not been tested to date in South India. Keeping this in view, this study aims at investigating the response of elevated ozone on popularly growing black gram varieties (Vigna mungo L.) with EDU application and potential of using remote sensing technique to identify ozone stress.

Plant materials and ozone exposure
The experiment was conducted in the Wetlands of Tamil Nadu Agricultural University, India (11.00° N, 76.92° E). The field experiment was conducted during February 2021 to April, 2021 (Season 1) and May, 2021 to July, 2021 (Season 2). Based on the prior screening experiment, four blackgram varieties (Vigna mungo L.) with different levels of ozone sensitivity were selected namely, VBN3 (sensitive), CO6 (moderately sensitive), VBN7 (moderately tolerant) and VBN8 (tolerant) (Dhevagi et al. 2021). The plants were grown under control (ambient) and elevated ozone condition in Open Top Chambers (OTCs) with a size of 2.7 × 2.7 m made up of poly carbonate sheets recommended for crop growth experiments (Figure 1). The factorial randomized block design was followed with twelve replications (N = 192). The treatments followed were control, control with ethylenediurea (EDU) application, elevated ozone and elevated ozone with EDU application. Meteorological conditions in the open top chamber during experimental period are given in Supplementary Table S1.
In elevated ozone treatment, blackgram varieties were exposed to weighted average ozone concentration of 100 ppb from 10.00-17.00 h over 10 days (from 31 days after sowing to 40 days after sowing) representing flowering stage. The ozone feed rate was fixed at 1.2 mg min −1 and the calculated AOT40 was found to be 4.4 ppm h and 4.2 ppm h for Season 1 and Season 2, respectively (Supplementary Figure S1). In control chamber, the ozone concentration was recorded to be <10 ppb. In EDU treatments, ozone protectant -EDU spray at 300 ppm concentration using a sprayer was applied twice during early hours at five days interval (30 DAS and 35 DAS) during ozone exposure to ensure continuous persistence of EDU in the leaf apoplast (Ashrafuzzaman et al. 2017). For non-EDU treatments, water was sprayed at five days interval.

Plant analysis
All the plant physiological, biochemical, antioxidant enzymes and growth measurements were taken after ten days of ozone exposure. Physiological traits, i.e. photosynthetic rate (A) and stomatal conductance (g s ) were measured using a portable photosynthesis system (ADC BioScientific LCpro-SD System, UK), while the chlorophyll content (Chl) was quantified using chlorophyll content meter (CCM-200+, USA). During photosynthetic measurements, the CO 2 concentration inside the leaf cuvette was 400 ppm, photosynthetic photon flux density (PPFD) was 1500 μmol m − 2 s -1 and leaf temperature was 28°C. The measurements were done on fully mature third leaves, from each variety and treatment. These measurements were performed between 10:00 and 14:00 hours. Visual O 3 induced leaf injury percentage was calculated according to the method followed by Chaudhary and Agrawal (2013) and details are given in Supplementary Table S2.
The biochemical and antioxidant enzyme measurements, i.e. malondialdehyde content (MDA) (Heath and Packer 1968), proline content (PRL) (Bates et al. 1973), ascorbic acid content (AsA) (Keller and Schwager 1977), phenol content (PHL) (Bray and Thorpe 1954), superoxide dismutase (SOD) (Fridovich 1975), peroxidise (POD) (Britton and Mehley 1955), catalase (CAT) (Aebi 1984), glutathione reductase (GR) (Schaedle and Bassham 1977), ascorbate peroxidase (APX) and total protein content (TP) (Lowry et al. 1951) were estimated at tenth day of ozone exposure (40 DAS). The growth indices  namely relative growth rate (RGR), net assimilation rate (NAR), leaf area ratio (LAR), specific leaf weight (SLW), specific leaf area (SLA), leaf weight ratio (LWR) and root shoot ratio (RSR) were calculated according to the formula given by Hunt (1982). In addition, ten days after ozone exposure spectral reflectance values of the blackgram varieties were measured using portable spectroradiometer (GER 1500) covering wavelengths ranging from 391.07-1093.50 nm with spectral resolution of 1.72 nm. The spectral measurements on ozone exposed blackgram varieties at fully expanded young leaves from the tip and the observation taken between 11.00 and 14.00 hours to ensure bright sunlight and avoid shadowing. During the field spectral measurements, fiber optic cable connected with lens was used and the measured leaf mid regions was larger than field-of-view of the sensor. A whiteboard was employed as reference and measured every 30 min during the measurements.
The yield traits i.e. plant height (PH), root length (RL), number of branches (NBP), pods (NPP) and leaves per plant (NLP), seeds per pod (NSP), pod length (PL), nodules (NNP), 100 grain weight (100 GW), plant weight (PW) and harvest Index (HI) were measured as per the standard protocol at harvest stage of the blackgram varieties. All the observed plant parameters were taken for principal component analysis (PCA), which was performed using R software (RStudio Version 3.5.1).

Statistical analysis
The ANOVA analysis was performed using SPSS (Version 16.0) wherein two-way ANOVA (Analysis of Variance) was used to test the significance of treatment; varieties and their interaction effect of plant traits and Tukey method was used to identify difference among treatment means. For the spectral indices identification, different combinations of spectral wavelengths were correlated using normalized difference spectral index (NDSI) (Equation 1) (Alexander 2020).
In equation (1), R is the spectral reflectance of elevated ozone exposed blackgram leaves and subscripts (i, j) are wavelengths (nm). Reflectance data obtained in a range from 391.07 to 1093.50 nm (i and j in nm) were used for examining the linear relationship between ozone injury percentages (OIP) and NDSI. Pearson's correlation arrays were generated using NumPy and heat maps of correlation coefficient were generated using Matplotlib, Jupyter Notebook (Version 6.0.1) in Anaconda Navigator (Van Der Walt et al. 2011). In addition, correlation coefficient of determination (R 2 ) between previously published spectral indices and observed physiological traits was studied (Supplementary Table S3). OriginPro 2019 (version 9.6.5) was used to plot the graphs.

Effect of EDU on physiological traits
Exposure to elevated ozone induced leaf injury like bronzing to crops compared to control. Relatively the ozone sensitive variety (VBN3) displayed the highest ozone injury percentage compared to ozone tolerant (VBN8) variety. In both the seasons, EDU application significantly reduced the leaf visible symptom in all four blackgram varieties. The variety VBN3 (Ozone sensitive) and VBN8 (Ozone tolerant) registered a leaf visible symptom of 40.33 and 21% during season 1 and 41.33 and 24% during the second season, respectively. The above results may be due to both direct and indirect effects on ROS scavenging efficiency of EDU, which reduces the leaf injury symptoms (Pandey et al. 2014). Similarly, EDU application reduced the ozone-induced visible leaf injury in clover cultivar (Singh et al. 2018b) compared to non-EDU applied treatment.
The blackgram cultivars showed significant reduction in photosynthetic rate under elevated ozone treatment, which ranged from 39.01 to 42.91% in season 1 and 40.99 to 46.35% in season 2. In both the seasons, the order of reduction follows VBN8< VBN7< CO6< VBN3. It was also observed that application of antiozonant (EDU) under ozone stress enhanced the photosynthetic rate. A significant increase by 40.78 and 37.21% in season 1 and by 43.03 and 35.77% in season 2 was noticed in VBN3 and VBN8, respectively ( Figure 2). In regard to two-way ANOVA results, photosynthetic rate showed significant variations among the varieties (V), treatment (T) and their interactions (V × T). Increase in photosynthetic rate reflects the efficiency of EDU in maintaining photosynthetic enzymes thereby resulting in higher photosynthesis and increased carbon fixation under ozone stress as reported by Fatima et al. (2019) in wheat cultivar (HD2987) and Xu et al. (Xu et al. 2019) in poplar.
Under elevated ozone treatment, reduction in stomatal conductance was observed in all test varieties in season 1 and 2. In this study, EDU treatment increased the stomatal conductance from 28.05 (VBN3) to 47.92% (VBN8) in season 1 and from 25.00 (VBN3) to 45.45% (VBN8) in season 2 ( Figure 2); which highlights the fact that EDU might influence the blackgram varieties by its protective antioxidant defense mechanism that would maintain the plant gas exchange properties. Consistently, ozone-mitigating effect of EDU was observed by significant increase in stomatal conductance under ozone stress in rice (Ashrafuzzaman et al. 2017) and wheat cultivars (Singh et al. 2009b). Results of ANOVA tests showed that the stomatal conductance of test varieties significantly differed among treatments (T) and varieties (V); while the interaction effect (V x T) was non-significant. Ozone-induced reduction in chlorophyll content varied from 39.58 (VBN7) to 46.47% (VBN3) in season 1 and from 36.25 (VBN7) to 42.47% (VBN3) in season 2. Furthermore, EDU supplement enhanced the chlorophyll content by 44.63 (VBN8), 47.28 (VBN7), 47.87 (VBN3) and 50.57% (CO6) in season 1 and 43.45 (VBN8), 48.57 (VBN7), 48.83 (VBN3) and 50.76% (CO6) in season 2 ( Figure 2). This increase might have been interpreted as improved antioxidants like carotenoids in chloroplast, which would impart a protection against oxidative stress by quenching the ROS (Agathokleous 2017). Similar type of observation was noticed in mustard cultivars, Kranti and Peelasona by Pandey et al. (2014) and in broad bean by Al-Qurainy (2008) under EDU supplement. Moreover, the threeway ANOVA results of chlorophyll content showed a significant variation among varieties (V) and treatment (T); while their interaction effect (V × T) was found to be insignificant.

Effect of EDU on biochemical traits
Elevated ozone exposure increased the malondialdehyde content (MDA) in all varieties compared to control in both the seasons (Table 1). EDU supplement decreased the MDA content by 24.40 and 25.44% (VBN3), 31.68 and 28.77% (VBN7), 33.46 and 33.80% (CO6), and 43.96 and 39.45% in VBN8 during season 1 and 2, respectively compared to ozone alone (Table 1). This might be attributed due to ozone-mediated lipid peroxidation, which was evidenced by significant lower MDA content compared to the treatment not receiving EDU (Ashrafuzzaman et al. 2017). Similar results were observed in clover (Singh et al. 2018b) and snapbean cultivars (Yuan et al. 2015). All the factors (V and T) and their interactions (V × T) exhibited significant variations.
The proline content was found to decrease in ozone + EDU treatment compared to ozone treatment (Table 1). The decrease was found to be 22.99, 11.55, 3.10 and 10.04% in season 1 and 20.66, 12.67, 2.88 and 8.25% in season 2 for VBN3, CO6, VBN7 and VBN8 respectively. Such decrease in proline content under EDU supplement suggests the efficiency of EDU in scavenging the singlet oxygen and hydroxyl radicals. This study was found to be in contrary with Rathore and Chaudhary (2019), who reported higher amount of proline accumulation in the castor cultivar Nidhi-999 by EDU treatment. The ANOVA results of proline content registered a significant influence in all individual factors (V and T) and their interactions (V × T). Ascorbic acid content exhibited an induction in EDU supplemented treatment under ozone exposed condition in all blackgram varieties as compared to ozone treatment, which ranged from 23.51 (CO6) to 83.33% (VBN8) in season 1 and 29.63 (CO6) to 87.50% (VBN8) in season 2. The reason may be due to significant increase in APX and GR activity under EDU treatment, which would lead to increase in ascorbic acid content. Moreover, increased ascorbic acid content upon EDU treatment in VBN8 has possibly provided better tolerance against ozone injury compared to other test varieties. Similarly, Burkey et al. (2003) reported that ozone tolerance correlated with increased apoplastic ascorbic acid content in snap bean genotypes. Furthermore, EDU mediated increment in ascorbic acid content have also been reported in wheat (Singh et al. 2009b) and mung bean cultivars (Agrawal et al. 2005). The two-way ANOVA tests of ascorbic acid content depicted a significant variation in varieties (V) and treatments (T); while their interaction effect (V × T) was insignificant.
The phenol content showed significant increase under EDU treatment compared to ozone treatment by 17.98% in ozone tolerant (VBN8) and 6.90% in ozone sensitive (VBN3) during season 1 and 16.28% (VBN8) and 6.59% in VBN3 during season 2 suggests that the tolerant varieties have higher balance in metabolic pathways compared to sensitive ones. This increase in total phenolics under EDU indicates maintaining the precursor (phenylalanine) availability for regenerating phenol content of the blackgram varieties. On contrary, reduction in phenolic content was reported in pea cultivars (Jabeen and Ahmed 2021)

in EDU-treated plants compared to non-EDU-treated plants. The results of ANOVA tests recorded significant differences in varieties (V), treatments (T) and their interactions (V x T) on phenol content.
Ozone-induced ROS modifies the structures of cellular proteins and induces the plant susceptibility to proteolysis in cells. A significant increase by 19.40% in total protein content was noticed in EDU treated sensitive variety (VBN3) over ozone alone treatment in season 1 and 18.59% in season 2. The observed higher protein content in EDU-treated plants under ozone stress suggests the role of EDU in protecting the foliar proteins from degradation. This protective effect is due to maintenance of higher level of antioxidative defense system that would quench the free radicals generated in plant cells, thereby maintaining membrane stability and sustain protein synthesis under ozone stress (Tiwari and Agrawal 2010). These findings corroborate with Singh et al. (2018a) on maize cultivars. The ANOVA results recorded a significant variation in varieties (V), treatments (T) and their interactions (V x T).

Effect of EDU on antioxidant enzymes
The activities of superoxide dismutase, peroxidase, catalase, ascorbate peroxidase, glutathione reductase were increased in all blackgram varieties under 100 ppb elevated ozone exposure during both the seasons. Furthermore, the increment was higher in ozone sensitive (VBN3) compared to tolerant one (VBN8).
EDU supplement improved the antioxidant defense system of blackgram varieties by increasing the activity. The increment of SOD, POD, CAT, APX and GR activity by 24.31,48.84,42.55,13.69 and 4.00%,respectively in VBN3 and 19.42,31.58,13.64,29.02 and 2.70%, respectively in VBN8 were recorded during season 1. Similarly in season 2, the SOD, POD, CAT, APX and GR activity increased by 22.59,24.86,44.44,11.32 and 9.38%,in VBN3 and 18.59,35.29,16.00,27.45 and 5.00%, in VBN8 (Figure 3), respectively. The activity of antioxidant enzymes was more pronounced in ozone sensitive (VBN3) compared to ozone tolerant (VBN8) variety. This result indicates that increased activity of SOD would convert O 2 − into H 2 O 2 and O 2 . Subsequently, increase in the activity of peroxidase and catalase detoxifies the H 2 O 2 into H 2 O which would help the plant to retain its normal state by maintaining dynamic equilibrium of ROS even under ozone stress. Increase in APX and GR activity also helps in regenerating the ascorbic acid content in plants system. Among the enzymes, peroxidase, catalase and superoxide dismutase were highly responsive towards EDU supplement in sensitive varieties. In consistent with these findings, the response of SOD towards EDU application was more effective in sensitive clover cultivar (Wardan) (Singh et al. 2018b) and wheat cultivar (HD 2987) (Fatima et al. 2019). Similarly, EDU supplement increased the peroxidase activity in ozonesensitive black gram cultivar (Azad-1) (Singh and Agrawal 2011), CAT activity in mung bean (Singh et al. 2010b), APX activity in mustard (Pandey et al. 2014) and pea cultivar (Jabeen and Ahmed 2021), and GR activity in Euramerican poplar (Dumont et al. 2014) and in maize genotypes (Gupta et al. 2020). The ANOVA tests of antioxidant enzymes showed significant variation among the varieties (V) and treatments (T); while their interactions (V x T) were significant only for SOD, POD and CAT.

Effect of EDU on growth indices
Among the observed growth indices, treatment and variety effect were significant for leaf weight ratio and root shoot ratio (Table 2). Under ozone stress, the ozone sensitive (VBN3) variety decreased leaf weight ratio by 4.43% in season 1 and 5.67% in season 2; whereas EDU supplement improved the leaf weight ratio by 2.48 and 3.12% in season 1 and 2, respectively. Similarly, root shoot ratio reduced by 9.08% in VBN3 in season 1 and 8.98% in season 2; while EDU application increased the ratio by 8.67% in season 1 and 9.35% in season 2. Similarly, in VBN 8 the root shoot ratio reduced by 5.42 and 5.97% during season 1 and 2; while EDU application increased the root shoot ratio by 4.63 and 4.12% in season 1 and 2, respectively.

Effect of EDU on yield traits
A significant variation was observed in plant height, number of branches per plant, pods per plant and leaves per plant, 100 grain weight and plant weight under ozone stress. EDU supplement depicted a significant increase by 9.04, 28.57 and 6.89% in VBN3 and 0.43, 20.00 and 5.71% in VBN8 during season 1 and by 5.70, 33.33 and 8.50% in VBN3 and 1.81, 25.00 and 8.33 in VBN8 during season 2 for plant height, number of branches per plant and pods per plant respectively. Similarly, number of leaves per plant, 100 grain weight and plant weight were also found to significantly increase by 9.75, 5.17 and 23.81% in VBN 3 and 6.21 5.56 and 17.75% in VBN 8 during season 1 and by 9.12, 5.70 and 25.52% in VBN 3 and 8.41, 8.40 and 18.75% in VBN 8 for season 2 respectively. This increment in yield traits of the blackgram varieties under EDU supplement might be due to improved cumulative carbon gain and equal partition of photosynthates to sink as suggested by Jing et al. (2016). Furthermore, supplement of EDU enhanced the photosynthetic traits and metabolic activity of the plants, which leads to increase in number of leaves, pods, seed and plant weight. This results corroborates with the findings of Singh et al. (2010a) in mung bean cultivars, Pandey et al. (2014) in mustard cultivars and Singh et al. (2018a) in maize cultivars upon EDU treatment. A significant varietal (V) difference was noticed in all the observed yield traits; whereas significant treatment (T) influence was observed in plant height, number of branches, pods and leaves per plant, 100 grain weight, plant weight and harvest index. In terms of variety and treatment interaction effect (V × T), plant weight was found to be significant (Table 2).

Principal component analysis (PCA)
Based on the PCA results, the total variance explained by first two components (PC1 and PC2) was 79.36, 74.98, 87.68 and 94.14% for control, control + EDU, ozone and ozone + EDU treatment, respectively (Figure 4). In control and control + EDU treatment, PC1 described that the physiological traits, antioxidant enzymes and yield traits were grouped together with positively correlation and showed higher loading. The growth indices depicted lesser loading in PC1 of the both control and control + EDU treatment. In ozone and ozone + EDU treatment, physiological and yield traits were clustered together with higher loading in PC1; while antioxidant enzymes depicted a lesser loading with a variance of 67.87 and 80.55%, respectively. Among the observed plant parameters, antioxidant enzymes showed distinct separation in comparison with control (control and control + EDU) and ozone exposed (ozone and ozone + EDU) treatments. Moreover, the antioxidant enzymes were positioned on opposite quadrants in control treatments (positive quadrant) and ozone exposed treatments (negative quadrant) indicating that plant antioxidant enzymes response towards ozone and EDU supplement. In addition, the plant physiological traits were spread in positive quadrant in all the treatments. The present result was in line with the report of Feng et al. (2010) who revealed that EDU-mediated plant response against ozone stress was biochemical rather than biophysical properties of the plants.

Ozone stress assessment using spectral measurement
Several non-destructive measurements have been used to assess ozone and EDU effects on plants.
Studying spectral reflectance of crop species poses more importance due to its precise and rapid technique to estimate ozone stress intensity (Gosselin et al. 2020). Plant physiological stress can be identified by increasing leaf spectral reflectance in the portions of the visible and near infrared range. In ozone treatment, the sensitive variety (VBN3) depicted higher reflectance in visible region (400 to 700 nm) compared to tolerant variety (VBN8) (Supplementary Figure S2), which might represent higher reduction in chlorophyll contents and lower photosynthetic capacity (Lawlor 2002). The variations in reflectance percentage under elevated ozone exposure might be related to the changes in physiological characteristics of blackgram varieties. Similar to this observation, Ghulam et al. (2016) revealed that influence of ozone stress on the reflectance pattern of soybean cultivars were more predominant in visible region. Near-infrared (NIR) radiation was reflected from the structure of spongy mesophyll tissue and cavities within the leaf. Ozone treatment displaying lower reflectance at infrared spectral region confirmed the destruction of mesophyll cells under oxidative stress (Govind et al. 2005). Moreover, lower reflectance in ozone sensitive (VBN3) variety at NIR region describes more destruction of mesophyll cells and internal leaf structure under ozone stress as compared to ozone tolerant one (VBN8). In EDU supplement under ozone stress, higher the absorption in visible region than in ozone treatment might be due to EDU-mediated increase in chlorophyll content of blackgram varieties (Supplementary Figure S2). Under EDU supplement, ozone sensitive (VBN3) variety displayed comparatively higher absorption in visible region and higher reflection in NIR region than in ozone tolerant one (VBN8), which might be associated with improved chlorophyll contents and cell structure. EDU supplement protected the cells from oxidative damage and maintained the cell structure which was also confirmed by spectral measurement of blackgram varieties grown under ozone + EDU applied treatment.
Heatmap of NDSI allowed the evaluation of combinations of different wavebands and detection of the most sensitive wavelength for ozone damage in observed blackgram varieties ( Figure 5). The correlation coefficient (r) ranged from −0.69 to 1. The extent of 400-450 nm (visible), 500-590 nm (visible), 700-780 nm (red-edge) and 800-950 nm (near infrared) spectral regions tended to have higher correlation with ozone injury percentage. Hence, in this study, a strong correlation in mid-500 nm, mid-750 nm and mid-900 nm were highly applicable for broad band satellite-based observations. Moreover, these regions are good indicators for photochemical and physiological stress identification studies. Similar to the current findings, Gosselin et al. (2020) reported spectral wavebands in mid-500 regions had the best correlations with visual chlorosis score in soybean genotypes and mid-900 nm and 660-700 nm range had the strongest correlation to necrosis score for snap beans and soybeans. Ghulam et al. (2016) identified 516-646 nm and 690-732 nm regions were responsible for ozone stress monitoring in soybean genotypes.
In this study, 566 and 573 nm as well as 564 and 586 nm (r > 0.94) were identified as the best wavebands having a strong correlation with ozone injury percentage. These wavebands are considered as sensitive wavelengths for ozone damage assessment in blackgram varieties. Similar to this observations, Sagan et al. (2018) [564,586] were more pronounced in ozone sensitive (VBN3) varieties compared to ozone tolerant (VBN8) which might be related to increase in dissipation of energy and maximum reflectance in VBN3 variety (Sukhov et al. 2021).
Relevant spectral indices from previous literatures and the indices developed in current study were compared for their correlation coefficient with respect to leaf physiological traits, i.e. photosynthetic rate, stomatal conductance and chlorophyll content ( Figure 6). Among the 13 indices, two developed in this study (NDSI [566,573] and NDSI [564,586]) represented strong correlation coefficient. NDSI [566,573] showed maximum coefficient of determination (R 2 ) with studied physiological traits, i.e. photosynthetic rate (0.61), stomatal conductance (0.51) and chlorophyll content (0.67). Similarly, NDSI [564,586] showed R 2 = 0.57 for photosynthetic rate, R 2 = 0.52 for stomatal conductance and R 2 = 0.58 for chlorophyll content. Furthermore, previously published Photochemical Reflectance Index (PRI586, mSR705, RERI, PRI519) had the next highest R 2 for chlorophyll content. Further, these indices with visible and red-edge spectral wavelengths were well-known to be susceptible for slight changes in biophysical and biochemical properties of leaves that could be used for ozone stress examination (Gitelson et al. 2001;Ghulam et al. 2016). In present study, the identified best fit mid-500 nm regions for ozone stress examination can be imaged by hyperspectral sensors for large scale application which detect hundreds of very narrow spectral bands. A hyperspectral sensor mounted on Unmanned Aerial Vehicle (UAV), for example drones have a potential application in agriculture and forestry for wide coverage of fields (Adão et al. 2017). This remote sensing approach of ozone stress identification can be used for large scale crop monitoring to ensure food security.

Conclusion
The current results clearly suggest that the elevated ozone (100 ppb) exposure significantly altered the physiological, biochemical, growth and yield traits of blackgram varieties; wherein VBN3 and VBN 8 was found to be ozone sensitive and ozone tolerant, respectively. Field experiment with ozone protectant reveals that Ethylene Diurea (EDU) application mitigates the ozone effects by inducing antioxidant defence system under ozone stress, especially in sensitive varieties. EDU supplement helped the blackgram varieties to stimulate growth and yield traits. The spectrums of 566 and 573 nm as well as 564 and 586 nm (r > 0.94) were identified as the best wavebands that have strong relationship with ozone injury percentage. Furthermore, this study concludes that EDU would be an appropriate tool for distinguishing the blackgram varieties towards ozone stress based on responsiveness ranking. Moreover, newly developed spectral indices could be a potential tool in ozone stress assessment and large-scale crop monitoring studies.