Effect of Different Levels of Sodicity on Soil Enzymes, Soil Microbial Biomass Carbon and Activity of Antioxidant Enzymes in Different Rice Varieties under Semi-arid Conditions

The majority of salt-affected soils are found in arid and semiarid regions. Increased salts accumulate in soils in these regions because evapotranspiration outpaces annual precipitation (Zhao et al., 2020). The biological, physical, and chemical characteristics of soils are severely affected by soils high salt concentrations (Ashrafuzzaman et al., 2022). Increased sodium-based carbonates and bicarbonates significantly deteriorate the physical characteristics of sodic soils (Ayers and Westcot, 1985; Minhas et al., 2007). For the exploitation and utilization of saline-alkaline soils, a comprehensive understanding of the variance in soil enzyme activity is required (Shi et al., 2019). The soil MBC makes up 1% to 3% of the soil total organic carbon, but it also has a high turnover rate and functions as a labile store for nutrients (Marumoto, 1984). The higher levels of sodium-based carbonates and bicarbonates in sodic soil and soil that has been irrigated with sodic water significantly worsen soil properties to raising pH, EC and exchangeable sodium percentage (ESP) (Minhas et al., 2007). As a result, it is anticipated that soil microbial populations and enzyme activities will behave differently in sodic environment.
       
Catalase (CAT) can prevent damaging effects of sodium by facilitating the peroxide generated during metabolism in plants (Guangming et al., 2017). Nannippori et al., (2011) stated that phosphatases play an essential role in converting organic P into inorganic forms that are available to plants. In order to guard against oxidative stress, plant cells produce antioxidant enzymes such peroxidase and catalase (DelRio et al., 2003) that break down H₂O₂ into water (Gara et al., 2003). Different researchers have previously reported on dehydrogenase and urease enzymes in relation to salinity and sodicity (Batra and Manna, 1997; Tripathi et al., 2006). Because dehydrogenases occur intracellularly in all living microbial cells, they are one of the most significant enzymes in the soil environment and are used as an indicator of overall soil microbial activity (Salazar et al., 2011). The specific objectives of the study were 1) To determine the effect of different sodicity levels on soil enzymes viz., urease, alkaline phosphatase and dehydrogenase activity 2) To evaluate the microbial biomass carbon content under different levels of sodicity 3) To assess the activity of plant catalase and peroxidase under increasing ESP levels.

During September 2022 to January 2023 a field experiment was conducted at Anbil Dharmalingam Agricultural College and Research Institute, Tiruchirappalli. The experiment was conducted in strip plot design with two replications at six different ESP levels  ie., 8, 16, 24, 32, 40 and 48 with six rice varieties V₁, V₂, V₃, V₄, V₅ and V₆ ie., TRY 1 (Trichy 1), CO 43 (Coimbatore 43), TRY 2 (Trichy 2), CSR 27 (Central Salinity research 27), TRY 3 (Trichy 3) and WP (White Ponni). The initial soil properties and ESP maintained were presented in Table 1. The ESP levels were maintained upto critical stage of rice (flowering stage). The soil samples were collected at flowering stage and analyzed for urease, Alkaline phosphatase (APH), Dehydrogenase (DHG) and soil Microbial Biomass Carbon (MBC). The plant samples collected at flowering stage were examined for activity of antioxidant enzymes viz., Catalase (CAT) and Peroxidase (POX).
 

 
Urease, alkaline phosphatase and dehydrogenase activities
 
The non-buffer method of Zantua and Bremner (1975) was used to measure the urease activity. Toluene and 9 ml of tris-hydroxymethyl aminomethane (THAM) buffer (0.05 M) at pH 9.0 were added to 5 g of 2 mm sieved soil in a 50 ml volumetric flask. For two hours at 370°C, the samples were incubated with 1 ml of a 0.2 M urea solution. KCl.Ag₂SO₄ solution was added, mixed and NH₄⁺-N was estimated in a 20 ml volume using the steam distillation method. The method of Tabatabai and Bremner (1969) was used to assess alkaline phosphatases, utilizing modified universal buffer (pH 11.0), p-nitrophenyl phosphate dibasic as a substrate and incubated at 35°C for one hour. Dehydrogenase activity was determined by 2, 3, 5 triphenyl tetrazolium chloride using 1 g of moist soil and expressed as µg of triphenylformazan (TPF) formed per gram of oven dry soil per 24 hours (Casida et al., 1964).
 
Soil microbial biomass carbon
 
According to Vance et al., (1987), the microbial biomass carbon was measured using the chloroform fumigation-extraction method. A 20 g of fresh soil was collected and it was fumigated using ethanol-free chloroform for 24 hours in a vacuum desiccator. After that, it was filtered and extracted with 0.5 M K₂SO₄. In a 250 ml conical flask, 10 ml of the filtrate was treated with 10 ml of 0.035 N K₂Cr₂O₇ and 20 ml of concentrated H₂SO₄. The substance was digested for 30 minutes on a hot plate at 150-1700°C before being cooled. It was titrated against 0.04 N FAS after the addition of 25 ml of distilled water and 5 ml of phosphoric acid using diphenylamine indicator. All the processes were completed, excluding fumigation, to create a non-fumigated set. The titration value was used to compute the carbon content. By deducting the extracted carbon from samples that had not been fumigated from samples, MBC was calibrated and expressed as µg g^-1 of soil.
 
Catalase and peroxidase activity
 
According to Gossett et al., (1994), the activity of the enzymes catalase and peroxidase was measured. Catalase activity was quantified as ìg of H₂O₂ min^-1 g^-1 and peroxidase activity as a change in absorbance at 430 nm min^-1 g^-1, respectively. For estimation of catalase activity 0.1 ml of enzyme extract solution and 1 ml of H₂O₂ free phosphate buffer (0.2 M) was taken and set as blank. 3 ml of H₂O₂ -phosphate buffer was added to it and mixed gently. Peroxidase was extracted in prechilled distilled water. 1 ml of the extract was used which is centrifuged at 2000 rpm. 2 ml of phosphate buffer, 0.5 ml of 1% pyrogallol and 0.05 N H₂O₂ was used for the determination of peroxidase. The change in optical density was recorded at 425 nm. 
 
Statistical analysis
 
The data obtained were analyzed statistically using SPSS software and test for significance (p = 0.05%).

Grain yield
 
The different ESP levels significantly affected the grain yield of rice crop. The mean grain yield of different rice varieties at different sodicity levels ranged from 5434 to 947 kg ha^-1 (Table 2). The highest grain yield was found at ESP 8 (5434 kg ha^-1) which is on par with ESP 16 (5204 kg ha^-1) followed by ESP 24, 32, 40 ie., 4477, 2343, 1659 kg ha^-1, respectively. The lowest grain yield found at the ESP level of 48 (947 kg ha^-1). The grain yield was found to be significantly differ among the rice varieties. The highest grain yield was recorded in TRY 3 (4693 kg ha^-1) followed by CO 43 (3393 kg ha^-1), TRY 1 (3437 kg ha^-1), TRY 2 (3134 kg ha^-1), CSR 27 (3161 kg ha^-1) and the lowest yield was recorded in WP (2246 kg ha^-1). Different rice varieties exhibit the sodicity tolerance differently in terms of grain yield at different ESP levels. The TRY 3 variety gave atleast 50% (as compared to ESP 8) grain yield upto 32 ESP. However, 50% yield was recorded upto 16 ESP only in case of WP. However, 50% yield was recorded upto 24 ESP in case of TRY 1, CO 43, TRY 2 and CSR 27. The grain yield decreases with increasing sodicity levels and the reason for yield reduction is the dominance of sodium ions causes depletion of enzymatic activity in soil. Due to its great sensitivity of WP variety, the yield has substantially reduced at higher sodicity levels. The results are in consistent with (Gao et al., 2007 and Singh et al., 2016). The rice is notably sensitive to sodicity at the early seedling stage and significant losses in yield have been recorded as a result of high mortality and poor crop establishment.
 

 
Soil enzymes
 
Urease   
 
The different ESP levels has significant effect on urease content and the mean value ranged from 14.1 to 5.2 µg NH_4­⁺-N g^-1 soil h^-1 (Table 3). The highest urease content was observed at ESP 8 (14.1 µg NH_4­⁺-N g^-1 soil h^-1) which is on par with ESP 16 (14.1 µg NH_4­⁺-N g^-1 soil h^-1) followed by ESP 24 (13.8 µg NH_4­⁺-N g^-1 soil h^-1), ESP 32 (10.7 µg NH_4­⁺-N g^-1 soil h^-1) and ESP 40 (8.57 µg NH_4­⁺-N g^-1 soil h^-1). The lowest urease content was found at ESP 48 (5.24 µg NH_4­⁺-N g^-1 soil h^-1). At ESP 8, 16, 24 and 32 the urease content slowly decreases with increasing sodicity. At highest ESP, the urease activity was drastically reduced. At ESP 40 and 48 only 60.69% and 37.11% urease activity was observed when compared to ESP 8. Liang et al., (2007), reported that urease encourages the hydrolysis of nitrogen-containing organic carbon into ammonium. At increasing sodicity levels low hydrolysis process may be a reason for decreased urease activity in the soil. There is no significant difference found between the plots of different rice varieties which indicate that the varietal character doesn’t affect the urease activity in soil. The interaction between the different ESP levels and different rice cultivated plots was also found to be non-significant.
 

 
Alkaline phosphatase
 
The data pertaining alkaline phosphatase was presented in Table 3. The alkaline phosphatase activity in soil decreases with increasing sodicity levels. The mean of APH activity at ESP 8, 16, 24, 32, 40 and 48 was found to be 156, 143, 94.2, 73.9, 52.7 and 25.9 µg PNP g^-1 soil h^-1, respectively. The results were comparable with the works of Batra et al., (2010) which confirmed that and the alkaline phosphatase decreased with increase in salt concentration. At ESP 40 and 48 only 33.82% and 16.61% of APH activity found while compared to the activity at ESP 8. The reason for low phosphatase activity may be that increasing salt concentration which affects enzyme activity by influencing the concentration of inhibitors or activators in the soil solution and the effective concentration of the substrate (Dick et al., 2000). There is no significant differences observed between the plots of various rice varieties. Additionally, no significance difference was also found in the interaction between the various ESP levels and various rice varieties cultivated plots.
 
Dehydrogenase
 
The increasing sodicity levels had a significant effect on DHG content of soil. The highest dehydrogenase activity was found at ESP 8 (27.4 µg TPF g^-1 soil 24 h^-1) followed by ESP 16 (25.5 µg TPF g^-1 soil 24 h^-1). From ESP 24, 32 and 40 a low DHG activity was recorded viz., 16.4, 10.2 and 2.60 µg TPF g^-1 soil 24 h^-1, respectively (Table 4). The lowest activity was observed in ESP 48 (1.12 µg TPF g^-1 soil 24 h^-1). Srivastava et al., (2014) reported that salt stress-induced soil physical qualities affect plants because DHG activity exhibited lower values in the case of sodic soil compared to normal soil. The reason behind the decreased dehydrogenase activity under increasing sodicity levels may be alterations in the catalytic site of enzymes, ionization-induced conformational changes and salting out effect (Tejada et al., 2006).
 

 
Microbial biomass carbon (MBC)
 
The results showed that the microbial biomass carbon decreases with increasing ESP levels (Table 4). The different ESP levels have significant effect on soil MBC and the mean value ranged from 235 to 158 µg g^-1. The highest soil MBC was observed at ESP 8 (235 µg g^-1) which is on par with ESP 16 (233 µg g^-1) followed by ESP 24 (193 µg g^-1), ESP 32 (174 µg g^-1) and ESP 40 (167 µg g^-1). The lowest soil MBC was found at ESP 48 (158 µg g^-1). Tripathi et al., (2006), observed that one of the causes of poor crop development in salt affected soils is likely to be a decline in MBC with increase in salt stress. However, the microbial activity in different rice varieties plots cultivated, interaction between ESP and different plots of rice varieties were found to be non-significant.
 
Activity of antioxidant enzymes in plants
 
Catalase activity
 
The different ESP levels have significant effect on catalase activity. The mean of Catalase (CAT) activity at different sodicity levels viz., ESP 8, 16, 24, 32, 40 and 48 was found to be 54.0, 55.3, 56.7, 58.9, 60.6 and 59.6 µg H₂O₂ g^-1min^-1, respectively (Table 5). The CAT activity increases with increasing sodicity levels. However, at higher ESP levels the activity was slightly reduced. It is in agreement with the studies of Tripathi et al., (2018). In case of different rice variety, the highest CAT activity was recorded in CO 43 followed by TRY 3, TRY 1, CSR 27, TRY 2 and the lowest activity was found in WP. The results are in accordance with Geetha et al., (2022), where the tolerant rice varieties recorded higher CAT activity than susceptible varieties. In interaction between the ESP levels and variety, the tolerant varieties CAT increases only upto ESP 40 and slightly decreased at ESP 48. However, in WP, the CAT activity increased upto 32 ESP only and then after it drastically reduced. Tolerable rice varieties at flowering phase of observation produced considerably more CAT activity upto ESP 40 than susceptible varieties which is also reflected in yield. Increase in CAT activity helps to achieve atleast 50% of yield upto ESP 32.
 

 
Peroxidase activity
 
Peroxidase (POX) activity increases with increasing sodicity levels in tolerant varieties. Different ESP level has a significant effect on POX activity. The different ESP levels viz., ESP 8, 16, 24, 32, 40 and 48 recorded the peroxidase activity of 10.6, 11.5, 11.9, 11.7, 11.5 and 11.1 min^-1g^-1, respectively (Table 5). Among the rice varieties, TRY 3 recorded the highest activity followed by TRY 1, CO 43, TRY 2, CSR 27 and the lowest in WP. Interaction between ESP levels and different rice varieties indicates that in rice variety TRY 3, the POX activity increases upto ESP 40 and decreases at ESP 48. In case of TRY 1, CO 43, TRY 2 and CSR 27 the POX activity increased upto ESP 32 and slowly declines at ESP 40 and 48 which are also reflected in grain yield. At ESP 32, increased POX activity enables us to reach up to 50% of yield. In WP initially at ESP 8 and 16 the POX activity increases and from ESP 24 to 48 the activity was decreasing at increasing sodicity levels. The percentage rise was higher in tolerant cultivars, demonstrating that they are naturally able to survive the stress situation. Comparable results were in accordance with Upadhay and Kumar (2022) stating that peroxidase activity was one among useful parameter for the identification of tolerant and susceptible genotypes.

The interaction between soil enzymatic activity and different rice cultivated plots was found to be non-significant. However, soil enzyme activity and soil MBC decreases with increasing ESP levels. A reverse trend was observed in antioxidant enzymes. The genotypes TRY 3 followed by TRY 1, CO 43, TRY 2 and CSR 27 significantly recorded higher catalase activity upto ESP 40 and peroxidase upto ESP 32 and decreases eventually after that. The increase in catalase and peroxidase increases the tolerance mechanism and produces a reasonable yield in tolerant varieties at higher sodicity levels. In view of antioxidant enzymes and grain yield, overall TRY 3 outperformed among other tolerant rice varieties studied and 50% of yield can be achieved upto ESP 32. At higher sodicity levels the antioxidant enzymes in tolerant varieties increased gradually and expose its tolerance capacity. So, improving the soil enzymatic activity, soil MBC and plant antioxidant enzymes will further increase the growth and yield of rice under higher sodicity levels.

The authors are thankful to scientists who helped technically in analysis.

The authors declare that there is no conflict of interest regarding the publication of this article.