Effect of zinc applications on physiological parameters in kharif rice (Oryza sativa L.) genotypes

Introduction

Rice (Oryza sativa L.) stands as the world's most vital food crop, serving as the primary dietary staple for over half of the global population and contributing 35% to 75% of the total caloric intake for more than 3 billion people across Asia. India, recognized as one of the original centers of rice domestication and cultivation, dedicates approximately 44.6 million hectares to this crop, yielding around 90.0 million tonnes. This volume constitutes 52% of the nation's total food grain production, operating at an average productivity of about 2.1 tons per hectare (Anonymous, 2017) [3] . Beyond its role as a fundamental energy source rich in carbohydrates, rice also serves as an important source of dietary protein. Despite its agronomic and caloric significance, polished rice is inherently a poor source of essential micronutrients, particularly zinc (Zn). Severe zinc deficiency in lowland rice ecosystems is a documented constraint across major global production zones, including India, Brazil, and the Philippines. Biochemically, zinc functions as an essential catalytic cofactor for numerous enzymes that govern crucial plant metabolic pathways (Fageria et al. 2011) [7] . These include glutamate dehydrogenase, which regulates nitrogen metabolism, and alcohol dehydrogenase, which maintains anaerobic metabolic pathways under waterlogged conditions. A global assessment by the Food and Agriculture Organization (FAO) indicates that approximately 30% of arable soils worldwide are zinc-deficient (Zinzala et al. 2019) [20,22] . Soil-borne zinc deficiencies restrict overall grain yield and compromise the ultimate nutritional quality of the harvest. Lowland rice fields are uniquely susceptible to this nutritional disorder; over 50% of global rice-growing areas are prone to zinc deficiency, making it one of the most critical nutritional stresses limiting rice production in Asia (Zinzala et al. 2019) [20,22] . Consequently, widespread soil zinc depletion not only drives agricultural yield stagnation but also directly correlates with systemic human micronutrient deficiencies.

Planting materials and treatments Experimental Site and Agro-Climatic Profile

The field experiment was conducted over two consecutive crop cycles (2017 and 2018) at the College Farm, Navsari Agricultural University, Navsari, Gujarat, India. The regional climate is typically tropical, characterized by deep, clayey, waterlogged lowland soils during the kharif monsoon window.

Experimental Design and Treatment Combinations

The trial was organized using a randomized block design (RBD) with a factorial concept, replicated three times. The experimental framework evaluated three distinct factors: varieties viz., IET-25450 (V 1 ), BPT-5204 (V 2 ) and IET-24766 (V 3 ), three soil base zinc application (S 1 ) control (00 kg ZnSO 4 ha -1 ), (S 2 ) 10 kg ZnSO 4 ha -1 at the time of transplanting and (S 3 ) 20 kg ZnSO 4 ha -1 at the time of transplanting followed by two foliar Zn application (F 1 ) 0% Zn EDTA and (F 2 ) 1% Zn EDTA spray at tillering and grain filling stage. Standard agronomic practices and recommended fertilizer schedules were maintained across all treatments.

Sampling and Physiological Measurements

Statistical Modeling

Data were analyzed using the standard Analysis of Variance (ANOVA) technique for factorial experiments (Gomez and Gomez, 1984) [11] . The significance of treatment effects was assessed using the F-test at a 5% probability level (α = 0.05). Variance dispersion was quantified via the Standard Error of Mean (S.Em.±), and treatment distinctions were resolved through Critical Difference (CD) formulations: C.D. = S.E. (d) × t 0.05, edf

Results and Discussion

Plant height Effect of different variety on plant height at 60 DAT, 90 DAT and at harvest

An evaluation of the chronological growth data presented in Table 1 indicates that plant height was significantly influenced by the chosen rice varieties across both individual experimental years and within the pooled analysis. As expected, vertical growth progressed continuously with increasing crop age. Among the evaluated cultivars, genotype IET-24766 (V

Interaction effect on plant height at 60 DAT, 90 DAT and at harvest

The interaction effects in respect of plant height between variety and soil base Zn application (V x S), variety and foliar Zn application (V x F), soil base Zn application and foliar Zn application (S x F) and variety, soil base Zn application and foliar Zn application (V x S x F) were did not show any significant effect in individual years of study and in pooled analysis at 60, 90 DAT and at harvest.

The first-order and second-order interaction effectsspecifically, the combinations of variety and soil-applied zinc (V x S), variety and foliar-applied zinc (V x F), soilapplied zinc and foliar-applied zinc (S x F), as well as the three-way interaction of variety, soil, and foliar zinc applications (V x S x F) did not exert a statistically significant influence on the plant height of rice. This lack of significance was consistent across both individual years of field experimentation and within the combined pooled analysis at all evaluated developmental stages, including 60 DAT, 90 DAT and at harvest. These results demonstrate that the structural elongation response of the crop to the individual main factors functioned independently, meaning that the vertical phenotypic expression of each genotype under specific zinc delivery methods did not rely on synergistic or mutually restrictive cross-factor dependencies.

Leaf area Effect of different variety on leaf area at 60 DAT, 90 DAT and at harvest

The data presented in Tables 2 demonstrate that varietal treatments had a significant effect on leaf area during both years of investigation, as well as in the pooled analysis. Among the evaluated genotypes, variety IET-24766 (V 3 ) established significantly higher leaf area magnitudes during consecutive years and in the pooled data over the remaining treatments. This performance was sustained chronologically at 60 DAT (44.72, 44.79, and 45.25 cm 2 , respectively), 90 DAT (47.69, 47.82, and 47.76 cm 2 , respectively), and at harvest (46.54, 46.68, and 46.61 cm 2 respectively). In contrast, the lowest phenotypic values for this functional canopy parameter were documented for BPT-5204 (V 2 ), which yielded minimum baselines at 60 DAT 60 DAT (36.90, 37.95 and 37.42 cm 2 , respectively), 90 DAT (39.82, 39.95 and 39.88 cm 2 , respectively) and at harvest (38.64, 38.44 and 38.69 cm 2 , respectively) across individual and combined years.

Effect of soil base Zn application on leaf area at 60 DAT, 90 DAT and at harvest

Statistical analysis presented in Tables 2 demonstrates that basal soil applications of ZnSO 4 at transplanting exerted a highly significant influence on the leaf area of rice across both individual year of experiment and within the integrated pooled data. Basal incorporation of 20 kg ZnSO 4 ha -1 (S 3 ) maximized the mean leaf area across both individual years and the pooled analysis at 60 DAT

Interaction effect on leaf area at 60 DAT, 90 DAT and at harvest

The first-and second-order interaction effects among the experimental factors specifically variety × soil zinc application (V x S), variety × foliar zinc application (V x F), soil zinc application × foliar zinc application (S x F), and the three-way variety × soil zinc application × foliar zinc application (V x S x F) did not exert any statistically significant influence on the leaf area of rice. This nonsignificant response remained completely consistent across both individual years of investigation and within the integrated pooled analysis at 60 DAT, 90 DAT, and at harvest.

Leaf area index Effect of different variety on leaf area index at 60 DAT, 90 DAT and at harvest

The results presented in Tables 3,4, and 5 reveal that varietal treatments exerted a highly significant effect on the leaf area index of rice across both individual years of investigation and within the pooled analysis. Across both experimental years and the pooled analysis, the rice genotype IET-24766 (V 3 ) consistently exhibited a significantly superior leaf area index relative to all other treatments at 60 DAT (1.59, 1.62 and 1.61, respectively), 90 DAT (1.92, 1.94 and1.93, respectively) and at harvest (1.71, 1.73 and 1.72, respectively). Conversely, the minimum mean leaf area index was systematically recorded in variety BPT-5204 (V 2 ) at 60 DAT (1.34, 1.35 and 1.35, respectively), 90 DAT (1.61, 1.63 and 1.62, respectively) and at harvest (1.43, 1.45 and 1.44, respectively) during both individual year and within the integrated pooled data.

Effect of soil base Zn application on leaf area index at 60 DAT, 90 DAT and at harvest

The statistical analysis presented in Tables 3,4, and 5 demonstrates that the leaf area index of rice was significantly influenced by varying levels of basal soil zinc application at transplanting across both individual experimental cycles and within the integrated pooled data. Basal application of 20 kg ZnSO 4 ha -1 (S 3 ) statistically maximized the mean leaf area index across both individual years and the pooled analysis at 60 DAT (1.67, 1.70 and 1.69, respectively), 90 DAT (2.04, 2.06 and 2.05, respectively) and at harvest (1.80, 1.82 and 1.81, respectively) relative to all other treatments. This treatment, alongside the moderate zinc rate, exhibited marked statistical superiority over the unfertilized control (S 1 ), which recorded the minimum leaf area index at 60 DAT (1.25, 1.27 and 1.26, respectively), 90 DAT (1.50, 1.52 and 1.51, respectively) and at harvest (1.36, 1.38 and 1.37, respectively) during both individual crop cycles and within the integrated pooled data.

Effect of foliar Zn application on leaf area index at 60 DAT, 90 DAT and at harvest Data presented in Tables 3,4, and 5 reveal that foliar zinc applications administered at the tillering and grain filling stage significantly influenced the leaf area index of rice across both individual experimental years and within the integrated pooled analysis. Foliar application of 1% Zn-EDTA (F 2 ) resulted in a significantly higher mean leaf area index across both experimental years and the pooled analysis at 60 DAT (1.62, 1.64 and 1.63, respectively), 90 DAT (1.96, 1.99 and 1.97, respectively) and at harvest (1.73, 1.75 and 1.74, respectively). Conversely, the minimum leaf area index was consistently documented in the control plots (F 1: 0% Zn-EDTA) at 60 DAT (1.31, 1.33 and 1.32, respectively) 90 DAT (1.57, 1.59 and 1.58, respectively) and at harvest (1.42, 1.43 and 1.42, respectively) during individual year and within the integrated pooled data.

Interaction effect on leaf area index at 60 DAT, 90 DAT and at harvest

The interaction effects in respect of leaf area index between variety and soil base Zn application (V x S), variety and foliar Zn application (V x F), soil base Zn application and foliar Zn application (S x F) and variety, soil base Zn application and foliar Zn application (V x S x F) were did not show any significant effect in individual years of study and in pooled analysis at 60 DAT but at 90 DAT the interaction effects in respect of leaf area index between variety and soil base Zn application (V x S), variety and foliar Zn application (V x F) and soil base Zn application and foliar Zn application (S x F) were show significant effect except variety, soil base Zn application and foliar Zn application (V x S x F) in individual years of study and in pooled analysis. Regarding the variety × soil zinc (V x S) interaction, the treatment combination V 3 S 3 (IET-24766 + 20 kg ha -1 ZnSO 4 ) statistically maximized the leaf area index across both year of experimental and in the pooled analysis at 90 DAT (2.26, 2.29 and 2.27, respectively) and at harvest (1.99, 2.02 and 2.01, respectively). Conversely, the minimum leaf area index was consistently documented under the V 2 S 1 combination (BPT-5204 + 00 kg ZnSO 4 ha -1 ) during both individual years and within the integrated pooled dataset (1.33, 1.35 and 1.34, respectively). With respect to the variety × foliar zinc (V x F) interaction at 90 DAT, the treatment combination V 3 F 2 (IET-24766 + 1.0% Zn EDTA) statistically maximized the leaf area index across both individual years and the pooled analysis (2.15, 2.17 and 2.16, respectively). Conversely, the minimum leaf area index was consistently documented under the V 2 F 1 combination (BPT-5204 + 0.0% Zn-EDTA) during both experimental cycles and within the integrated pooled data (1.39, 1.40 and 1.39, respectively). Treatment receiving interaction effect of (S x F) soil Zn application (20 kg ZnSO 4 ha -1 ) and foliar Zn application (1.0% Zn EDTA) (S 3 F 2 ) produced significantly higher leaf area index during first and second year of experiment as well as in pooled data at 90 DAT (2.22, 2.25 and 2.23, respectively) and at harvest (1.94, 1.97 and 1.95, respectively). Conversely, minimum mean leaf area index were recorded in S 1 F 1 Control at 90 DAT (00 kg ZnSO 4 ha -1 + 0.0% Zn EDTA) and at harvest in both years and in pooled data at 90 DAT (1.36, 1.37 and 1.36, respectively) and at harvest (1.25, 1.26 and 1.25, respectively). Plant height increased with advancement of crop age and maximum plant height was recorded with IET-24766 followed by IET-25450 and BPT-5204. However, the percent increase in plant height was highest up to 60 DAT thereafter increases at decreasing rate during both years. The increases in plant height among varieties are mainly due to differences in their genetic makeup (Singh et al. 2013) [18] . Similar results were also reported by Arif et al. (2018) [4] , Kumar and Singh (2018) [12] , and Oahiduzzamana et al.

(2016) [14] . The leaf area and leaf area index tested varieties increased up to 90 DAT, there after it decreased. It might be due to the start senescence and drying of leaves. The higher leaf area and leaf area index was recorded under the IET-24766 in comparison to other varieties. It might be due to wider and longer leaves, greater source size and more number of tillers per hill. Similar results were also reported by Gautam et al. (2008) [9] , Dore et al. (2018) [6] , Ali et al.

(2014) [2] and Shukla et al. (2014) [17] .

Plant height increased with advancement of crop age and maximum plant height was recorded with IET-24766 followed by IET-25450 and BPT-5204. However, the percent increase in plant height was highest up to 60 DAT thereafter increases at decreasing rate during both years. The increases in plant height among varieties are mainly due to differences in their genetic makeup (Singh et al. 2013) [18] . Similar results were also reported by Arif et al. (2018) [4] , Kumar and Singh (2018) [12] , and Oahiduzzamana et al.

(2016) [14] . The leaf area and leaf area index tested varieties increased up to 90 DAT, there after it decreased. It might be due to the start senescence and drying of leaves. The higher leaf area and leaf area index was recorded under the IET-24766 in comparison to other varieties. It might be due to wider and longer leaves, greater source size and more number of tillers per hill. Similar results were also reported by Gautam et al. (2008) [9] , Dore et al. (2018) [6] , Ali et al.

(2014) [2] and Shukla et al. (2014) [17] . Plant height increased with advancement of crop age and maximum plant height was recorded with IET-24766 followed by IET-25450 and BPT-5204. However, the percent increase in plant height was highest up to 60 DAT thereafter increases at decreasing rate during both years. The increases in plant height among varieties are mainly due to differences in their genetic makeup (Singh et al. 2013) [18] . Similar results were also reported by Arif et al. (2018) [4] , Kumar and Singh (2018) [12] , and Oahiduzzamana et al.

(2016) [14] . The leaf area and leaf area index tested varieties increased up to 90 DAT, there after it decreased. It might be due to the start senescence and drying of leaves. The higher leaf area and leaf area index was recorded under the IET-24766 in comparison to other varieties. It might be due to wider and longer leaves, greater source size and more number of tillers per hill. Similar results were also reported by Gautam et al. (2008) [9] , Dore et al. (2018) [6] , Ali et al.

(2014) [2] and Shukla et al. (2014) [17] . Plant height increased with advancement of crop age and maximum plant height was recorded with IET-24766 followed by IET-25450 and BPT-5204. However, the percent increase in plant height was highest up to 60 DAT thereafter increases at decreasing rate during both years. The increases in plant height among varieties are mainly due to differences in their genetic makeup (Singh et al. 2013) [18] . Similar results were also reported by Arif et al. (2018) [4] , Kumar and Singh (2018) [12] , and Oahiduzzamana et al.

(2016) [14] . The leaf area and leaf area index tested varieties increased up to 90 DAT, there after it decreased. It might be due to the start senescence and drying of leaves. The higher leaf area and leaf area index was recorded under the IET-24766 in comparison to other varieties. It might be due to wider and longer leaves, greater source size and more number of tillers per hill. Similar results were also reported by Gautam et al. (2008) [9] , Dore et al. (2018) [6] , Ali et al.

(2014) [2] and Shukla et al. (2014) [17] . An inspection of the data revealed that plant height continued to increase with advancement in age but rapid increase in height was recorded up to 90 DAT thereafter it increased with slow rate. The slow growth of plant after 90 DAT might be due to diversion of photosynthates towards sink i.e. the grain and reproductive parts. The application of 20 kg ZnSO 4 ha -1 at the time of transplanting was recorded significantly taller plant over rest of other treatment. This was might be due to availability of zinc by two ways i.e. soil by xylem and foliar applied zinc by phloem. As a result of high photosynthetic and hormonal activity, the meristematic growth in the apical region resulted in the sufficient elongation of the plants due to Zn application. Ghatak et al.

(2005) [10] , Fageria et al. (2003) [8] , Shivay et al. (2010) [16] have reported a positive response of plant height in rice to Zn application. Zn-induced availability of growth regulators such as gibberellins, kinetin, and indole-3-acetic acid and enhancement in the ability of plant to cope with an adverse environment. This result corroborates the finding of Meena et al. (2018) [13] , Alam and Kumar (2015) [1] , Boonchuay et al. (2013) [5] and Zinzala et al. (2016) [21] .

It is apparent from the data that the leaf area and leaf area index increased up to 60 DAT and after that it started declined from 90 DAT. Between 30-60 DAT the rate of leaf formation was higher and after 60 DAT the rate of leaf drying was very high which led to decrease the leaf area and leaf area index. It was further noticed that higher leaf area and leaf area index was recorded in (S 3 ) 20 kg ZnSO 4 ha -1 at the time of transplanting and it showed significant superiority over rest of the treatments at 60, 90 DAT and at the time of harvesting. The maximum leaf area and LAI is obtained due to providing sufficient amount of zinc through appropriate sources ZnSO 4 that are essentially required to photosynthesis, sufficient cell expansion and elongation rate of leaves due to increase in both number and duration of epidermal cells. This is also due to increase of plant height as well as number of tillers. Similar observations were made by Dore et al. (2018) [6] , Singh et al. (2012) [19] , and Rehman et al. (2012) [15] .

Conclusions

This study demonstrates that combining soil zinc applications with targeted foliar sprays is an effective strategy for managing zinc limitations in lowland paddy systems. The rice genotype IET-24766 showed high genetic responsiveness, developing an optimal crop height, individual leaf area, and functional canopy index. To maximize the Leaf Area Index and maintain a functional canopy through late growth stages, the recommended treatment is an integrated application of 20 kg ZnSO 4 ha -1 soil base incorporation combined with 1% Zn-EDTA foliar sprays at the tillering and grain-filling stages.

Future Research Directions

To build on these physiological findings, future molecular studies should investigate the regulation of zinc transporter gene families and carbonic anhydrase expression levels. This will help clarify the genetic mechanisms behind variation in zinc use efficiency across different rice genotypes.

References

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