Iron toxicity in lowland rice influenced by application of high potassic fertilizer with suitable cultivars enhanced productivity and climate resilience

Iron poisoning in low - land rice in India develops gradually and is primarily caused by anaerobic conditions in submerged rice fields. A high concentration of ferrous ions in the soil solution disrupts the potassium balance in rice plants, leading to adverse effects on crop growth. In the 2021 – 2022 period, an experiment was conducted in non - saline, iron - rich soil (pH – 4.82, Fe– 458.6 mg kg – 1 ) to mitigate iron toxicity in rice cultivars through potassium nutrition. The experiment involved 4 potassium application doses (K - 40, K - 80, K - 100 and K - 120) and 32 rice cultivars, replicated twice using a split - plot design. Higher potassium doses led to increased tiller counts, but gradually decreased root length. Notably, cultivars like Kanchan, Indravati, Jagabandhu, Santepheap and Salibahan exhibited the lowest iron concentration in their grains compared to susceptible cultivars. Administering K - 120 resulted in a yield increase of over 36.70 q ha - 1 . Grain yield increased with higher K dosage, although it did not affect total iron content. However, K doses did influence specific fractions of iron in the soil. Hence, potassium nutrition appears crucial in managing iron toxicity in inceptisols, especially when paired with cultivars tolerant to iron toxicity.


Introduction
Rice serves as a fundamental food source for nearly half of the world's population, providing essential calories.To sustainably feed an estimated 9.1 billion people by 2050, an additional 100 million tonnes of rice production is required (1).However, despite favorable conditions, the genetic potential for rice grain yield has plateaued, leading to decreased productivity.Abiotic stressors such as soil toxicity, salinity, acidity and nutrient deficiencies significantly impede grain yield across all rice-growing environments (2).Consequently, considerable efforts are directed towards developing rice varieties resilient to iron-poisoned soil.The physiological response of rice plants to low-lying soil profoundly influences their ability to tolerate high iron concentrations.The rhizosphere, where roots interact with soil, exhibits higher oxidation levels compared to the surrounding growing media.This is facilitated by rice roots transporting molecular oxygen into the root medium through air chambers and aerenchyma present in leaves, stems, nodes and roots.Moreover, ferrous iron in the soil solution oxidizes to Fe (III), forming visible deposits on the surface of rice roots.Rice roots exhibit greater oxidizing capacity at growth points and elongating portions compared to basal regions.Iron uptake for rice plants primarily occurs from the soil, necessitating readily available ferrous ions for healthy growth and development.However, excessive iron in the soil disrupts several physiological processes in the rhizosphere, contributing to iron poisoning affecting approximately 18 % of global soil.Elevated iron concentrations in toxic environments can range from 200 to 1000 ppm.To mitigate iron toxicity, rice cultivars accumulate excess iron in vacuoles and apoplasts, facilitating detoxification through reactive oxygen species production and the activation of antioxidant enzymes.
Rice roots possess various mechanisms to combat iron toxicity, including their ability to repel iron at the root surface, thereby preventing its uptake into the roots.Potassium has been implicated in both iron exclusion at the root level and its subsequent translocation from roots to shoots, indicating its crucial role in iron toxicity mitigation (3,4).When plants receive an adequate supply of potassium, the enhanced redox potential becomes more apparent.As iron concentrations decrease and bronzing intensity diminishes, there is an increase in potassium concentration and accumulation in rice shoots.This dilution effect amplifies dry matter production.Consequently, efforts have been directed towards integrating rice cultivars resilient to iron toxicity with effective potassium management strategies.This endeavor aims to generate insights that can enhance rice yields in low-lying soil environments afflicted with iron toxicity.

Experimental site
The current field experiment was conducted at the Central Experimental Farm of the Odisha University of Agriculture and Technology, located in Bhubaneswar, Odisha, India (200 15' N latitude, 850 52' E longitude, elevation 25.9 m above mean sea level), during the wet season of 2021 and 2022.This area falls within the east and southeastern coastal plain agroclimatic zone of Odisha and the east coastal plain and Hills zone of humid tropical India.The experimental plot featured sandy loam soil with a pH of 4.82 and an organic carbon content of 0.54 % (Table 1).

Experimental details
The experiment was designed using a split-plot layout, featuring 32 common rice varieties assigned to the main plots and 4 potassium treatment levels (control with K2O at 40 kg ha -1 , K2O at 80 kg ha -1 , K2O at 100 kg ha -1 and K2O at 120 kg ha -1 ) assigned to the subplots.Each treatment was replicated twice.Although mid-season draining of the field is an effective management strategy for iron toxicity, this particular field had poor drainage facilities.

Plant, Soil sampling and analysis
After harvesting the crop, rice grains from different cultivars were collected and prepared for Fe and K analyses.The grains were ground into a fine powder.For the analysis, 0.5 g of the powdered samples were placed in 5 mL of concentrated nitric acid for pre-digestion overnight.Subsequently, the samples were digested in a diacid mixture (3:2 HNO3: HClO4).The digested solution was filtered and made up to a volume of 50 mL.The Fe content was analyzed using an Atomic Absorption Spectrometer (AAS), while K was measured using a flame photometer.
To determine the DTPA-extractable Fe, 20 mL of DTPA was added to 10 g of soil and shaken for 2 h using a mechanical shaker.The solution was then filtered and the Fe content was measured using AAS.Sequential fractionation of Fe was performed using polypropylene centrifuge tubes containing 5 g of sieved soil (5).Since the extraction process is sequential, careful attention was required at each stage to extract a specific fraction without losing any soil.This meticulous process was maintained throughout the extraction.The following procedure was used to extract each chemical fraction: Exchangeable Fe-After adding 25 mL of 1 M NH4NO3 at a pH of 7.0 to a 5 g soil sample in a centrifuge tube, the mixture was agitated for 2 h using a mechanical shaker.The sample was then centrifuged for 10 min at 1776 rpm and the supernatant was filtered for analysis.The residue was retained for extraction of the second fraction.

Carbonate bound Fe-
The residue was then combined with 25 mL of 1M sodium acetate adjusted to pH 5.0 with acetic acid and shaken for 5 h.After the shaking process, the suspension was centrifuged and the supernatant filtered for examination.

Iron/manganese oxides bound Fe-
The residue from the previous step was treated for 5 h in a water bath with 50 mL of 0.04M NH2OHHCl in 25 % acetic acid.After allowing the mixture to settle, it was centrifuged and filtered for analysis.The residue was then retained for further examination.
Organic matter bound Fe-The residue was treated with 5 mL of 30 % H2O2 and 3 mL of 0.02 M HNO3 and then placed in a water bath for 3 h.After cooling, 50 mL of NH4NO3 was added and the mixture was shaken for 2 h.Subsequently, it was filtered, centrifuged and analyzed.
Residual Fe-After drying and crushing the residue, the organic Fe was removed.A 50 mL Teflon beaker was filled with 1.0-0.5 g of soil.To this, 5 mL of hydrofluoric acid, 1 mL of perchloric acid and a few drops of concentrated H2SO4 were added.The mixture was then heated on hot plates until the soil completely dissolved.This procedure was repeated as necessary.The dissolved material was then transferred to a 100 mL volumetric flask, filtered and analyzed.The experiment detailed in this publication was conducted during the Kharif seasons of 2021 and 2022.Consistent trends and outcomes were observed each year.Although absolute levels varied due to seasonal effects, the responses to different cultivars and soil management strategies remained consistent.The data presented here represents the experiment from the 2022 season.

Statistical analysis
The data were analysed statistically using a split-plot design (6,7).Statistical analysis, correlation coefficients and scatter diagram were generated uisng Microsoft Office Excel 2019.Cluster analysis of the rice cultivars was performed using R Software (R.4.3.0)(8).

Effect on tiller number, root length and SPAD value at maximum tillering stage
In all 32 rice cultivars, the number of tillers per plant increased with potassium nutrition from 40 kg to 120 kg K2O ha -1 at the maximum tillering stage.The plants treated with 120 kg K2O ha -1 exhibited the highest tiller count among the cultivars and this increase was found to be significant for both the cultivars and the potassium treatment doses.There was also a statistically significant interaction between the potassium applications rates and the variations among cultivars.The lowest tiller number was observed in all cultivars treated with 40 kg K2O ha -1 and this difference was also significant.Similarly, root length at the maximum tillering stage was measured, revealing that iron toxicity influences root growth.However, root growth was more significantly impacted at 120 kg K2O ha -1 than at 40 kg K2O ha -1 , except in the cases of Kanchan, Mahalaxmi and Savitri, where the lowest root growth was observed at 40 kg K2O ha -1 .The dosages, variety and interaction of the roots were crucial.Similarly, the chlorophyll concentration, expressed in SPAD values as an indicator of nitrogen content, showed a gradual increase with the increased application of K2O.This increase was significant for most cultivars, except for Urbasi, Hasant, Mahalaxmi, Manika, Ashutosh, Mahanadi, Salibahan and Savitri, where the SPAD values increased significantly as potassium application increased from 40 to 120 K2O ha -1 (Table 2

Effect on iron and potassium content in rice grain
The iron concentration in grain was significantly impacted by treatment, cultivar diversity and their interaction.Potassium treatments resulted in a progressive decrease in iron concentration, from 40 kg K2O to 120 kg K2O ha -1 .Cultivars such as Kanchan, Indravati, Jagabandhu, Santepheap and Salibahan exhibited lower iron contents at 120 kg K2O ha -1 compared to other cultivars.Additionally, the potassium concentration in the grain increased in all cultivars across treatments ranging from K-40 to K-120 and this increase was found to be significant.Indravati Swarna, MTU-1010 and Mahanadi had higher K concentrations, ranging from 0.31 to 0.37 %, compared to other cultivars (Supplementary Table 2).

Effect on grain yield and straw yield
The grain yields of all cultivars increased with potassium application, with K-120 resulting in larger yields compared to the other treatments.Although grain output was lowest in K-40, it was observed that grain yield was significantly influenced by the 4 potassium treatment doses, cultivar diversity and their interaction.The highest grain yield, measured in kg K2O ha -1 , was observed in Indravati (45 q ha -1 ), followed by Mahanadi, Manika, Tejaswini and Upahar with yield ranging between 42 and 43 q ha -1 .Conversely, straw yield exhibited the opposite trend, with varieties treated with K-40 showing maximum production, except for Kanchan, Urbasi, Upahar, MTU-1010 and Savitri.Additionally, it was demonstrated that straw yield was affected by the treatments, the varieties and their interactions.

Effect on Harvest index
The potassium treatments produced mixed results regarding the harvest index, which was determined to be noteworthy.Among the cultivar, Santepheap displayed a particularly strong harvest index in response to potassium treatments (Table 3).

Correlation coefficient
The results were further confirmed by Karl Pearson's Correlation analysis, which showed a strong positive correlation (p< 0.01) between grain yield and the uptake of both iron and potassium.Similarly, straw yield also correlated with the uptake of iron and potassium at different doses of potassic fertilizer (Table 4).

Screening of cultivars based on grain yield, iron and potassium content
The scatter diagram analysis of the relationship between grain yield and iron uptake (Supplementary Figs.1-4) revealed that nine rice cultivars in quadrant 3, classified as the very high priority group, yielded more than 36.07q ha -1 when treated with the maximum potassium levels.
Similarly, the scatter diagram analysis of the association between potassium uptake and grain yield (Supplementary Figs.5-8) showed that 12 cultivars in quadrant 3, categorized as the extremely high priority group, absorbed more than 10.27 kg ha -1 of potassium.Among these, the Indravati, Upahar, Tanmayee and Bina rice cultivars exhibited similarities in their uptake of Fe and K concerning grain yield under optimal potassium nutrition (K-120).Further cluster analysis, based on grain/straw yield, harvest index, iron/potassium content and uptake, was conducted using R software and identified 2 naturally occurring data clusters (Fig. 1).Within these clusters, cultivars such as Indravati, Santepheap and Kanchan demonstrated high performance, followed by Salibahan with moderate ability.Conversely, Jagabandhu exhibited susceptibility in iron-toxic soil treated with potassium nutrition.

Fractionation of soil iron
Potassium application after rice harvest affected the soil's iron content in various chemical forms.Sequential fractionation, as illustrated in Fig. 2, showed that exchangeable iron constituted only 2.02 % of the total iron.Carbonate-bound iron contributed 1.45 to 2.49 % and exhibited an increasing trend with higher potassium application.Oxide-bound iron, which ranged from 16.42 to 28.4 %, also increased with higher potassium doses, following a similar trend to carbonate-bound iron.This form of iron was 1000-2000 times greater than exchangeable iron.Organic-bound iron accounted for 4 to 11 % of the total iron, but did not show a consistent trend with varying potassium doses.Residual iron made up the largest portion of total iron, ranging from 57.6 to 78 %, but showed a declining tendency with increasing potassium doses.
The total iron content, which is the sum of these 5 fractions, ranged from 1963 to 2274 ppm and was not affected by the potassium application doses.Exchangeable iron concentration was found to decrease with increasing potassium doses.Potassium doses significantly negatively correlated with the exchangeable fraction of iron and positively correlated with carbonatebound, oxide-bound and organic iron as well as DTPA-extractable iron (Table 5).Additionally, there was a significant reduction in DTPA-extractable Fe in postharvest soil compared to the initial soil Fe levels (Table 1).

Effect on tiller number, root length and SPAD value at maximum tillering stage
Iron plays a crucial role in several physiological metabolic processes in plants, including the growth of chloroplasts, chlorophyll production, electron transport and redox reactions.At K-40, certain cultivars exhibited signs of iron toxicity, such as a significant increase in tiller number due to altered potassium nutrition, a gradual decrease in root length with higher potassium application and irregular SPAD values.Despite increased potassium treatment, iron uptake progressively declined as potassium application increased.These results suggest that iron acquisition in the roots may facilitate potassium uptake, thereby promoting iron toxicity and reducing the number of tillers.Previous research has also reported a decrease in root length under similar conditions (9).Exposure to excessive iron ions can lead to an imbalance in free radical generation, causing oxidative stress in plants (10).

Effect on iron uptake, potassium uptake, grain yield and straw yield
The study revealed that grain yield increased with the potassium fertilization dose, peaking at cultivar K-120.This suggests that potassium deficiency may also result from high iron levels in low-lying areas and poorly drained soil during the wet season (11), a condition that can be mitigated by applying potassium fertilizers.The varied responses of cultivars to potassium management techniques indicate that the issue of iron toxicity can be addressed by combining suitable tolerant varieties with effective soil management practices.Tolerant cultivars that respond positively to potassium nutrition may help mitigate iron toxicity by reducing nutrient uptake by plants.Furthermore, we found a strong correlation between grain production, straw yield, potassium and iron uptake in response to potassium nutrient management.

Grouping of cultivars based on grain yield, iron and potassium content
Cluster analysis revealed 2 naturally occurring groups among the cultivars, categorized based on their priority regarding nutrient uptake and grain yield.Cultivars such as Savitri, Jagabandhu, Hiranmayee, Moti, Pratikshya, Ranidhan, Hasant and Upahar were identified as susceptible to iron-toxic soil conditions, resulting in low yields.On the other hand, Tanmayee, Upahar, Bina dhan-11 and Indravati exhibited extremely high tolerance to iron, with increased yields in response to potassium nutrition.Prachi and Kanchan were classified in the high priority group.

Fractionation of soil iron
After the rice crops were harvested, it was observed that the total iron content in the soil remained unaffected by varying potassium application doses, with concentrations ranging from 1963 to 2274 parts per million (Fig. 2).However, the fractions of iron present in the soil were significantly influenced by the application of potassium.The majority of the iron existed in the residual form, suggesting that it was acquired near the rhizosphere soil and not absorbed by the plant; instead, it facilitated potassium absorption by the crop.High concentrations of iron in the root zone affected root length, although there was an increase in grain yield at K-120.This increase may have been caused by a higher % of carbon translocated to the grain and a corresponding decrease in carbon delivered to the roots.Potassium application doses led to a decrease in exchangeable iron, with this form being closely linked to potassium doses.However, compared to other soils, the content of exchangeable iron was found to be extremely low, indicating a positive correlation between potassium fertilization and a reduction in soil iron content.Additionally, potassium had a significant impact on reducing soil iron content, with an increase in potassium content leading to a decrease in plant-available forms of iron, particularly exchangeable iron (12).

Conclusion
These finding confirm that to sustain the crop productivity in iron-toxic low-land rice fields, it is essential to enrich the soil with high-potassium fertilizer and employ suitable rice cultivars.This approach aids in fostering rice growth and maximizing production.

Table 1 .
Details of plant materials

Table 1 .
Initial and post-harvest soil properties

Table 2 .
).Effect of potassium doses on tiller no. and root length and SPAD value of rice cultivars grown under iron toxic soil at maximum tillering stage

Table 3 .
Effect of potassium doses on grain yield and Straw yield and harvest index of rice cultivars grown under iron toxic soil

Table 4 .
Simple correlation analysis Supplementary Fig. 1.Scatter Diagram (Relationship between grain yield and iron uptake in grain of treatment K-40).**

Table 5 .
Correlation coefficient among forms of Fe and other parameters **