Release behaviour of iron and zinc in different textured soil and its distribution in rice plant ( Oryza sativa L.) in North West of India

The present study aimed to assess the relationship of soil properties with extractable zinc (Zn) and iron (Fe) in soil as well as rice plant at different incubation period. The soil and plant samples were collected from 10 districts (Yamuna Nagar, Sonipat, Jind, Panipat, Karnal, Panchkula, Kaithal, Rohtak Ambala, and Kurukshetra) of RWCS (Rice wheat cropping system) of northwest Haryana of India. The effects of soil properties especially soil texture with the micronutrient in soil and rice plant at different incubation period have not been well studied. In the lab-cum-survey study, the analysis of grain samples collected from different districts of Haryana under varied soil texture showed a positive correlation between Zn/Fe concentration in grain and mean release rate of Zn/Fe in soil (Zn – 0.80; Fe – 0.98). The highest Fe concentration in grain was found in clayey soils (59.74–60.41 mg/kg) having a maximum mean soil Fe release rate (17.98–18.03 mg/kg). Likewise, the highest Zn concentration in grain was recorded in clayey soils (29.71– 30.57 mg/kg) of Yamuna Nagar and Panchkula, which has the highest mean soil Zn release rate (1.03–1.14 mg/kg). Univariate and multivariate analysis under principal component analysis (PCA) was carried out to determine the linear relationship between soil properties and extractable soil Zn and Fe concentration as well correlogram correlation matrix using for soil properties with grain Zn and Fe concentration. Hence, the study concluded that the detection for Zn and Fe are more successful in soils with a higher proportion of clay particles than in sandy soils. Plant uptake potential is highly influenced by soil micronutrient interactions with soil properties, especially soil texture, which can be predicted by extractable soil micronutrients

application. A plant's components can be analyzed to evaluate the functional connections between metal transport, accumulation, and uptake (Eskandari, 2011;Efe and Yapuzi, 2011,). In this study, Fe and Zn have been studied during different stages of plant growth, as well as their accumulation within the plant and dependency of soil texture (Cakmak and Kutman, 2017).
As inorganic nutrients are generally limited to the soil, there is an important relationship between nutrient application and accumulation that controls plant productivity and diversity. It has not been extensively studied how nutrients are collected or redistributed between different organs of rice plants throughout the year. In addition to providing information on the uptake of potentially dangerous elements, which have become increasingly important contaminants in recent years, this information will ensure more efficient use of fertilizers by more precisely matching supply and demand of these nutrients (Bailey, West and Black, 2015).
Micronutrients also play a significant role in the physiology of plants because they show intricate interactions that affect their availability for absorption and accumulation (Kiwan, Watfa and Saleem, 2014). A study was undertaken to investigate whether micronutrients accumulate in the tissues before and after seed reserves are mobilized.
Plant productivity will be lower and the quality of the produce will be lower if soils are not able to supply micronutrients to plants. Changing land use, soil management, and cropping systems all affect plant availability of soil micronutrients. Micronutrient availability is also influenced by soil properties including pH, soil texture, organic matter, and other nutrients (Talukdar, Basumatary and Dutta, 2009;Dhaliwal, 2019). A soil's texture has a significant impact on plant accessibility to micronutrients (Vijaya, Arokiaraj and Martin, 2011;Sachan and Krishna, 2018). There are several competing reactions that affect the solubility of micronutrients in soil, such as precipitation, adsorption and even chelation with clay (Alloway, 2008). Therefore, from an agricultural standpoint, it is more important to have micronutrients in ‚the plant available' form than in their total form.
As a result, in this study, the following key questions were addressed: 1. determine the concentration of plant-available micronutrients, as determined by DTPA at different incubation days, in the rice wheat cropping systems of northwest India; 2. how does soil texture affect the concentration of micronutrients available to plants; 3. how strong is the correlation between soil and plant micronutrient content (Zn and Fe) and soil properties.

Sampling
To explore the release behavior of micronutrients under diverse textured soils, three surface soil samples (0-15 cm) were collected from each North-Eastern district (10) Figure  1. These samples were brought to the laboratory of CCS HAU, Hisar for further analysis. The incubation study was carried out without the addition of these nutrients (Zn, Fe) by maintaining soil moisture at field capacity. Also collected the rice plant samples from the same field of soil survey. The sampling was done at periodical intervals for the estimation of DTPA (Diethylene Triamine Penta Acetic Acid) -extractable micronutrients (Fe and Zn) at periodical incubation of 0, 10, 20, 30, 40, 50, 60 days and at harvest.

Soil sample preparation and analysis
Form farmer field, one common soil sample after crop harvest was collected from the top 15 cm soil with the help of a soil sampling auger. For this, about 3 samples per districts (cores) were taken randomly from different physiographic positions of the farmer field in a zig-zag manner and mixed to make a representative (composite) sample to cover the entire field The collected soil samples were thoroughly mixed on a clean polythene sheet and the bulk was reduced by the quadrating so that about 0.5 kg of composite sample was retained and kept in a clean polythene bag after tagging and labelling. The collected samples were spread out on plastic paper in the shade for air drying. After air-dried soil samples were crushed gently with a wooden mortar and pestle and sieved through a 2 mm sieve. The top 15 cm soil of farmer field had a pH determined with a calibrated Glass electrode pH meter (10 g soil: 25 mL deionized H 2 O) (Jackson, 1973), EC (1 : 2) determined by Conductivity bridge meter (Richard, 1954), organic carbon analyzed by Walkley and Black,1934, soil texture determined with help of International Pipette Method (Piper 1966), available nitrogen (N), phosphorus (P) and potassium (K) were analyzed with Subbiah and Asija, 1956;Olsen et al., 1954;and Jackson, 1973, repsectively. DTPAextractable Zn and Fe were analyzed with DTPA (Lindsay and Norvell, 1978) extraction method. Atomic Absorption Spectrophotometer (Model: Varian AA240z) was used for the determination of Zn and Fe.

Plant sample preparation and analysis
Proper plant sampling is the key to reliable plant analysis results. For the lab study, plant samples were collected (from the same field where Zn and Fe release behaviours were studied) under the RWCS periodically (10,20,30,40,50,60,80 DAS,at harvest) for analysis in the laboratory to understand the partitioning of Zn and Fe mineral nutrients to grain and plant. The samples were kept in the oven for drying at 65 °C for 24-72 hours to obtain a constant weight. The mechanical grinding of plant materials was carried out with stainless mills usually to pass a 60-mesh sieve. The collected grinded samples were used for further laboratory analysis for different nutrients. Zn and Fe were determined by Atomic Absorption Spectrophotometer (Model: Varian AA240z).

Statistical analysis
For each of the ten rice wheat cropping systems, the mean differences of soil properties were tested using a one-way analysis of variance. A normal distribution assumption was tested for the data. The descriptive statistics (mean, median, standard deviation, variance and coefficient of variation, skewness, and kurtosis) were calculated for all soil parameters using R Studio v1.1.463. To remove redundant variables and extract factor components, the principal component analysis (PCA) was used. To examine the relationship among the 13 soil properties with relation to soil extractable Zn and Fe, we used the PCA with Varimax rotation to statistically group them into four principal components (PCs). The pairwise Pearson's correlation between the soil chemical properties with relation to grain Zn and Fe concentration was calculated in R Studio v1.1.463.

Figure 1
Sampling sites in different districts of Haryana

Ethics approval
This study was approved by the CCSHAU, Haryana for ethical purposes. While collecting soil and plant samples, the researchers protected the confidentiality of respondents. The norm is to access and take soil and plant samples from sampling sites after presenting CCSHAU's (Haryana) approval proposal for village farmers and introducing the research's objective. Farmer permission was obtained before soil and plant samples were taken.

Statistical description of soil properties (13) within the study area
The results of the Kolmogorov-Smirnov test indicated that most variables had a normally distributed distribution, confirming their normality. It is also evident that the constraint values differ between the fields. There are essentially differences in the mean and median values of soil properties measured in the study area, which indicates dominant measures of central tendency in the soil properties (Table 1). According to the coefficient of variation, soil properties with the highest variability are CaCO 3 , CEC, electrical conductivity (EC), exchangeable N, K and available zinc, iron and clay (CV ≥30%). Instance, the higher variability (CV ≤5%) was measured for soil pH, while the moderate variability (CV = 5-30%) was measured for the remaining soil properties, according to the guidelines of Warrick 1998 for soil properties variability. In addition, skewness and kurtosis indices for soil variables differed noticeably from the 0 and 3 standard values, respectively, at 0-15 cm. A number of factors account for these variations in chemical properties, including soil management practices in the study area, soil parent material, and the role of groundwater depth and irrigation water quality (Anderson, Mitsch,and Nairn, 2005;Wani, Shaista and Wani, 2017). Numerous other studies have shown the same results, such as Cambardella and Karlen, 2000.

Release behaviour of DTPA-extractable iron in soil
The soil samples collected from ten different districts of Haryana was analysed in the laboratory for analysing release of Fe at 0, 10, 20, 30, 40, 50, and 60 days after incubation (DAI) (Figure 2

Release behaviour of DTPA-extractable zinc in soil
As the period of incubation increased, a gradual decrease in DTPA-extractable Zn in soils was noticed up to 60 DAI (Figure 3) Zn at the later period of incubation especially between 40-50 days of incubation (3.29 to 0.13 mg/kg) was observed to be more or less at par in all the soil except soils of Panipat, Kaithal and Kurukshetra soils where it was noticed between 50-60 days of incubation (2.4 to 0.11 mg/kg). The decrement in soil Zn was observed to be more pronounced between 0 to 10 or 15 DAI in the soils under study. It was also observed that a decrease in soil Zn was highly consistent with an increase in the incubation period in most of the soils under study. Based on the results, the release pattern of Zn metal was initially rapid but then slowed significantly varied.
The magnitude of the inherent release of Fe and Zn may be governed by many factors, i.e., type and amount of clay, micronutrient status, alternate wet and dry cycles, soil pH, moisture content etc. The results, by and large, are in line with those of Zheng and Zhang (2011), who

Figure 3
Release of DTPA-extractable Zn at different days after incubation

Figure 2
Release of DTPA-extractable Fe at different days after incubation found that metals in the exchangeable form fraction decreased consistently during 150 days of incubation in all different moisture regimes. Sachan and Krishna, (2018) noticed that there was a sharp increase in exchangeable-Zn found only up to 15 DAI but quickly reduced at 30 DAI. During early incubation, higher release rates were obtained, but they gradually declined as incubation progressed to 90 days. The results of our study also showed a very similar trend for all the studied soils. The release rate and extent of release for Fe were greater than Zn. This trend suggests that the studied soils are more capable of replenishing soil solution Fe than Zn. The finding indicated that the clay loam soils (Panchkula soils) release Zn and Fe at much higher rates than sandy clay soils (Yamuna Nagar soil) because of their higher initial values of Zn and Fe. There is evidence that an early cycle may be associated with enhanced nutrient uptake and use, as the earliest fruit maturation may facilitate a greater need for metabolic sinks, thereby intensifying transport of photo-assimilates, affecting plant nutrient concentration (Ma and Rao, 1997).

Iron and zinc concentration in plant at different stages
Fe concentration slowly increased in the micronutrient's evaluations (July-September/October) due to a seasonal variation in the micronutrients. After that, the concentration decreased, in November and December (Figure 4). During August of the pre-flowering period, Fe concentrations increased (15.09 to 72.76 mg/kg) and reached their highest level at 80 days after sowing, but then steadily decreased during the grain-filling period (72.76 to 71.36 mg/kg). It has been observed that maximum Fe concentration was reported in plant samples (without panicle) of Kurukshetra (27.56 mg/ kg) followed by Karnal (24.63 mg/kg) and Ambala (23.76 mg/kg) as compared to the rest of the soils. Whereas minimum concentration of Fe was reported in Jind plant samples (15.09 mg/kg). About 40-50% of the iron absorbed during the vegetative and reproductive stages was present in the shoots, while only 25-35% of the iron absorbed during the harvesting (ripening) stage was found in the shoots. The highest Zn concentration (32.40 mg/kg) was noted at 80 DAS, and the trend in the Zn concentration in the plant was virtually steady throughout the evaluation period after 80 DAS (11.3 to 32.4 mg/kg) had decreased the concentration ( Figure 5). Comparative studies were conducted between plant samples taken at different times and soil release of Fe and Zn from the same area of soil. According to our analysis, the highest Zn and Fe content was found in grain and plant parts on different days when higher Zn and Fe had been released in different soil districts. Nearly half of the zinc absorbed during the vegetative and reproductive stages was present in the shoots, whereas only 23-35% of the zinc absorbed during the ripping (harvesting) stage was found in the shoots due to Zn content transferred from shoots to grain. More than 80% of the zinc absorbed was found in the shoots regardless of the growth stage.
Coarse-textured soils provide easier passage through their aggregation, retaining less nutrients and releasing most of the nutrients initially at one time, therefore may not meet the needs of the plants, particularly during the grain development stage. In contrast, clay soil holds more nutrients than sandy soil, and release nutrients less at one time but release continuously and thus provides a satisfactory amount of nutrients to plants to store in grains. Clay minerals affects soil fertility by regulating nutrient availability and supplies, carbon sequestration and stabilization, micro-aggregate formation, and soil microbial functionaries and population (Alloway, 2008). Dissolution-precipitation and adsorption-desorption processes are the key processes associated in these relationships. Mineral adsorption reactions are more crucial in regulating plant nutrient availability as compared to the release of nutrient elements by mineral weathering. While the concentration of Zn and Fe in soil solution are primarily controlled by the oxidation and precipitation reactions. Plants trigger biochemical responses such as the release of reducing and chelating chemicals, as well as rhizosphere acidification, which can enhance the availability of Zn, Fe, and other micronutrients (Singh and Schulze, 2015). In the RWCS of Haryana, the same result was observed when micronutrients (Fe and Zn) were evaluated in clay and sandy soil.
A linear correlation study was carried out between average release rate of Zn and Fe under different textured soils with mean Zn and Fe buildup in rice grain (Figure 6 and 7). It was found that the Zn and Fe concentration was higher in the grains of the same soil in which average Zn and Fe release rate was more. In this regard, the Fe concentration in grain under clay sand (60.41 mg/kg) and clay loam (59.74 mg/kg) was highest having maximum mean Fe release rate of 18.03, and 17.98 mg/kg, respectively. The minimum Fe content in grain was recorded 51.92 mg/kg in sandy soils having lowest Fe release rate (3.75 mg/kg). Furthermore, the highest Zn concentration in grain was recorded in clay loam (30.57 mg/kg) and clay sand (29.71 mg/kg) soils of Yamuna Nagar and Panchkula, respectively having a respective Zn release rate of 1.14, and 1.03 mg/kg. The minimum Zn concentration (22.42 mg/kg) in rice grain was noted in sandy soils of Jind which has the lowest mean Zn release rate (0.47 mg/kg). There was a prefect positive correlation between Fe concentration in grain and average Fe release rate in soil (0.98), as well as in Zn content in grain and average Zn release rate in soil (0.80).

Figure 6
Iron concentration in rice grain under diverse textured soils of different districts of Haryana

Analysis of soil properties variability with soil extractable Zn and Fe by principal component analysis (PCA)
Principal component analysis (PCA) and partial correlation were used to analyze the effects of multicollinearity and group soil properties into four principal components (PCs  Brejda et al. (2000) reported that PCs with at least 5% variance explaining the best variability of a factor component.
Basically, the left and bottom axes are the PCA plot -use them to read the PCA scores of the samples. Loading plot axes on top and right indicate how strongly a characteristic impacts the principal components. We calculated the PCA using the prcomp command of the R statistical software. The PCA was applied in order to observe possible correlations between soil physical properties and soil extractable micronutrient Zn and Fe at different locations. When compared, Zn and Fe soil concentration showed sand% and EC of soil, while some positive interaction with clay%, SOC and CEC was observed (Figs 8-9).
Most of the information has already been captured by the first three to four PCs, so we can ignore the rest without losing anything. According to the scree plot, each PC captures a certain amount of variation from the data. The y axis represents the eigenvalues, which in essence represent the amount of variation. Use a scree plot to select the principal components to keep. The ideal curve should be steep, bend at an elbow -this is your cutting-off point -and then flatten out. The scree plot ( Figure 10) can be used as a graphical tool to choose

Correlogram correlation of soil properties with relation to grain Zn and Fe
Correlations that are positive are displayed in blue while correlations that are negative are displayed in red. The correlation coefficients determine the color intensity and the size of the circle (Figure 11). Correlogram's correlation test showed that significant positive correlation (Grain Zn & Fe ~ clay (%), CEC, SOC, extractable soil Zn; p <0.005) and negative correlation (Grain Zn & Fe ~ sand (%), EC, pH; p <0.005). Srinivasan et al. (2017) studied that Micronutrient like Mn, and Cu showed significant correlations with chemical properties (pH, EC, CEC, and OC).

Figure 8
Principle component analysis load plot of soil properties in relation to soil extractable Zn concentration

Figure 9
Principle component analysis load plot of soil properties in relation to soil extractable Fe concentration

Figure 10
Illustration of PCA scree plot of soil properties with relation to soil extractable Zn and Fe

Conclusion
North western India was found to have significantly different soil properties among rice wheat cropping systems (RWCS). There are essentially differences in the mean and median values of soil properties measured in the study area, which indicates dominant measures of central tendency in the soil properties. In view of this, the following outlines are drawn from the present study: 1. The DTPA-extractable Zn concentration gradually decreased from the first day of incubation up to 60 days as Zn does not have redox potential.
2. The DTPA-extractable Fe generally increased initially up to 30 days after incubation and then reduced throughout incubation up to 60 days due to its redox potential. Also, the rate and extent of release were greater for Fe than Zn.

Figure 11
Correlation matrix using correlogram of soil properties with relation to grain Zn and Fe