Introduction

Widespread Zn and Fe deficiency in lowland rice fields not only hampers crop yield but also produces Zn-deficient food products derived from the grains. This can lead to Zn deficiency in the population (Alloway 2008). Zinc malnutrition problems affects one-third of the population of the developing and underdeveloped countries of Africa, Asia and South America (Cakmak 2008; Harvest plus 2012). Zinc dense cereal grains may be attained by (1) applying Zn fertilizer in different growth stages of crop, i.e. agronomic biofortification (Cakmak 2009; Saha et al. 2015a), (2) Screening of elite cultivars having higher Zn sequestration potential (Saha et al. 2015b) and (3) modifying transporters to improve Zn translocation in the plant by genetic manipulations (Palmgren et al. 2008). Rice, a major staple in Southeast Asia (Timsina et al. 2010), has limited contents of Zn as well as Fe and undergoes substantial loss of these elements on processing to final food products (Saha et al. 2015b; 2017a, b). The distribution of minerals in rice kernels is not uniform. About 50% of the mineral content is located in the bran layer and 10% in the embryo (Lu et al. 2013); both will be removed at the penultimate stage of consumption. In addition, rice contains phytic acid, potential anti-nutrients, obstructing the availability of divalent minerals like Zn2+ and Fe2+. Phytic acid (inositol hexaphosphate) forms complexes with mineral ions, such as Fe2+, Zn2+ and Ca2+ and reduces their bioavailability (Gupta et al. 2015; Lucca et al. 2001; Mendoza 2002). Screening suitable cultivars with low phytic acid content may be the option to secure Zn bioavailability in human diets. Zinc loading in the cereal grains, however, can succeed through agronomic biofortification of Zn with some subsequent changes in other parameters like (1) a possible depletion in Fe content (Saha et al. 2015b; 2017a, b), (2) a reduction in phytate content (Hussain et al. 2012; Saha et al. 2017a, b) and (3) an increase in protein content (Cakmak et al. 1989).

Our present study, therefore, hypothesized that application of Zn fertilizer in rice through basal and/or foliar is able to produce Zn dense grain associated with some alterations in Fe, phytic acid and crude protein.

Materials and methods

Experimental site

The study was conducted in kharif season (Summer) for two consecutive years (2011 and 2012) at Central Research Farm, Bidhan Chandra Krishi Viswavidyalaya, West Bengal, India, which is situated at 22°58.114′ N latitude and 88°29.543′ E longitude.

Weather conditions viz. rainfall, temperatures (Tmax and Tmin) and relative humidity (RH) during June to September were 1265 mm, 32.0 °C, 26.3 °C and 97% in 2011 and 987 mm, 32.6 °C, 25.1 °C and 94% in 2012, respectively, prevailed over that location during the crop growth period. Soil samples were collected at a depth of 0.2 m from the experimental plots before transplanting of rice. The soil was clay loam in texture with (1) neutral pH, i.e. 6.73 (soil/water:: 1:2.5), (2) higher in oxidizable organic carbon, i.e. 8.9 g kg−1 (Walkley and Black 1934), (3) higher in DTPA extractable Fe, Cu and Mn contents, i.e. 215.8, 6.0 and 16.0 mg kg−1, respectively, but (4) marginally deficient in DTPA extractable Zn content, i.e. 0.70 mg kg−1.

Details of the cultivars tested

The seeds of twenty six rice varieties with a wide genetic variation including two locals, four aromatic, seventeen high yielding varieties (HYVs) and three hybrids (Table S1) were collected from the gene bank maintained by ICAR-National Rice Research Institute, Cuttack, India. We selected the higher number of high yielding varieties due to their wide adoption and popularity in India.

Crop establishment

The collected seeds were raised in a nursery bed following standard methods in the middle of June for both the years. The seed bed was saturated with water for first four days and then the water level was increased gradually up to 5 cm during seedling growth. Twenty-one-day-old seedlings were transplanted in the main plots having strip plot design with three replications following standard protocols. Chemical fertilizer N-P-K @ 80-17.5-31 kg ha−1 was applied on the basis of soil test results. One half of the N and entire amount of P and K were applied at the time of sowing and the other half of N at maximum tillering stage (~ 21 DAT) of the crop. Water level up to 5 cm height was maintained from transplanting to grain filling stage. The role of applied Zn to boost up growth and yield of the cereals in soil with marginally deficient or deficient in Zn is well established (Cakmak 2008; Phattarakul et al. 2012). In the present experiment, Zn was applied in the soil and/or through foliar methods with an aim to produce higher yield along with significant Zn loading in the grains. Three Zn treatments viz., (1) no Zn (Zn0), (2) Zn at the rate of 20 kg ha−1 in the form of zinc sulphate heptahydrate (ZnSO4.7H2O) through basal (Zn1) and (3) Zn1 + two foliar sprays in the form of zinc sulphate heptahydrate (ZnSO4.7H2O, 0.5% aq. solution) at pre-flowering and grain forming stages (Zn2) were designed to screen out the most efficient Zn treatment for rice. Zinc fertilizer was applied in the soil at the time of final land preparation through broadcasting followed by surface incorporation. Foliar application of Zn was done with a knapsack sprayer (volume 14 L). The rice varieties and Zn treatments were considered as the main plot and the subplots, respectively, for statistical analysis. Brown plant hopper (Nilaparvata lugens) and Gundhi Bug (Leptocorisa acuta) during early crop establishing stage and milky stage of rice were controlled by applying Imidachloprid @100 mL ha−1 and Carbaryl @ 25 kg ha−1 through foliar spray.

Sample collection, preparation and storage

At maturity, the rice cultivars were harvested plot-wise and representative samples of grains were collected, washed thoroughly in tap water followed by distilled water and 1 M HCl solution to remove the dust and other impurities. The samples, after washing, were dried through winnowing and collected separately in different brown paper envelopes according to the varieties and Zn treatments. These dried grains were dehusked with a laboratory-scale hulling machine (THU-35C, Satake, Japan) to form the brown rice. Each cultivar was assessed in triplicates for thousand-kernel weight (TKW), yield of brown rice (YBR) and breakage from hulling. The brown rice was further milled for 45 s with a laboratory-scale milling machine (TM05C, Satake, Japan) to obtain white rice. The white rice was then measured for yield and subsequently ground through a Willey mill grinder for further analysis.

Analytical procedure

Zinc and iron content

Brown rice grain samples were ashed at 650 °C in a muffle furnace for 4 h, and the ash was dissolved in 5 mL of 1 M HCl. The content was diluted to 25 mL using distilled water and filtered into conical flask through Whatman No. 42 filter paper. These solutions were analyzed for Zn and Fe by recording absorbance at 213.7 nm and 248.3 nm, respectively, by using atomic absorption spectrophotometer (GBC Avanta, Model 912). The detection limit of Zn and Fe was 0.005 mg Zn L−1. The Zn uptake in brown rice was estimated by multiplying the Zn concentration and the dry weight of brown rice.

Phytic acid

Phytic acid levels in brown rice were determined after extraction in 100 g L−1 Na2SO4–HCl (1.2%) concentration on an anion exchange column and were analysed spectrophotometrically at 500 nm after reacting with 0.3% sulphosalicylic acid by using a UV–Vis spectrophotometer following the method outlined by Ma et al. (2005). All the samples were analysed in triplicates.

Statistical analysis

Analysed data of the experimental trial were pooled for both the years (2011 and 2012). All variables presented here are quantitative in nature, are large in number and assumed to follow a normal distribution. The significance of the effects of the treatments and cultivars was evaluated by two-way ANOVA using SPSS 14.0 statistical software. The mean effects of three replicates were further subjected to post hoc test (Duncan multiple range test) to identify the homogenous means at p < 0.05.

Results

Influence of Zn fertilization on Zn concentration in brown rice

Results showed that the concentration of native Zn in brown rice was higher in aromatic cultivars (32.0 mg kg−1) followed by HYVs (29.3 mg kg−1), locals (29.0 mg kg−1) and hybrids (25.9 mg kg−1). On average, Zn concentration in brown rice of the tested cultivars varied from 22.9 to 79.7 mg kg−1 with a mean value of 40.3 mg kg−1 (Table S2). Results also showed that Zn content in brown rice varied significantly not only among the cultivars but also within the selected categories (local, aromatic, high yielding and hybrid). Application of Zn showed a significant variation within the categories, and the enrichment of Zn was higher on its application through soil plus foliar than only soil. Among the cultivars, Zn content in brown rice was higher in the hybrids followed by local, HYV and aromatic on application of Zn through soil plus foliar (Fig. 1a). Zinc application through soil enriched the brown rice with Zn up to 5.0 mg kg−1 (17%) over the control; whereas soil plus foliar applied Zn triggered such enrichment up to 27.8 mg kg−1 (95%) over control.

Fig. 1
figure 1

Influence of Zn fertilization on a Zn and b Fe content in brown rice

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Influence of Zn fertilization on Fe concentration in brown rice

Results (Table S3) showed that Fe content in brown rice varied from 50.0 to 62.9 mg kg−1 with a mean value of 54.4 mg kg−1. Native Fe content in brown rice of the tested rice cultivars was highest in the aromatics (72.0 mg kg−1) followed by locals (66.9 mg kg−1), hybrids (58.0 mg kg−1) and HYVs (56.4 mg kg−1) (Fig. 1b). Results further showed that application of Zn caused a significant decrease in Fe over the control, irrespective of the cultivars. Such depletion in Fe was higher when Zn was applied through soil plus foliar (17.4%) compared to only soil application (9.4%).

Influence of Zn fertilization on phytic acid content in brown rice

Results showed a wide variation in phytic acid content of brown rice among all the rice cultivars tested. It varied from 4.05 g kg−1 in Swarna to 10.45 g kg−1 in GB2 with a mean value of 7.31 g kg−1 (Table S4). On average, native phytic acid content in brown rice was higher with HYV (8.67 g kg−1) followed by hybrid (8.40 g kg−1), aromatic (7.97 g kg−1) and local (7.72 g kg−1) cultivars. Results also showed that application of Zn caused a significant decrease in phytic acid content over the control among the cultivars tested and such a decrease was maximized when Zn was applied through soil plus foliar (31.2% over the control). Thus, Zn fertilization would be a useful strategy to reduce the levels of phytic acid in brown rice. As per the phytic acid content upon soil plus foliar application of Zn, the cultivars followed the following trend: hybrid > local > HYV > aromatic (Fig. 2).

Fig. 2
figure 2

Phytic acid content in brown rice upon Zn fertilization

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Influence of Zn fertilization on phytic acid/Zn molar ratio (P/Zn) in brown rice

The phytate-Zn (P/Zn) molar ratio in brown rice showed a wide variation among the rice cultivars tested (Table 1 and Figure S1a). On average, the P/Zn molar ratio in brown rice varied from 6.36 to 44.95 with a mean value of 20.43. The results also showed that the native P/Zn molar ratio in brown rice was higher with hybrid (32.05), followed by HYV (29.44), local (26.19) and aromatic (24.94) varieties. Application of Zn significantly decreased the molar ratio over the control in all the cultivars tested. The magnitude of such decrease was higher on application of Zn through soil plus foliar (64%) than only soil application (23%). It has been observed that such a decrease was maximized with the cultivars GB2, GB1, PHB 71 and KRH 2 and PA 6444.

Table 1 Phytic acid/Zn molar ratio (P:Zn molar ratio) and phytic acid/Fe molar ratio (P:Fe molar ratio) in brown rice upon Zn fertilization
Full size table

Influence of Zn fertilization on phytic acid/Fe molar ratio (P/Fe) in brown rice

The phytate iron molar ratio (P/Fe) is an indicator of Fe bioavailability to humans. Phytic acid has a strong binding capacity for Fe. Results showed that the P/Fe molar ratio in brown rice varied from 7.96 in Gayasur, an aromatic cultivar, to 17.29 in GB 2, a HYV with a mean value of 11.50 (Table 1). Among the different categories, the native P/Fe molar ratio in brown rice was higher in HYV (13.09) followed by hybrid (12.37), local (9.79) and aromatic (9.41) varieties than in the control.

Application of Zn through soil plus foliar significantly decreased the P/Fe molar ratio of brown rice in all the cultivars, except Kalma 222 and PHB 71. Soil application of Zn, on the other hand, showed an erratic trend on the P/Fe molar ratio of the cultivars. The results showed a 17% decrease in the P/Fe molar ratio on soil + foliar Zn application, increasing Fe bioavailability in humans. Results also showed that the magnitude of decrease in this ratio was maximized with Swarna sub1, Triguna, Khitish, IR 36, Sabita and Satabdi than the rest of the cultivars tested. Among the different categories, such a decrease was higher with aromatic followed by local, HYV and hybrid categories Figure S1b).

Influence of Zn fertilization on crude protein content in brown rice

On average, crude protein content in brown rice varied from 7.91 to 11.18 g 100 g−1 with a mean value of 9.28 g 100 g−1. Results also showed that among the different categories, crude protein content was higher in the aromatics (10.0 g 100 g−1) followed by locals (9.50 g 100 g−1), HYVs (8.73 g 100 g−1) and hybrids (8.56 g 100 g−1). Zinc application results in an increase in its amount over the control and such an increase was higher when Zn was applied through soil plus foliar (10%), irrespective of the cultivars tested. Results also showed that the increase in crude protein content was maximized in the aromatic followed by local, HYV and hybrid categories (Figure S2).

Discussion

Influence of Zn fertilization on Zn concentration in brown rice

External application of Zn caused a significant variation within the categories of cultivars, and the enrichment of Zn was higher on its application through soil plus foliar than only soil. This may be attributed to the considerably low plant available Zn concentration in the studied soil, i.e. 0.67 mg kg−1 which is marginally deficient in Zn (Mandal and Mandal 1990; Sims and Johnson 1991), therefore, applied Zn through soil and soil plus foliar application which enhanced the Zn concentration in brown rice. Moreover, soil plus foliar application was found superior as compared to the sole soil application in enhancing zinc concentration in grain as it (soil plus foliar) overcomes the limitation of zinc immobilization in soil in flooded environments (Wissuwa et al. 2008). Our previous experiment also showed wide variations in whole grain Zn concentration across the tested cultivars (Saha et al. 2017a, b). Results from the present study demonstrate that grain Zn enrichment efficiency of the rice cultivars varied widely upon Zn applications owing to their variation in genetic make-up which was also confirmed by previous other workers viz. Martinez et al. (2010) and Phattarakul et al. (2012).

Influence of Zn fertilization on Fe concentration in brown rice

Fe resides mainly in the surface bran layer and embryo in the rice kernel. Hence, brown rice may be considered as a good source of Fe. Iron profiling in brown rice among different cultivars may be a useful tool for breeding programs aimed at the production of Fe-rich transgenic rice. The present study indicated that Fe in brown rice varied significantly among the cultivars and also across different categories. Maximum Fe loading in brown rice of the local and aromatic cultivars may be obvious due to (1) high iron chelating phytosiderophore secretion from roots (Cakmak et al. 1996; Rengel et al. 1998) and (2) efficient utilization of iron from high-iron containing soils (more than 100 mg kg−1 DTPA extractable Fe) of this region. Application of Zn either through soil or soil plus foliar reduced the Fe concentration in brown rice showing an antagonism between Zn and Fe loading in grains of brown rice. An established competition between Zn and Fe for ZIP (zinc iron transport proteins) family proteins through transport channels in the plant (Palmgren et al. 2008) as well as interference in the chelation process between two metals during their translocation (Kabata-Pendias and Pendias 2001) supported our present study to the observations revealing an antagonism between Zn and Fe in rice.

Influence of Zn fertilization on phytate as well as phytate/Zn and phytate/Fe molar ratio in brown rice

The phytic acid/Zn (PA/Zn) and phytic acid/Fe (PA/Fe) molar ratios are considered to be the indicators for Zn and Fe bioavailability in edible foods (Morris and Ellis 1989; Hussain et al. 2012). Phytic acid is also a major inhibitor for absorption of Zn and Fe in humans as it strongly chelates with Zn and Fe to form an insoluble salt referred as phytate or phytin (Mitchikpe et al. 2008), and its content in brown rice is always higher than polished rice (Itani et al. 2002). In general, rice cultivars varied widely in phytic acid content in their seeds/grains. In the present study, emphasis was given to making a profile for phytic acid among the tested rice cultivars and also to evaluate the effect of Zn application on it (phytic acid). That application of Zn fertilizers reduced the level of phytic acid in brown rice might be due to the inverse relationship between P and Zn uptake (Mandal et al. 1980; Husssain et al. 2012) with a concomitant increase in grain Zn content. This, in turn, reduced the P/Zn molar ratio after Zn fertilization below 15:1 in all the rice cultivars tested to improve Zn bioavailability in humans.

On the other hand, the decrease in the P/Fe molar ratio upon soil plus foliar application of Zn in almost all the tested rice cultivars excepting Kalma 222 and PHB 71 is due to greater decrease in phytic acid as compared to the net decrease of Fe concentration in rice grains. However, the decrease in the P/Fe molar ration in brown rice of the tested cultivars upon only soil application of Zn was inconsistent possibly due to concomitant decrease of both phytic acid as well as the Fe concentration in the grains. The present study, therefore, revealed that on average the P/Fe molar ratio in brown rice significantly decreased upon either soil or through soil plus foliar application of Zn which is corroborated by the previous findings of Erdal et al. (2002), Saha et al. (2017a, b).

Influence of Zn fertilization on crude protein content in brown rice

Results of the present study showed a wide variation of crude protein content in brown rice among all the tested cultivars. Zn application is useful to increase protein content in the edible plant parts as Zn is involved in protein metabolism through several enzyme systems (Fang et al. 2008), though its content may vary significantly with different genetic make-up and management practices (Liu et al. 2005). The highest increase in protein content in brown rice was found when Zn was applied through the soil plus foliar mode of application. The increase in protein content in processed rice due to Zn application (Horino et al. 1983) can also improve the desired quality characteristic of rice grains.

We found that Zn fertilization is an effective tool to increase grain Zn concentration and productivity of rice across the different categories (Locals, aromatic, HYV and hybrid) particularly in the areas deficient or marginally deficient in plant-available Zn. Zinc application through soil plus foliar method caused a significant decrease in phytate/Zn as well as phytate/Fe molar ratios; therefore, it can be successfully adjusted to the required limit by external Zn application in rice. Such an application of Zn through different modes also increased crude protein content in brown rice. Thus, soil plus foliar zinc fertilization is an effective agronomic strategy that may complement Zn bio-fortification programme in rice.