Nitrogen and Zinc Interaction Improves Yield and Quality of Submerged Basmati Rice ( Oryza sativa L . )

Nitrogen (N) and zinc (Zn) are the two major yield-limiting factors of flooded rice cultivation systems. Both nutrients interact and affect availability of the other in alkaline calcareous soils. In order to evaluate the interactive effects of N and Zn on yield and quality of Basmati rice, a field study was conducted at Sheikhupura (Site I) and Sargodha (Site II), Pakistan. Nitrogen treatments (0, 40, 80, 120 and 160 kg/ha) were kept in main blocks while zinc levels (0, 8, 10, 12 and 14 kg/ha) were kept in sub blocks. The main effects of N and Zn levels were significant for grain yield and yield attributes. N and Zn interaction significantly improved the grain yield, yield components and all kernel quality parameters, except kernel amylose contents. Combined application of 120 kg N/ha and 14 kg Zn/ha produced the maximum grain yields at both sites (6.12 and 5.78 t/ha). This combination also yielded the maximum kernel lengths and widths, water absorption ratio and kernel protein contents. There was a significant positive correlation between grain yield and total dry matter, panicle-bearing tillers, spikelets panicle, grain weight, and harvest index. Application of 160 kg N/ha was detrimental to yield and quality attributes and reduced the agronomic efficiency of N use at both sites. Site comparison showed that soil pH and soil nutrient contents play a significant role in determination of the optimum nitrogen and zinc fertilizer doses for maximum yields.


Introduction
Rice is staple food for more than half of the world's population (Fageria et al., 2008).In Pakistan, rice with an average yield of 2398 kg ha -1 (Anonymous, 2013) is ranked as the second most essential food of the human diet.Basmati (aromatic) rice is known for its long fluffy grains with minimum kernel dimension, aroma intensity, texture of the cooked rice, high elongation during cooking, palatability and longer shelf life (Ahmad et al., 2005).Most of the Basmati rice in Pakistan is produced in the Punjab province.Both yield potential and quality characteristics of new Basmati varieties such as 'Super Basmati', 'Basmati 2000' and 'Shaheen Basmati' has been improved by rigorous breeding efforts.There is a big difference in the yield potential of different rice varieties and the actual yields obtained by the farmers.The yield potential of Basmati varieties ranges from 4.5 to 6 t ha -1 while the farm yields vary between 2.0 to 2.8 t ha -1 (Ahmad et al., 2005; Shivay et al.,   2010).Basmati rice is also an important export commodity of Pakistan.So, quality of Basmati rice is next to rice production.Among major constraints of quality and paddy yields in rice, imbalanced nitrogen fertilization (Wang et al., 2008) and zinc deficiency (Shivay et al., 2010) in soils are the most important variables.
Zinc (Zn) deficiency in staple foods is a major threat in combating malnutrition related health problems in many developing countries (Impa et al., 2013).After nitrogen, zinc is the second most yield-limiting nutrient in rice (Quijano-Guerta et al., 2002).Most of the rice crop in Pakistan is cultivated in alkaline calcareous soils with high clay contents.Due to these soil characteristics, most of the zinc (Zn) applied as fertilizer is adsorbed and only little is recovered by rice crop during the growth period (Tahir et al., 1992;Rahman et al., 2002).High soil pH is considered to be the most crucial factor in causing Zn deficiency in Pakistani soils (Qadar, 2002).In addition, Zn deficiency is also wide spread in these soils (Rashid et al., 1999).Zn 5 th and 7 th July of both growing seasons at Sheikhupura and Sargodha, respectively.Before transplantation, half of the total nitrogen and half of the total Zn (doses as per treatments), 80 kg/ha phosphorus and 60 kg/ha potassium were applied in the puddle field in the form of urea, ZnSO4, SSP and SOP, respectively.The remaining doses of nitrogen and zinc fertilizers were applied during active tillering stage.Butachlor (60% EC) @800 ml/ha was applied in standing water for weed control (Reddy, 2004).To control borers and leaf folders, Carbofuran (10% GR) @25 kg/ha was broadcasted at 55 day after transplantation.Irrigation water was maintained at a depth of 3-4 cm at transplantation and one week afterwards at a depth of 5-6 cm throughout the growing season till one week before harvesting.An area of 7.5 m 2 was harvested from each plot and 20 plants were randomly selected for determination of yield attributes. Final grain and dry matter yields were estimated from 7.5 m 2 .Manual threshing was done separately for each plot.Agronomic efficiencies of N and Zn were calculated as: AEN = grain yield (kg/ha) with N application -grain yield (kg/ha) without N application / N application (kg/ha) AEZn = grain yield (kg/ha) with Zn application -grain yield (kg/ha) without Zn application / Zn application (kg/ha) Kernel dimensions (length and width) of 100 normal kernels out of each treatment were measured using a dial caliper and then averaged.Water absorption ratio of kernels was determined by dividing the weight of cooked rice with that of raw rice.Protein contents were calculated by micro-Kjeldahl digestion method to estimate nitrogen content, which was multiplied with factor 5.9 in order to convert into protein (Jacobs, 1958).Kernel amylose contents were determined according to the method of Juliano (1971) by using Spectrophotometer.The data collected were subjected to Fischer's ANOVA using MSTAT-C program.The treatment means were compared using Duncan Multiple Range Test at 5% probability level.

Paddy yield components
Statistical analysis showed that year effect for different parameters studied in this work at both experimental sites was not significant.Therefore data presented in this work represent the averages of both years (2009 and 2010) for either site.Tillering of Basmati rice was significantly influenced by nitrogen (N) application.Comparison of means (Tab. 1) showed that N significantly increased the number of paniclebearing tillers up to 120 kg N/ha; thereafter panicle bearing tillers were decreased at a higher dose of N (160 kg/ha).Similar trend was observed at both sites.Promotion of growth and tillering in rice by N application has been reported by many studies (Mae, 1997;Samonte et al. 2006).Reduction in number of productive tillers at higher N dose could be the result of excessive vegetative growth (Gebrekidan and Seyoum, 2006).On the other hand, significant effect of Zn on tiller production was only evident at site I (Sheikhupura) where 12 kg Zn/ha increased the number of panicle bearing tillers in comparison to control (Tab.1; 0 kg Zn/ha).While at site II (Sargodha), increase in Zn rates did not significantly affect the number of panicle-bearing tillers.These varying responses to fertilization has also been shown to affect kernel protein contents of rice (Mirzavand, 2007).Since Zn is a costly fertilizer, its economic use is important from the point of view of poor farmers.
Nitrogen (N) has been an important yield determinant of the flooded rice production systems.High prices, food shortages in developing world and the adverse effects of heavy N losses from flooded fields however necessitate optimal use of N. Nitrogen losses through ammonia volatilization, leaching and runoff have been recorded from 20-80% in the rice production (Singh et al., 1998;Griggs et al., 2007;Norman et al., 2009).Economic use of nitrogen fertilizer is especially important for small growers as urea prices are continuously on rise.The response of rice crop to nitrogen fertilizers have been well documented, however the effects of N interaction with zinc application rates on yield and quality of rice are lacking.Non-judicious use of nitrogen fertilizer could further aggravate Zn deficiency in alkaline soils and suppress paddy growth and yield (Rashid, 1996).Nitrogen fertilization is found to enhance zinc contents of flooded rice, where urea performed better than NH4NO3 in increasing Zn contents (Chaudhry et al., 1977;Singh and Singh, 1981).They observed that higher Zn contents in plant tissues by nitrogen application were due to better growth, increased Zn solubility and increased root efficiency for Zn uptake.Under submerged conditions, urea is more efficient as compared to nitrate containing fertilizers.Urea-N application lowers soil pH and in addition it forms NH3-Zn complex and thus increases Zn availability under alkaline conditions (Gao et al., 2012).Keeping in view the possible N × Zn interactions in alkaline soils, present study was aimed to identify optimum doses of N and Zn for higher grain yields and better quality of Basmati rice.
Rice nursery (cv.'Super Basmati') was sown during first week of June using hand drill at both sites.Nursery seedlings of 28-30 days old were manually transplanted in puddled fields by keeping a row to row and plant to plant distance of 22.5 cm on Zn supply at two sites of the present experiment could be related to differences in soils characteristics.Zn nutrition has been shown to have positive influence on tiller production of rice (Impa et al., 2013;Sarwar et al., 2013).The interaction between N and Zn regarding panicle-bearing tillers was 374 significant (Tab.2).Highest number of panicle-bearing tillers hill -1 (13.8 and 13.1) at sites I and II were produced by 120 N + 14 Zn and 120 N + 12 Zn (kg/ha), respectively.
The number of spikelets panicle -1 varied from 88.1 to 110.7 across treatments, sites and years.At both sites, an increase in the rate of N application up to 120 kg ha -1 progressively increased the number of spikelets panicle -1 over control (Tab.1).Gebrekidan and Seyoum (2006) revealed that increase in spikelet number by N is the result of increase in panicle length.
In a recent study Ding et al. (2014) showed that the increase in the number of spikelets panicle -1 by nitrogen fertilization is associated with an increase in cytokinen contents.Whereas at 160 kg N/ha, the decrease in spikelets panicle -1 as compared to that of 120 kg N/ha (Tab. 1) may be because of increased competition among tillers for carbohydrate supply (Wu et al., 1998).Different Zn application rates also significantly affected the number of spikelets panicle -1 at both sites.Though spikelets panicle -1 at Zn rates of 12-14 kg/ha were significantly higher over control, the gain at higher Zn doses was relatively small at both sites (Tab.1).Zinc deficiency affects floral development of plants by lowering enzyme activity (Pandey et al., 2001).Improvement in different grain yield components with an increase in Zn levels could be attributed to adequate Zn supply that might have affected the metabolic and enzymatic activity (Hafeez et al., 2010).Combined effect of N and Zn on spikelets panicle -1 was also significant.Maximum number of spikelets panicle -1 at both sites were recorded where 120 N + 14 Zn (kg/ha) were applied (Tab.2).While control plots (where N and Zn were not applied) showed the lowest number of spikelets panicle -1 .Grain weight of rice kernels is an important yield attribute, which is mostly determined by genetic potential rather than environmental conditions (Ashraf et al., 1999).Data showed that thousand grain weight significantly increased with an increase in N fertilizer up till 120 kg/ha, then declined at 160 kg N/ha (Tab.1).Similar response was evident at both sites.Plots not supplied with N showed the lowest grain weights.Limited availability of nitrogen during grain filling stages affects carbohydrates supply to developing seeds and therefore decreases grain weight (Mae, 1997).Mean comparisons showed that Zinc application significantly increased the grain weight.At site I (Sheikhupura) increment in Zn fertilizer progressively increased the thousand grain weight, whereas at site II (Sargodha) grain weight was significantly increased till 10 kg Zn/ha and then remained static onwards (Tab.1).Combined application of N and Zn also had a significant positive effect on thousand grain weight at both sites.Highest thousand grain weight was found where 120 N + 14 Zn (kg/ha) was applied (Tab.2).

Total dry matter, paddy yield and harvest index
The nitrogen fertilizer significantly increased the total dry matter (TDM) yield with increasing rate of N application at both sites up to 120 kg ha -1 , thereafter TDM was slightly decreased at 160 kg ha -1 and difference between the two treatments were significant only at Sheikhupura site (Tab.1).Increase in dry matter yield by N application is apparently the result of growth promotion by nitrogen.Similarly, increasing rates of Zn application significantly increased TDM up to 10 kg ha -1 at both sites (Tab.1).Increment in Zn application from 10 kg Zn/ha onwards did not significantly affect the TDM yield.Wissuwa et al. (2006) observed that Zn deficiency caused stunted growth and reduced the TDM yield of many rice cultivars.They further exhibited that lower TDM yields under Zn deficiency are well correlated with lower Zn contents in rice plants.Interaction between N and Zn application rate affecting when N and Zn were not applied (Tab.3).Site comparison showed that grain yields and HI at all Zn levels were slightly higher at Sargodha (Site II) than that of Sheikhupura (Site I) despite relatively lower soil Zn contents at the former site.Lower relative soil pH at Site II might have increased Zn solubility and thus produced better grain yields.Khan et al. (2007) compared the response of paddy yields to Zn fertilizer applied under different soil conditions.They noticed that the soils having low pH and lower CaCO3 contents gave the highest yield response to Zn.

Agronomic efficiency
Agronomic efficiency of nitrogen use (AEN) or zinc use (AEZn) describes the capability of yield increase per kg nitrogen or zinc applied, respectively.Agronomic nutrient use efficiency depends on various factors such as nutrient availability, soil conditions, fertilizer source and dose, and most importantly crop acquisition efficiency (Mengel and Kirkby, 2001).Nitrogen application increased AEN up to 120 kg N/ha, thereafter further increase in nitrogen rate (160 kg N/ha) reduced AEN at both sites (Fig. 2).The minimum AEN 7.0 and 14.1 kg grain/ kg N were obtained with the application of 40 kg N/ha whereas the maximum AEN 23.8 and 22.3 kg grain/ kg N were recorded at application of 120 kg N/ha at site I and site II, respectively (Fig. 2).Agronomic efficiencies for N use (AEN) for irrigated rice varied between 11.5 kg grain/kg N in on-farm studies (Dobermann et al., 2002)  N in research trials (Ladha et al., 2005) due to greater variability in soil and climatic conditions and incidence of insect/pest attack at farmer's field.Conversely, increase in Zn rates reduced the agronomic efficiency of Zn use (AEZn) at both sites.
Highest AEZn were recorded at lower Zn doses at both sites (Fig. 2).High Zn use efficiency and high agronomic Zn efficiency at lower Zn doses have also been reported for barley (Genc et al., 2002) and rice (Muthukumararaj and Sriramachandrasekharan, 2012), respectively.Comparison of both sites showed that AEZn values were higher at site II (Sargodha) than site I (Sheikhupura) under all Zn treatments.This varied response could probably be due to differences in inherent soil zinc contents at both sites.It seems that lower Zn contents (0.32 mg Zn/ kg soil) at site II as compared to site I (0.51 mg Zn/ kg soil) caused the relatively better yield response to applied Zn at site II.

Kernel quality
Combined application of N and Zn significantly improved the kernel dimensions (kernel length and width) at both sites (Tab.5).Nitrogen application significantly increased the kernel length at each increment up to 120 kg N/ha where significant Zn response on kernel length was only evident at the highest dose of the experiment (14 kg Zn/ha).Application of 120 N + 14 Zn (kg/ha) produced the highest kernel lengths 7.04 mm and 6.31 mm at site I and II, respectively (Tab.5).On the other hand, increase in both N and Zn progressively increased the kernel widths at both sites.Kernel width varied between 1.07 to 2.99 mm across years, N and Zn treatments and sites with maximum values obtained at 120 N + 14 Zn (kg/ha) and minimum values at 0 N + 0 Zn (kg/ha).An improvement in kernel dimensions (kernel length and kernel width) by increased N fertilization has also been shown by Maqsood et al. (2013).Both Khan et al. (2009) and Shivay et al. (2005) reported that Zn application @ 10-15 kg/ha improved the yield and quality of Basmati rice in Pakistan and India.Water absorption ratio and protein contents of kernels were also significantly improved by combined application of N and Zn at both sites.Tab. 5. Interactive effect of nitrogen and zinc rates on kernel quality of Basmati rice (Oryza sativa L.) The maximum kernel protein contents were recorded with the application of 120 N + 12 Zn (kg/ha) at site I while at site II with the application of 120 N + 14 Zn (kg/ha; Tab. 5).Poor N availability during flowering can severely affect rice protein contents (Perez et al., 1996).To attain high paddy yields with high protein contents, sufficient N supply is necessary (Perez et al., 1996).High protein contents at higher N doses (120 kg N/ha) indicate that plants were well supplied with N and sufficient N was mobilized from vegetative parts to grains (Samonte et al., 2006).This study is also in agreement with the findings of others (Schinir et al., 1990;Ali et al. 1992;Ahmad et al., 2009) who reported a significant improvement in kernel dimensions, kernel water absorption ratio, protein contents and milling quality of rice by optimum N fertilization.It was observed that both nitrogen and zinc treatments did not affect kernel amylose contents at both sites.It is interesting to note that kernel protein contents were higher at site II (Sheikhupura) as compared to site I (Sargodha) in almost all treatments (Tab.5).While kernel amylose contents were slightly lower at site II than that of site I in most of the treatments.It seems that differences in soil factors such as pH, organic matter and available phosphorus and potassium other than treatments at both experimental sites produced the contrasting differences in these two quality parameters.
. 1. Main effect of nitrogen and zinc rates on yield and yield components of Basmati rice (Oryza sativa L.) Tab. 2. Interactive effect of nitrogen and zinc rates on yield components of Basmati rice (Oryza sativa L.) N = nitrogen; Zn = zinc; TDM = total dry matter; GY = grain yield; PBT = panicle-bearing, tillers; S = spikelets; GW = grain weight; HI = harvest index; NS = Not Significant.Treatment means were separated with Duncan Multiple Range Test at 5% probability.Different letters on treatment means within each column show statistical differences at P≤0.05.TabN = nitrogen; Zn = zinc; PBT = panicle-bearing tillers; S = spikelets; GW = grain weight; NS = not significant; *, **: Significant at 0.05 and 0.01, respectively