Effect of micronutrient foliar application on morphology, yield and iron and zinc grain concentration of durum wheat genotypes

Durum wheat has a comparative adaptive advantage over bread wheat under hot
 and dry conditions. Accordingly, it feeds millions of people in the Middle
 East and North Africa. Under these conditions, the deficiency of nutrients,
 including micronutrients, is a major concern for many reasons, including
 calcareous soil under drought stress conditions. Therefore, growth, yield,
 iron (Fe) and zinc (Zn) concentration in durum wheat cultivar grains were
 investigated. A factorial experiment based on a randomized complete block
 design with three replications was conducted in the Dryland Agricultural
 Research Institute (DARI) - Moghan. The first factor comprised spraying at
 four levels, including the control and foliar spraying with Fe, Zn, and
 Fe+Zn and the second factor consisted of genotypes at four levels: Dehdasht
 (G1), Seymareh (G2), and two new genotypes (G3 and G4). Solutions of Fe and
 Zn fertilizers were sprayed at the tillering, early ear emergence, and milk
 stages, with a ratio of 2 and 1.5 g fertilizer/1000 ml solution (W/V),
 respectively. The results showed that genotypes G1, G3 and G4 produced
 higher grain yield per square meter than G2. This increase was due to the
 higher weight of 1000 grains in G3 and G4 genotypes and 1000-grain weight
 with a higher grain number in G1. G1 and G2 had greater spike length, number
 of grains per spike and spikelet than G3 and G4 genotypes. In all studied
 traits, except Fe and Zn concentration, the combination of Fe+Zn showed the
 highest and control had the lowest performance. Also, the application of Zn
 was superior to Fe. The highest Fe concentration of G1, G2, G3, and G4 was
 observed at Fe+Zn, control, Zn, and Fe levels, respectively. The highest Zn
 concentrations were observed in the G3 genotype when only Zn was used or in
 combination with Fe. According to the results, the Fe and Zn spray
 application increased durum wheat yield on Fe and Zn deficient soil.


Introduction
Wheat is the most important crop grown in Iran and provides more than 45% of protein and 55% of the calories needed by the people (Malakouti, 2007). Compared to bread wheat, durum wheat (Triticum turgidum L.) tends to store more Zn and Fe in grains (Conti et al., 2000). Durum wheat, as the hardiest wheat species, is well adapted to semi-arid and dryland climates and it is superior to bread wheat in hot and dry conditions (Elias and Manthey, 2005). The reasons for the low availability of Fe and Zn are high pH, high calcium carbonate content, heavy texture, low organic matter and low soil moisture (Cakmak et al., 1996). Most of these factors are present in rain-fed conditions. It is estimated that 80 percent of the Iranʼs farms are potentially deficient in Zn (Malakouti, 2007). In another study, 37% of wheat fields had severe Fe deficiency and 40% of wheat fields had severe Zn deficiency (Dorostkar et al., 2013). Recent researches support the hypothesis of declining the concentration of micronutrients in new wheat cultivars over time (Fan et al., 2008). In developing countries, the decline in micronutrient concentration is more intense due to poor crop management and soil degradation. The average Zn concentration of modern wheat is low compared to the early and wild wheat (Cakmak, 2000). For instance, Nikolic et al. (2016) concluded that the levels of Zn and Fe in the grain of two bread wheat cultivars grown in Serbia were rather low, whereas only 13% of the soil samples were Zn deficient and none was Fe deficient. However, the study of 57 durum wheat cultivars grown under field conditions in Italy showed a low genetic variation in Zn (29 to 46 mg kg -1 ) and Fe (34-67 mg kg -1 ) (Ficco et al., 2009). The amount of Zn in durum wheat grains was 8 to 12 mg kg -1 under lower Zn availability and 15 to 25 mg kg -1 under higher Zn availability conditions (Erdal et al., 2002). The concentration of Zn may also be reduced to 10 mg kg -1 , which is not enough to meet human needs (Cakmak, 2008). Research over the past two decades has proven that there is a close relationship between healthy soil, healthy plants and healthy humans, and malnutrition is often associated with illnesses in humans (Sanchez and Swaminathan, 2005). The use of micronutrients to improve crop yield and human health is greatly increased (Alloway, 2008).
Fe deficiency disrupts the synthesis of chlorophyll, electron transfer chain, photosynthesis (Ziaeian and Malakouti, 2006), and decreases leaf green pigments (Kumar and Sool, 2000), and the aboveground growth (Mohamed and Ali, 2004). These traits have a close relationship with the yield of crops. The application of Zn increased leaf and stem growth (Brennan, 2007), the number of grains per spike (Yilmaz et al., 1997) and 1000-grain weight of wheat (Malakoti and Hasanpor, 2003;Yilmaz et al., 1997). Seadh et al. (2009) reported that among the micronutrient elements, Zn had a major role in plant height, spike length, number of spikelets per spike, number of grains per spike, 1000-grain weight, grain yield, straw yield, protein and carbohydrates. Ziaeian and Malakouti (2001) have Effect of micronutrient foliar application on morphology and yield of durum wheat genotypes 227 described that the application of micronutrient elements increases grain yield, straw yield, 1000-grain weight and protein content of the grains. The experiment conducted on wheat crop in 25 locations of Iran included Zn, Fe, manganese (Mn) and copper (Cu) showed that Zn application significantly increased grain yield (about 15%), 1000-grain weight, grain number per spikelet, Zn concentration and protein content (Ziaeian and Malakouti 2001). Hussain et al. (2005) have stated that the use of a micronutrient spray at the tillering, boot and milk stages increases wheat grain yields by increasing plant height, number of grains per spike and 1000grain weight. The soil and foliar application of Zn-containing fertilizers improved Zn concentrations in bread and durum wheat (Cakmak, 2008). The application of zinc sulphate in Zn-deficient soil increased Zn concentration and grain yield (Yilmaz et al., 1997). An increase in Zn concentrations was reported by Graham et al. (1992) and Shivay et al. (2008) under field conditions. Ming and Yin (1992) have found that Zn application reduces Fe concentration. Cakmak et al. (1999) concluded that lines with high Zn efficiency had higher Zn uptake by roots, but not higher Zn concentration in grains because increased Zn uptake is used to increase dry matter production. An increase in grain Fe concentration with a foliar spray of FeSO4 was reported by Zhang et al. (2010) and Singh et al. (2004). On the other hand, Gupta (1991) identified that foliar Fe fertilizers did not affect grain Fe concentration. There are mostly antagonistic (Saha et al., 2015;Tiwari and Pathak, 1982) and seldom synergistic (Zeidan et al., 2010) relationships between Zn and Fe concentrations in cereals. Monasterio and Graham (2000) and Grusak and Cakmak (2005) believe that grain Fe and Zn concentrations are positively correlated in cereals, biofortification is independent of environment, and raised grain Fe and Zn concentrations can be combined with improved agronomic traits. Thus, breeding for biofortification of Fe and Zn in cereals is feasible. Generally, tetraploid (ssp. durum) varieties showed less genetic variability for Fe and Zn concentrations (Monasterio and Graham, 2000;Grusak and Cakmak, 2005). An increase in inorganic concentrations might be a consequence of slower growth, reduced yield, low harvest index or smaller seeds (Monasterio and Graham, 2000). Yield increase led to a decline in nutrient concentration named the yield dilution effect.
The purpose of this study was to investigate the effect of Zn and Fe foliar application on the yield and quality of durum wheat grains.

Material and Methods
This study was carried out as a factorial experiment in a randomized complete block design with three replications at the Dryland Agricultural Research Institute (DAIR) -Moghan during the 2015-2016 cropping season. Soil characteristics of the study farm are given in Table 1. The DTPA micronutrient extraction method was used to estimate the potential soil availability of Zn and Fe. Critical levels of soil Fe and Zn were 8.5 and 0.55 mg/kg, respectively (Feiziasl, 2006). Therefore, the level of the two elements was lower (Table 1). The first factor comprised foliar spraying at four levels including control and spraying with Fe, Zn and Fe+Zn. The second factor consisted of different genotypes of durum wheat at four levels including Dehdasht (G1), Seimareh (G2) and two new genotypes (G3 and G4). The genealogy and the code of the genotypes are shown in Table 2. Each experiment plot consisted of 6 rows with 5-meter length and 20-cm inter-row space. The seeds were disinfected with carboxin thiram then cultivated with an automatic planter (Wintersteiger) at depths of 5 to 7 cm. Sowing time and harvest time were 6 November 2015 and 5 June 2016, respectively. Seed density within one square meter was 300. Approximately 28.5 kg/ha N and 12 kg/ha P were utilized before planting.  Table 2. The pedigree of genotypes cultivated for the experiment. The Zn and Fe nano-chelate fertilizer of Khazra Company was sprayed with a ratio of 1.5 and 2 g fertilizer/1000 ml solution (w/v), respectively, at the tillering, early ear emergence and milk stages. Main stems from one square meter were harvested and then plant height, peduncle length, spike length, number of spikelets per spike, number of grains per spike, number of grains per spikelet, and 1000grain weight of main spikes were assessed. Grain yields comprised the weight of grains of all plants harvested in a square meter. After cleaning the grain samples, they were dried at 70°C for 2 h, then were milled into flour and passed through a sieve of one millimeter. All samples of flour were digested by using the HNO 3 -HCl mixture, and Fe and Zn concentrations were measured by an atomic Effect of micronutrient foliar application on morphology and yield of durum wheat genotypes 229 absorption spectrometer (Shimdzu, AA-6300) at wavelengths of 248.3 and 233.9 nm, respectively.
SAS software was used for the analysis of variance (ANOVA). Duncan's multiple range test at p < 0.05 was used to determine differences between treatment means.

Results and Discussion
Stem height Stem height was affected by spraying and genotypes (Table 3). Mean comparisons showed that Zn and Fe+Zn treatments gave significantly greater stem height in comparison to control. The difference between Zn and Fe treatments was insignificant. Genotype G2 had the lowest stem height, and G3 and G4 showed a significant advantage over others (Table 4). El-Magid et al. (2000) concluded that the Fe and Zn foliar application increased the height of the wheat plants. Khan et al. (2008) achieved a similar result by the zinc sulfate soil application for wheat. Seadh et al. (2009) reported that the highest wheat plant height was related to Zn treatment among micronutrient elements applied.

Peduncle length
The effect of spraying and genotypes on peduncle length was significant ( Table 3). The foliar application of Fe+Zn gave the largest peduncle length and Zn, Fe and control treatments had lower values, respectively. Genotypes G1, G3 and G4 had the greatest peduncle length and genotype G2, with the lowest stem length, produced the minimum peduncle length (Table 4). Genotypes G3 and G4 also had the greatest stem height.

Spike length
Plants from control plots produced the least spike length, and there were no significant differences between control and Fe treatment (Table 4). Zn and Fe+Zn foliar application meaningfully increased spike length compared to control, and the greatest spike length was observed for Fe+Zn application. Genotypes G3 and G4 had the lowest spike length, and the highest length was related to the genotype G1. Genotypes G3 and G4 produced the least length of the spike, although they had the greatest stem and peduncle length. Hemantaranjan and Grey (1988) also observed that soil Fe and Zn application increased spike length. Sultana et al. (2016) reported that 0.02% and 0.006% Zn foliar application significantly increased spike length in comparison to control.

Number of spikelets per spike
Comparison of the means (Table 4) showed that the control level produced the lowest number of spikelets per spike. Fe, Zn and Fe+Zn foliar sprays significantly increased the spikelet number per spike over the control. Combined Fe and Zn foliar application caused the highest number of spikelets in each spike. The genotype G4 formed the lowest number of spikelets per spike, and the highest spikelets belonged to the genotype G1. The genotypes G2 and G3 were intermediate. Seadh et al. (2009) found that Zn treatment produced the highest number of spikelets per spike. Khan et al. (2008) also stated that zinc sulfate application increased the number of spikelets per spike in wheat.

Number of grains per spike
Control treatment had the least number of kernels per spike (Table 4). Foliar application of Fe, Zn and Fe+Zn significantly increased the number of kernels per spike over the control. Fe+Zn treatment, which had a significant difference with Fe and Zn sprays, produced the greatest number of kernels per spike. The genotype G4 produced the lowest number of kernels per spike, but genotypes G2 and G1 which had greater spike length and spikelet number per spike significantly improved the number of kernels per spike. An increase in the number of kernels per spike (Hemantaranjan and Grey, 1988;Yilmaz et al. 1997;Malakoti and Hasanpor, 2003) as a result of Zn application has been reported. Seadh et al. (2009) found that Zn application produced the highest number of kernels per spike in wheat.

Number of grains per spikelet
The results of the analysis of variance (Table 5) and mean comparisons (Table  6) are presented. The results showed that the control had the least number of kernels per spikelet and the application of Fe alone and especially in combination with Zn significantly amplified the kernel number. Genotypes G1 and G2 had significantly more kernels than genotypes G3 and G4. A significant increase in the number of kernels per spikelet was reported by Ziaeian and Malakouti (2001).

The weight of 1000 grains
The results showed that the genotype G2 had the lowest 1000-grain weight (32.6) and the other genotypes G1 (35.7), G3 (39.3) and G4 (43.1) significantly enhanced 1000-grain weight ( Table 6). The genotype G4 had fewer kernels per spikelet and spike than the rest of the genotypes (Table 4), and as a result, fewer kernels absorbed more photosynthetic material and increased their weight. The high yield of genotypes G3 and G4 was associated with higher kernel weight of them. Hussain et al. (2005) have reported that a micronutrient spray increases the weight of 1000 grains at the tillering, boot and milk stages. In the present study, as the application of the micronutrients has increased the number of spikelets and the number of kernels in both spikelets and spikes, more kernels as a sink have been produced and photosynthetic materials have been allocated among more kernels and thus there was no significant difference among spray treatments. Grain yield per square meter Plants from control plots had the lowest grain yield. Spraying with Fe and Zn significantly improved grain yield compared to control. The highest grain yield was obtained from plants sprayed with Fe+Zn, which had a significant advantage over Fe and Zn foliar spray. Genotypes G1, G3 and G4 had significantly higher grain yield than the genotype G2 which had the lowest grain yield. Ziaeian and Malakouti (2001) reported that wheat grain and straw yields were increased by the foliar spray of Fe and Zn. Experiments on wheat in 25 locations in Iran with treatments including Zn, Fe, Mn and Cu revealed that Zn application significantly improved grain yield (about 15%). Seadh et al. (2009) found that among the micronutrient elements, the Zn treatment produced the highest grain yield. Hussain et al. (2005) reported that the micronutrient spray at tillering, boot and milk stages enhanced wheat grain yield by increasing plant height, number of kernels per spike and 1000-grain weight. Pahlavan-Rad and Pessarakli (2009) stated that the application of 80 kg zinc sulfate per hectare increased the number of kernels per spike and grain yield. Different varieties of wheat not only have different growth potentials, but may also be different in terms of the response to fertilizers Effect of micronutrient foliar application on morphology and yield of durum wheat genotypes 233 (Hemantaranjan and Gray, 1988). Khoshgoftarmanesh et al. (2005) have concluded that wheat cultivars respond differently to the application of zinc sulfate. Rengel and Graham (1995) also showed that Zn fertilizer increased wheat grain and straw yield, and the wheat cultivars showed different responses.

Fe grain concentration
Grain Fe concentration was affected by the interaction of spraying × cultivars ( Table 5). The G1 and G2 genotypes accumulated more Fe in comparison with the G3 and G4 genotypes when they were not sprayed with micronutrient fertilizers. The application of Fe and Zn increased the Fe concentration in genotypes G3 and G4 over control but decreased their concentration in genotype G2. A single spray of Fe and Zn in comparison with control reduced the Fe content of grains in the G1 genotype, but Fe combined with Zn increased it. Therefore, in new genotypes, contrary to cultivars, the spray of micronutrients increased the Fe concentration ( Figure 1). Findings of previous studies differ in Fe concentration. Abbas et al. (2009) found that the application of 8 kg Zn/ha increased the amount of Fe absorbed, while the higher levels had a decreasing effect. Khan et al. (2014) reported the highest concentrations of Fe at Fe treatment and Yassen et al. (2010) at the micronutrient combination treatment.

Zn grain concentration
In the G1 genotype, the application of Fe alone and in combination with Zn concentration significantly reduced grain Zn concentration compared to control. Also, Zn concentrations for Zn treatment and control were similar. The Zn and Fe application significantly reduced the Zn concentration of the genotype G2 in comparison with control, but the combined application of Zn and Fe caused a slight reduction in grain Zn concentration. Spraying of micronutrients, especially Zn alone or in combination with Fe, significantly increased grain Zn concentration of the genotype G3 in comparison to the control. Grain Zn concentration of the G4 genotype was the same among different levels of the foliar spray ( Figure 2). Wang et al. (2012) found that the Zn foliar application, not soil application, increased the amount of Zn in wheat grains. Ravi et al. (2008) concluded that the combined application of Zn and Fe increased grain Zn concentration. It has been stated that micronutrient concentration in wheat grains mainly depends on environmental factors and interactions between the genotypes and the environment (Morgounov et al., 2007;Nan et al., 2002). Biofortification strategies include the application of mineral nutrients and the development of genotypes that take up more Zn from the soil and collect it in edible organs (White and Broadley, 2011). There are genotypes with a higher concentration of nutrients (White and Broadley, 2005). In our study, foliar spraying treatments clearly increased Zn concentration of G3 over control. It is notable that G3 along with G1 and G4 had higher grain yield. Therefore, there was no relation between grain nutrient concentration and grain yield. A decrease in nutrient concentration at sprayed treatments might be due to the dilution effect since grain yield has been increased by foliar treatments at the tillering stage.