The results of combined analysis of variance of biennial data showed that the effect of micronutrient foliar application treatment was significant at 1% probability level for traits such as seed Zn content, plant dry weight, chlorophyll a and b, carotenoids, leaf greenness, grain yield, 1000-grain weight and harvest index were (Table 4). In this regard, the interaction of foliar application and year for seed Zn content, chlorophyll b, carotenoids and 1000-grain weight was significant at 1% probability level. Also, the simple effects of micronutrients foliar application on chlorophyll a, carotenoid content, plant dry weight, seed Zn content, grain yield and 1000-grain weight were significant (Table 4). The statistical results showed that the highest number of tillers per plant T4 (24), the highest plant height T4 (156.8 cm), the highest dry weight of plant T7 (44.4 gr / m2), the highest number of seeds per cluster T3, T4 (149) and the highest cluster lengths T3 (30.8 cm) were obtained when the plants received micronutrients through the leaves. On the other hand, the lowest index of grain yield and plant dry weight during two years of experiment was related to the control (Table 3).
Seed Zn content
The results of combined analysis of variance showed that foliar application of micronutrient fertilizer on the concentration of zinc in rice grain was significant at the level of 1% and also the interaction of year and foliar application was significant in this regard (Table 4). In this regard, the comparison of the biennial mean showed that the highest concentration (about three times the normal limit) was related to T6 (67.02 mg.kg− 1) and the lowest was related to the control (Fig. 1a). It seems that foliar application of zinc-containing micronutrient fertilizer at different vegetative and reproductive stages of rice has significantly increased the content of zinc in the grain in foliar application treatments compared to the control. In this regard, Jiang and his colleagues (Jiang et al., 2008) reported that this could be related to the connections between the xylem and phloem vessels in the wheat panicle and the exchange of elements between them. Also Ishimaru and his colleagues (Ishimaru et al., 2005) expressed that in the study of zinc transport within the rice plant tissue, it was reported that this element is stored in the plant after being absorbed through the stomata and transferred to the leaves. This causes the transfer of zinc from decaying leaves at the end of the growth period through the phloem to the seed and thus plays a role in increasing the amount of this element in the seed.
Leaf chlorophyll content
The content of chlorophyll a was significantly affected by the use of micronutrients at a probability level of 1% in foliar treatment compared to the control treatment based on statistical results (Table 4). The highest number of chlorophyll a was related to T4 with 0.56 and four times foliar application and the lowest was related to T2 with 0.29 with mg.g-1fwt unit (Fig. 1b). Considering the significance of the results of the mentioned trait, it is possible that the foliar spray of micronutrients that play a crucial role in the synthesis of chlorophylls such as zinc, iron and copper could have a significant effect on increasing the chlorophyll content of cell. In a similar study by Server and his colleagues (Sarwar et al., 2013), they reported phenological response of rice to different levels of micronutrients under calcareous soil conditions in all treatments was significant, especially in tillering stage except full flooding treatment increased in both years of research. The use of zinc alone or in combination with boron increased the chlorophyll content of rice.
As can be seen in Table 4, the chlorophyll b content was affected by the foliar application of micronutrients, so that the difference between the content of chlorophyll b and the control was significant at the level of 1% probability. The interaction effect of year and foliar application was also significant for this trait at 1% probability level. The comparison of mean data in two consecutive years showed that the highest number of chlorophyll b was related to T4 with 1.42 and the lowest was related to T6 with 0.88 with mg.g-1fwt unit (Fig. 1c). The simultaneous supply of essential micronutrients affecting the formation of chlorophyll pigments seems to increase the chlorophyll content of rice through foliar application during the tillering and stem formation stages, which increases light absorption and accelerates photosynthetic processes. In a similar study by Zayed and his colleagues (Zayed et al., 2011) expressed chlorophyll content (SPAD index) was significantly improved as a result of receiving micronutrients compared to the control when the rice plant received micronutrients and also the treatment of iron, zinc and manganese was found to have the highest value among the studied traits.
According to Table 4, the amount of carotenoids increased significantly (at a probability level of 1%) compared to the control when micronutrients were used, and the interaction of foliar application and year was also significant at the same level. The highest number of carotenoids was related to T4 with 0.429 and the lowest number was related to T2 with 0.253 with mg.g-1fwt unit based on the comparison of the mean of statistical data during two years of experiment (Fig. 1d). Due to the role of micronutrients used, the activation of enzymes responsible for the synthesis of proteins such as zinc along with copper, which is an essential element for the formation of pigments, may have played a role in the structure and activation of vital enzymes and facilitating intracellular reactions. According to the Gomez-Garcia and Choa-Alejo (2013) opinion, it seems that in line with the theories proposed in this field, increasing the amount of carotenoids in the studied foliar spraying treatments compared to the control is due to the determinative role of these elements. Pursuant to most accepted theories, carotenoids are synthesized by the three genes cl, c2, y, as well as several enzymes involved in the synthesis of carotenoids in chili peppers, although little is known about the molecular mechanism of this process.
Plant height, panicle length and plant dry weight
The statistical results indicate the influence of foliar application of micronutrient fertilizer on plant height was not significant (Table 4). In this relation, the interaction effect of foliar application and year was not significant, too. The results of comparing the mean data over two years show that the highest plant height was related to T4 (156.8 cm) with four sprays of micronutrient liquid fertilizer and the lowest was related to the control (113 cm) without foliar application (Fig. 1e). The increment of plant height in foliar treatments was probably due to a significant increase in stem length due to an increase in internode distance in result of enhanced production of plant growth hormones, which plays a critical role in zinc content that increased cell division and photosynthesis rate. Arif et, al (2012) reported that integrated application of zinc and boron has been the best fertilizer balance for further growth and yield response of rice and also increased plant height, number of tillers, panicle length, number of seeds per plant, number of fertile spikes, and dry weight.
Regarding the panicle length, which is directly related to the number of grains, the results of combined analysis of statistical data during two consecutive years of experiment showed that the effect of micronutrient fertilizer foliar application treatment on the length of panicle was not significant (Table 4). This was while the interaction effect of foliar application and year also had no significant influence. Also, according to the results of comparing the two-year average of the data, the highest cluster length was related to T3 (30.8 cm) and the lowest was related to the control (28.1 cm) (Fig. 1f). It seems that increasing the transfer of nutrients and minerals and increasing the productivity of these elements along with regulating plant hormone levels, enhancement of cell division due to increasing concentration of plant growth regulators within plant tissue has a significant influence on cluster length in micronutrient foliar application treatments. Also in a similar study by Zayed et. al (2013) reported that plant dry weight, leaf area index, chlorophyll content along with plant height and cluster length showed a significant increase compared to the control after receiving micronutrients.
According to the outlets, there was a significant difference (at the level of 1% probability) between plant dry weight of foliar and control treatments as a result of combined analysis (Table 4). The highest plant dry weight was related to T5 and the lowest amount was related to the control (Fig. 1g). Obviously, increasing plant dry weight is directly related to increasing plant height, increment of the number of tillers per plant and also increasing the volume of the root system, which can be caused by improving the growth rate of rice plant as a result of receiving micronutrients through leaf that affect the plant metabolism. In a search in the present study, Hossein Abadi et al. (2006) also reported that the results showed that micronutrients can only partially increase plant dry weight by improving growth conditions and affecting wheat yield.
The difference in leaf color diagram number was significant according to the results of combined analysis of variance at the level of 1% probability (Table 4). But the interaction of foliar application and year was not significant for this trait. Based on the results of comparing the average data during two years, T4 (number 3.84) had the highest number of leaf color diagram for Shiroodi cultivar and the lowest value was related to the control (number 2.5) (Fig. 1h). The facilitating processes related to nitrogen metabolism, improving the rate of decomposition and synthesis, as well as accelerating nitrogen reduction reactions are some of the factors that have contributed to the significance of leaf color diagram in foliar spraying treatments compared to the control. The results indicate an increase in the productivity of basal nitrogen fertilizers that used in the investigation. In this regard, Maralian et al. (Maralian et al., 2008) also reported that micronutrient foliar application had a significant effect on traits such as LCC, number of tillers and number of fertile tillers at a probability level of 1 percent.
Grain yield and its yield components
Total grain yield was significantly lower (at the level of 1% probability) compared to micronutrient treatments when nutrients were not used (Table 4). This trend was similar to the statistical results on traits such as number of tillers and number of panicles per plant. However, there was no significant difference between grain yield of micronutrient treatments. The comparison of the biennial average of the data showed that the highest grain yield was related to T4 (4257 kg / ha) with four sprays and the lowest was related to the control (3499 kg / ha) (Fig. 1i). In connection with the effective factors in increasing the grain yield of rice plants, in which the foliar application of micronutrients may have played a direct role, it can be facilitated by the inoculation process, which is manganese roles, grain formation and maturation which boron it is effective, activation of enzymes responsible for protein synthesis which is zinc role. Furthermore, facilitate the reduction of nitrate and sulfate processes, which are the role of iron, and to accelerate the metabolism of carbohydrates in which copper is effective. In this regard, Mahendra et al. (2017) stated that the application of zinc sulfate significantly affected the number of tillers, plant height, number of seeds per panicle, 1000-seed weight, harvest index and biological yield. There has also been a significant increase in the availability of primary and secondary macronutrients.
The 1000-grain weight was also affected by zinc, iron, manganese, boron and copper and a significant difference was observed between foliar and control treatments at the level of 1% probability (Table 4). Also, the interaction effect of foliar application and year showed a significant difference. In this regard, comparison of data averages over the two years showed that the highest 1000-grain weight was related to T4 with (29.8 gr) and the lowest was related to control (25 gr) (Fig. 1j). It is likely that increasing the productivity of macronutrients in which iron, boron and manganese directly affect, and increasing the fertility of clusters, have played the largest role in enhancement the 1000-grain weight and significance of micronutrient treatments. In this regard, Hosseinzadeh et al. (2012) also reported that in the study of micronutrient foliar application time, it was shown that foliar application time had a positive and significant effect on the number of filled grains per panicle, 1000-grain weight and maximum grain yield. The foliar application 40 days after transplanting had the greatest effect. In a investigation to study the effect of zinc on maize Sadeghi et al. (2017) reported that the comparison of the mean of treatments showed that with increasing the amount of element application, 1000-grain weight, the number of seeds per panicle, the amount of available Zn in corn grain, grain protein percentage and grain yield increased.
In this experiment, the effect of different times of micronutrients foliar application on the rice harvest index was significant at the level of 1% probability (Table 4). The highest harvest index was related to T4 (42%) which was not significantly different from other foliar spraying treatments. The lowest harvest index was related to the control (34%) (Fig. 1k). By affecting the yield-related indices and yield components of rice, it is obvious that the harvest index, which is directly related to grain yield, has also been affected by micronutrient spraying. Therefore, it is possible that increasing grain yield as well as 1000-grain weight had a direct influence on the significance of the harvest index. In this regard and in a similar study, Ghasemi et al. (2013) reported that the highest harvest index and maximum 1000-grain weight were obtained under the interaction of three factors for treatment with zinc, iron and manganese sulfate fertilizers.