Iodine biofortification of wheat, rice and maize through ...

Iodine biofortification of wheat, rice and maize through fertilizer strategy

I. Cakmak, C. Prom-u-thai, L. R. G. Guilherme, A. Rashid, K. H. Hora, A. Yazici, E. Savasli, M. Kalayci, Y. Tutus, P. Phuphong, et al. Plant and Soil An International Journal on Plant-Soil Relationships ISSN 0032-079X Plant Soil DOI 10.1007/s11104-017-3295-9

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Author's personal copy Plant Soil DOI 10.1007/s11104-017-3295-9

REGULAR ARTICLE

Iodine biofortification of wheat, rice and maize through fertilizer strategy I. Cakmak & C. Prom-u-thai & L. R. G. Guilherme & A. Rashid & K. H. Hora & A. Yazici & E. Savasli & M. Kalayci & Y. Tutus & P. Phuphong & M. Rizwan & F. A. D. Martins & G. S. Dinali & L. Ozturk

Received: 5 April 2017 / Accepted: 22 May 2017 # Springer International Publishing Switzerland 2017

Abstract Aim Iodine (I) deficiency is distinct from other micronutrient deficiencies in human populations in having a high endemic prevalence both in well-developed and in developing countries. The very low concentration of iodine in agricultural soils and cereal-based foods is widely believed to be the main reason of iodine deficiency in humans, especially in developing countries. In the present study, the possibility of using iodine containing fertilizers for agronomic biofortification of cereal grains with iodine was studied. The aim was to establish the best application method (to the soil or as foliar spray), the best form of iodine (potassium iodate or potassium iodide) and the optimal dose of iodine. Additionally, experiments were conducted to study

transport of iodine in plants and localization of iodine within the grains. Materials and methods Experiments were conducted both under greenhouse conditions and in the field on wheat (Triticum aestivum) grown in Turkey and Pakistan, on rice (Oryza sativa) grown in Brazil, Thailand and Turkey and on maize (Zea mays) grown in Turkey. The iodine concentration in the grain, localization of iodine in different grain fractions of wheat (i.e., endosperm, bran and embryo) and iodine concentration of both brown rice and polished rice was analyzed. In short-term experiments, the translocation of iodine from older into younger leaves was also studied. Inductively coupled plasma mass spectrometry (ICP-MS) was used for analysis of iodine in plant and soil samples.

Responsible Editor: Philip John White. I. Cakmak (*) : A. Yazici : Y. Tutus : L. Ozturk Faculty of Engineering and Natural Sciences, Sabanci University, 34956 Istanbul, Turkey e-mail: [email protected] C. Prom-u-thai : P. Phuphong Agronomy Division, Department of Plant and Soil Sciences, Faculty of Agriculture, Chiang Mai University, Chiang Mai 50200, Thailand L. R. G. Guilherme : G. S. Dinali Department of Soil Science, Federal University of Lavras, 3037, Campus UFLA, Lavras, CP 37200-000, Brazil A. Rashid Pakistan Academy of Sciences, Islamabad, Pakistan

K. H. Hora SQM EUROPE N.V, Houtdok-Noordkaai 25a, B-2030 Antwerpen, Belgium E. Savasli : M. Kalayci Transitional Zone Agricultural Research Institute, 26002 Eskisehir, Turkey M. Rizwan Soil Science Division, Nuclear Institute for Agriculture and Biology, Faisalabad, Pakistan F. A. D. Martins State of Minas Gerais Agricultural Research Corporation, Epamig. Fazenda Experimental de Sertãozinho, Zona Rural, CP 135, Patos de Minas 38700-970, Brazil

Author's personal copy Plant Soil

Results In greenhouse experiments on wheat, soilapplied potassium iodide (KI) and potassium iodate (KIO3) at increasing rates (i.e., 0, 0.1, 0.25, 1, 2.5, 5, 10 and 20 mg I kg−1 soil) both iodine forms substantially increased iodine concentration in the shoot, with the highest shoot iodine resulting from the KI treatments. However, these soil treatments did not affect iodine concentrations in the wheat grain, with the exception of the highest iodine rates (i. e., 10 and 20 mg I kg−1 soil) which also depressed the grain yield. In contrast to the soil applications, foliar spray of KI and KIO3 at increasing rates during heading and early milk stages did enhance grain iodine concentrations up to 5- to 10fold without affecting grain yield. Including KNO3 or a surfactant to the iodine containing foliar spray further increased the grain iodine concentration. In a short-term experiment using young wheat plants, it was found that iodine is translocated from older into younger leaves after immersion of the older leaves in solutions containing KI or KIO3. Adding KNO3 or a surfactant in the immersion solution also promoted leaf absorption and translocation of iodine into younger leaves. Field experiments conducted in different countries confirmed that foliar application with increasing rates of iodine significantly increased grain iodine concentrations in wheat, brown rice and maize. This increase was also found in the iodine concentration of the endosperm part of wheat grains and in polished rice. Conclusions The results of the present study clearly show that foliar application of iodine containing fertilizers is highly effective in increasing grain iodine concentrations in wheat, rice and maize. Presented results suggest that iodine is translocated from shoot to grain by transport in the phloem. Spraying KIO3 up to the rate of 0.05% w/v is suggested as the optimal form and rate to be used in agronomic biofortification with iodine. The substantial increase in grain iodine concentrations could contribute to the prevention of iodine deficiency in human populations with low dietary iodine intake. The reasons behind the higher effectiveness of foliar-applications compared to the soil applications of iodine fertilizers in improving grain iodine concentration are discussed.

Keywords Agronomic biofortification . Iodine . Maize . Potassium iodate . Potassium iodide . Potassium nitrate . Rice . Surfactant . Wheat

Introduction Despite increasing access to sufficient food for all, and significant achievement in reducing global hunger, micronutrient deficiencies including zinc (Zn), iodine (I) and iron (Fe) deficiencies still represent a global public health problem, affecting around 2 billion people (Bailey et al. 2015). A range of serious health complications and chronic diseases can be attributed to an inadequate intake of micronutrients, leading to micronutrient deficiencies that may even cause death when not diagnosed and treated. In contrast to many other nutritional deficiencies, iodine deficiency is special in being highly prevalent both in developing and well developed countries (Pearce et al. 2013; Gonzali et al. 2017). Based on urinary iodine data collected from school-aged children it has been estimated that globally about 1/3 of these children has an insufficient intake of iodine (De Benoist et al. 2008; Andersson et al. 2012). Europe is identified as the region with the highest prevalence of iodine deficiency in human populations (Zimmermann and Andersson 2011). Iodine deficiency is associated with various health complications such as endemic goitre, intellectual and mental impairments, growth retardation, and increased pregnancy loss and infant mortality (Pearce et al. 2013; Lazarus 2015). Recent reports also highlight the importance of a high prevalence of mild iodine deficiency in humans, particularly among pregnant women, and there is growing evidence on close associations between mild maternal iodine deficiency and cognitive impairment in their children (Pearce et al. 2016). Although the implementation of programs and interventions based on iodized salt or iodine supplements has been successful in preventing severe iodine deficiency over the past decades, inadequate iodine intake is still a growing health concern today, with a widespread occurrence of global iodine deficiency in human populations (Andersson et al. 2012; Gonzali et al. 2017). This may be due to a number of factors, including constraints in availability of iodized salts for all households, instability of iodine during storage or cooking, lack of monitoring of iodine content in iodized salts, food manufacturers not using iodized salt in processed foods or the increasing attention to minimize the daily sodium intake. For example, about 60% of the human population living in Pakistan do not use iodized salt (Zia et al. 2015). Today, minimizing the extent of iodine deficiency in human populations is a growing challenge among


human nutritionists, plant scientists and agronomists. Several published reports are available indicating that the high prevalence of human iodine deficiency is often associated with the regions where soils contain very low amounts of iodine (Fuge and Johnson 1986). High consumption of staple foods such as cereal-based foods with very low iodine concentration is often proposed as a particular reason for the iodine deficiency in humans (Johnson 2003; Fuge and Johnson 2015). On a global average three major cereal crops, i.e., wheat, rice and maize, are responsible for up to 60% of the daily energy intake of human populations (Tilman et al. 2002). In the case of developing countries, contribution of cereals to the daily caloric intake is much greater than the world average, most likely exceeding 75%, especially in the rural areas (Hossain et al. 2008; Cakmak et al. 2010a). Grain crops grown for human nutrition and also livestock feed, commonly contain extremely low levels of iodine. For example, cereal grains collected from 38 locations in Austria (Shinonaga et al. 2001) and 86 locations in Pakistan (Zia et al. 2015) contained very low iodine concentrations, ranging from 2 to 30 μg kg−1. In Germany, various grain samples of barley and maize used for cow-feed contained, on average, ≤9 μg kg−1 and 13 μg kg−1 iodine, respectively (Schöne et al. 2017). The low concentration of iodine found in grains can be associated with the low iodine content in the soil. In most cases, agricultural soils are very low in iodine because the parental materials/rocks involved in soil formation are naturally low in iodine. The major source of iodine in soils is suggested to be iodine contained in oceans or seawaters from where iodine is volatilized into the atmosphere and transported onto soils and plants through rain or aerial deposition (Shetaya et al. 2012; Fuge and Johnson 2015). Several soil chemical factors are known to increase adsorption and fixation of the iodine existing in soils and thus limit the availability of iodine for root uptake (Johnson 2003; Fuge and Johnson 2015). Soil factors involved in depressing availability of iodine in soils for root uptake include high levels of organic matter, clay minerals and Al- and Fe-oxides. Iodine might be also leached from the soil profile in the form of iodide (I−) (Horel et al. 2014). Besides the low content of iodine in soils there are also plant-related factors which limit iodine accumulation in edible parts of food crops such as the seeds/ grains. A major part of the iodine absorbed by the roots

may remain in the roots and cannot be transported into shoot tissues (Hong et al. 2008). Very low iodine concentrations in grains compared to leaves led to the commonly assumed idea that iodine has a poor phloem mobility (Muramatsu et al. 1995; Mackowiak and Grossl 1999; Tsukada et al. 2008). By contrast, results presented in more recent publications indicate the possibility of transport of foliar applied iodine into roots from leaves of lettuce plants (Smolen et al. 2014) or from older leaves into new leaves in tomato plants (Landini et al. 2011). Iodine concentration of 10 μg iodine per kg cereal grains is far too low to meet the daily requirement of the human populations. Reported daily iodine requirement for adults ranges between 150 to 200 μg (Zimmermann 2007; Swanson and Pearce 2013). In a given typical developing country where daily per capita consumption of rice or wheat is about 400 g, and assuming an iodine concentration in grain of 10 μg per kg, the contribution of these major staple grains to daily iodine intake would be no more than 4 μg; less than 2,5% of the daily human iodine demand. Reduced dietary intake is widely believed to be the root cause of the iodine deficiency problem in the world (Fuge and Johnson 2015; Gonzali et al. 2017). Therefore, enrichment (biofortification) of cereals with iodine is nowadays an important nutritional research topic and humanitarian challenge. An agronomic biofortification approach, i.e. the application of iodine-containing fertilizers, as has been demonstrated for zinc and selenium, could be an effective tool in improving iodine concentration of cereals (Lyons and Cakmak 2012; Cakmak 2008; White and Broadley 2009; Welch et al. 2013). Published research indicates that iodine biofortification of food crops through application of iodine-enriched fertilizer works very well. However, these promising results were shown mainly in vegetable crops (Weng et al. 2008; Kiferle et al. 2013; Mao et al. 2014; Smolen et al. 2016; Lawson et al. 2015) and very little or no information is available for field-grown cereal crops. Major aim of the designed experiments in the present paper is to generate information about the effectiveness of soil- and foliar-applied iodine fertilizers on grain iodine concentrations of wheat, rice and maize grown under field conditions in Turkey, Thailand, Brazil and Pakistan. To gain insight on the uptake of iodine by cereals, additional experiments were conducted under greenhouse conditions to monitor shoot accumulation and grain deposition of


soil- and foliar-applied iodine. In a short-term experiment, translocation of iodine from older into younger leaves was also studied. Studying the distribution of iodine in endosperm, embryo and bran parts of wheat grains biofortified with iodine was an additional goal of the presented study. Finally, iodine concentration of brown and polished rice was investigated in fieldgrown rice plants with and without foliar spray of iodine.

Materials and methods Different greenhouse and field experiments were established to study the response of bread wheat (Triticum aestivum), rice (Oryza sativa) and maize (Zea mays) plants to the applications of iodine in form of potassium iodide (KI) or potassium iodate (KIO3). In greenhouse experiments, only wheat (Triticum aestivum, L. cv. Tahirova 2000) was used, while in case of the field experiments wheat, rice and maize were used as described below.

Greenhouse experiments Bread wheat plants were grown in 3 kg plastic pots filled with a calcareous soil (18% CaCO3) having an alkaline pH (pH 8.0 in distilled H2O) and low organic matter (1.5%). The experimental soil used had a clay loam texture and contained 3.2 mg of total iodine per kg soil, measured as described below. The following nutrients were added per kg airdry soil: 200 mg N in the form of Ca(NO3)2.4H2O, 25 mg S in the form of K2SO4, 100 mg P in the form of KH2PO4 and 2.0 mg Zn in the form of ZnSO4.4H2O. Based on long-term experience with this soil, the amount of these nutrients is sufficient for plant development, and no other minerals are required. Twelve seeds were sown per pot and after emergence the number of plants per pot was thinned to 8. During the booting stage, plants were topdressed with 200 mg N kg−1 soil in the form of Ca(NO3)2. The plants were grown until grain maturation. The pots were completely randomized in the greenhouse and irrigated with deionized water if required. Five replications (i.e., 5 independent pots) were used in the experiments.

Effects of soil-applied iodine on shoot and grain iodine concentration To investigate the effect of soil-applied iodine on shoot and grain accumulation of iodine, KI or KIO3 were applied into the soil at these increasing rates of iodine: 0, 0.1, 0.25, 1, 2.5, 5, 10 and 20 mg iodine kg−1 soil. In this experiment, plants were harvested at two dates; the first harvest was realized during the stem elongation stage when the plants were 39-days-old, and the 2nd harvest was made at grain maturation. At the first harvest, the shoot parts of the plants were harvested and subjected to determination of straw dry matter production and iodine concentration in the shoot. At the second harvest, the grain yield and iodine concentration in the grain was determined. Grain samples collected after hand-threshing were washed shortly (for about 15 s) under running deionized water to remove any dusts or soil particles from the surface of grains. Effects of foliar spray of iodine on grain iodine concentration Investigating the effect of foliar application of KI and KIO3 on grain iodine concentration was the next topic of the greenhouse experiments. The following rates of the KI were applied with foliar spray: 0, 0.005, 0.010, 0.025, 0.05, 0.10 and 0.25% w/v salt in spray solution. The resulting iodine concentration as was applied with KI was also applied with KIO3. The foliar treatments were applied until most of the leaves were wet with the spray solution but without run-off from leaves, and realized at the heading and early milk stages. During the spray operations, the soil surface of pots was covered in order to avoid soil contamination from the foliar iodine sprays. Plants were harvested at grain maturation. As described above, grain samples collected were washed thoroughly under running deionized water for about 15 s to remove any dust or soil particles from the surface of grains. In an additional experiment, KI and KIO3 containing foliar sprays were applied to plants either with or without KNO3 and/or a non-ionic surfactant (a tenside provided by SQM), to study the effect of these additives on grain iodine concentration. The KNO3 and the surfactant used were added in the spray solution at the rates of 1% (w/v) and 0.05% (v/v), respectively. The iodine compounds were sprayed to foliar at the rates of 0.05% (w/v) for KI and 0.065% (w/v) for KIO3 twice,


once at the heading and then at the early milk stages. Final spray solutions of the chosen KI and KIO3 treatments had the same iodine concentrations. The addition of KNO3 to the spray solution was tested in anticipation of future implementation of agronomic iodine biofortification through including iodine in existing foliar fertilizer spray programs. Plants were harvested at grain maturation, and the grain samples were prepared for iodine determination as described above.

Translocation of iodine from older into younger leaves in wheat In a short-term experiment young wheat plants were used to follow iodine translocation from older leaves into younger leaves. In this test, as shown in Fig. 1, 8 cm long tips of the first leaves were immersed twice daily into an iodine-containing solution for 10 s. and this was done on 4 consecutive days. Two days after the last leaf treatment, the youngest – untreated - leaves were harvested separately and analyzed for iodine. The treatment solution contained either KI or KIO3 at the rates of 0.25% (w/v) and 0.32% (w/v), respectively. Also in this experiment, KI and KIO3 were applied with or without 1% (w/v) KNO3 and 0.02% (v/v) of the non-ionic surfactant. Due to repeated immersion of the leaves, the concentration of the non-ionic surfactant in the treatment solution was reduced from 0.05% (v/v) to 0.02% (v/v) to avoid potential leaf damage.

Translocation of iodine from first leaves into young leaves Shoot part analyzed for iodine Iodine Treatments

Fig. 1 Treatment of the first leaf of wheat with the immersion solutions containing iodine with or without additives

Field experiments To study effectiveness of foliarly applied KI or KIO3 on grain iodine concentration in practice, field experiments were established on rice in Thailand (cv: Sanpatong-1), Brazil (cv: Relampago) and Turkey (cv. Osmancık 97), on wheat in Pakistan (cv: Faisalabad-2008) and Turkey (cv: Bezostaja) and on maize (cv: Kerbanis F1) in Turkey. In the experiments conducted in Thailand and Brazil, both KI and KIO3 were used, while in Pakistan and Turkey only KIO3 was applied. The field experiments in Brazil, Thailand and Pakistan were designed in randomized blocks with four replications. The applied spray solutions contained 0.05% (w/v) detergent powder BSurf^ in Pakistan, 0.005% (v/v) Toptech in Thailand and 0.5% (w/v) Assist in Brazil. The plot sizes were 3 m × 3 m in Thailand, 4 m × 2.5 m in Pakistan and 5 m × 2 m in Brazil. The total iodine concentration, pH, texture and total amount of organic matter of the soils used in the field experiments are presented in Table 1. The soil iodine concentrations, measured as described below, showed a large variation among the countries and ranged from 0.48 to 3.24 μg kg−1 soil. There was also considerable variation between the locations concerning the other soil parameters which were measured by using standard methods. In field studies conducted in Thailand and Brazil two forms of iodine (KI or KIO3) were applied to plants at 5 different rates for KI: 0, 0.010, 0.025, 0.05 and 0.10% KI w/v salt in spray solution. The same amounts of iodine applied as KI were also applied as KIO3 (i.e., 0, 0.013%, 0.032%, 0.065% and 0.129% KIO3 w/v salt in spray solution). The iodine salts were applied twice, first at the heading and then at the early milk stages. The leaf sprays were realized either very late in the afternoon or on cloudy days to avoid any possible damage on leaves that might be caused by high day temperature or high sunlight. The mentioned iodine rates were sprayed in 600 to 800 l water per ha depending on plant size to achieve optimal leaf coverage. At harvest, grain yield was determined and the grains collected were used for iodine analysis as described below. After threshing wheat grains by hand, wheat grains were washed under running deionized water for about 15 s to remove any dust or soil particles. In case of rice, both the hulled seed samples from field and then the de-hulled seeds (brown rice) were washed. Thereafter, the washed grains were dried at 40 °C and milled using an agate cup mill prior to analysis.

Author's personal copy Plant Soil Table 1 Total iodine concentration, soil pH, texture and total amount of organic matter of soils used in the field experiments Country

Crop tested

Total soil iodine (μg kg−1)

Soil pH

Soil Texture

Organic matter (%)

Thailand

Rice

0.48

5.5

Sandy loam

1.5

Brazil

Rice

2.81

5.3

Clay

3.4

Pakistan

Wheat

1.5

8.1

Loam

0.7

Turkey-I*

Wheat

2.11

7.8

Clay

1.5

Turkey-II

Wheat + Maize

1.56

8.0

Clay

1.2

Turkey-III

Rice

3.24

6.5

Sandy clay loam

0.6

Turkey-IV

Wheat

2.14

7.8

Clay loam

1.1

*First location in Turkey was used for the wheat experiment conducted in the Eskisehir site and its results are presented in Table 10; the 2nd location used for the experiments conducted on wheat and maize in the Eskisehir-Toprak Su site (Fig. 2), the 3rd location was used for the rice experiments conducted in the Edirne province (Fig. 2) and the 4th location was in Sakarya province and used for additional wheat experiment (Table 10).

In Turkey, the trials were conducted in 4 locations on large plots (about 150 m2). Wheat, rice and maize were agronomically biofortified with iodine by foliar spray of 0.05% (w/v) KIO3 including 0.05% (v/v) of the nonionic surfactant to improve shoot coverage of the replace by sprayed solutions. The water volume used per ha varied between 600 to 800 l depending on plant size in the plots. Foliar spray of KIO3 to wheat, rice and maize was realized three times as following: once during the heading stage for rice and wheat and silk stage for maize and then 2 times during the milk and early dough stages for all 3 crops. In this particular experiment with large plots, the number of replications was 4 for wheat, 3 for maize and 3 for rice. The harvesting of the grains and preparation of grains for iodine analysis were conducted as described above. De-husking and polishing of rice from these experiments were realized by using a Zaccaria laboratory mill (PAZ/1-DTA, Brazil).

Iodine localization in wheat seeds Localization and concentrations of iodine in endosperm, embryo and bran parts of wheat seeds were investigated on selected seed samples differing in iodine concentrations (from 18 to 1706 μg kg−1 seed). Fractionation of grain parts was conducted as described by Cakmak et al. (2010b). First, whole embryos of seeds were isolated by using a surgical blade. Then, de-embryonated seeds were milled by a vibrating agate cup mill for 20 s at 700 rpm, and the resulting flour was sieved with a 100 μm mesh sieve, and the sieved part was considered as endosperm part. The part remaining on the sieve was

sieved again with a 1000 μm mesh plastic sieve in order to separate the bran part.

Iodine measurement Plant samples Tetramethylammonium hydroxide (TMAH) is widely used in extraction of iodine from a variety of plant based materials including wheat flour. A large range of TMAH concentration (i.e. 5% to 25%), duration of time (i.e. 1 h to 12 h) and temperature (i.e. 60 °C to 200 °C) are used in extraction protocols for analysis of iodine in different samples (Mello et al. 2013). After performing a few optimization tests by using different reference materials with known iodine concentrations, we found that a microwave extraction by using 1.25% TMAH (v/v) at 90 °C for 1 h is useful for measurement of iodine, particularly for starch-rich grain samples. This optimized method minimized physical and chemical interferences (i.e. matrix effect) in particular for grain samples, increased lab efficiency by reducing time and energy consumption and brought a safer occupational environment with reduced TMAH consumption. It also allowed us to detect very low iodine concentrations in grain samples (i.e. below 10 μg kg−1 seed). Thus, 250 mg (±10 mg) dried and ground leaf or grain samples were extracted in 20 ml of 1.25% TMAH at 90 °C for 1 h using a closed-vessel microwave reaction system (MarsExpress, CEM Corp., USA). Following the extraction step, the samples were cooled down to room temperature, centrifuged at 5000 g for 20 min and the


supernatant was diluted 1:1 by volume with ultra-pure water (18.2 MΩ. cm at 25 °C) prior to iodine analysis. Soil samples For extraction of iodine from soil samples the extraction protocol of Zia et al. (2015) was followed with some modification. Soil samples were initially dried at 35 °C until a constant weight and sieved through 2 mm nylon mesh. The sieved samples were then ground with agate mortar and pestle to pass through 125 μm nylon mesh. 250 mg of soil sample was then added with 5 mL of 5% TMAH (v/v) and incubated at 70 °C for 3 h in a shaking water bath. Following extraction, all samples were added with 5 mL of ultra-pure water (18.2 MΩ.cm at 25 °C) and centrifuged at 5000 g for 20 min. The resulting supernatant was diluted 1:5 by volume with ultra-pure water (18.2 MΩ) prior to iodine analysis. Iodine analysis of all plant and soil samples was performed by inductively coupled plasma mass spectrometry (7700 Series, Agilent Technologies, USA).

Statistical analysis Statistical analysis was performed using JMP statistical software (JMP, SAS Institute, Cary, NC). Significant differences were assessed with ANOVA, using Generalized Linear Model (GLM), followed by student’s t test to compare means wherever ANOVA indicated significant effects. Means were considered significantly different from one another at α = 0.05.

Results Greenhouse experiments In the first experiment, the effects of increasing soil application of KI and KIO3 on shoot growth, grain yield and iodine concentrations of plants were studied. Generally, the plants grew better when KIO3 was added in the soil compared to the KI treatment (Table 2). Shoot dry matter production tended to increase with increasing rates of KI or KIO3 up to iodine rate of 0.25 mg kg−1 soil, and then reached a plateau. However, at the highest iodine rate (i.e., 20 mg kg−1) shoot dry matter decreased, especially in case of the KI treatment (Table 2). The detrimental effect of KI on shoot growth at the highest

rate was statistically significant (Table 2). Visible phytotoxic effects of the highest iodine rate were observed to develop on older leaves as leaf chlorosis and necrosis mainly on the leaf tips. Grain yield was not affected by the treatments up to the rate of 5 mg I kg−1. Depressed grain yield was found at the rates higher than 5 mg kg−1, more pronounced in the KI-treated soils. The interaction of the effects of iodine dose and form on grain yield was statistically significant because the yield reducing effect of the highest rates was statistically significant only in the case of the KI treatment, and not for the treatments with KIO3 (Table 2). Iodine concentrations of the shoots were not affected by the soil-applied KI and KIO3 until the rate of 2.5 mg I kg−1 soil. At higher rates the shoot iodine concentrations showed substantial enhancement with the increasing application rates, particularly with the KI treatments (Table 3). At iodine application rates higher than 2.5 mg I kg−1, application of the same rate of iodine as KI increased shoot iodine concentration at least by 2fold compared to application as KIO3 (Table 3). The results of analysis of the grain iodine concentrations of plants that received increasing doses of iodine in the soil are interesting (Table 3). The iodine rates applied up to 5 mg kg−1 did not affect grain iodine concentrations compared to the control. However, grain iodine concentrations were increased at the higher levels of 10 and 20 mg I kg−1, with the highest value found at the 20 mg I kg−1 application. These were the same rates at which grain yield was depressed (Table 2). Higher grain iodine concentrations at the last 2 iodine rates are most probably resulting from the Bconcentration effects^ due to lower yields. The iodine form did not have a statistically significantly different effect on grain iodine concentrations, and no statistically significant interactions between I form and application rate were observed. In the second experiment, the effect of foliar applied KI or KIO3 on grain iodine concentration was studied at different application rates as shown in Table 4. The grain yield of the experimental plants was not affected by foliar spray of either iodine form up to and including the rates of 0.05% w/v KI and 0.065% w/v KIO3. However, at the highest 2 rates of KI (i.e., 0.1 and 0.25%, w/v KI) grain yield was significantly lower compared to the other rates and the control. For KIO3 this reduction in yield was less pronounced. In contrast to the soil applications, foliar iodine applications markedly affected

Author's personal copy Plant Soil Table 2 Effect of increasing rates of soil iodine applications in the form of KI and KIO3 on shoot dry matter production at early stem elongation stage (i.e. 36-days-old plants) and grain yield at full

maturity of bread wheat plants grown under greenhouse conditions. Means sharing the same letter are not significantly different from one another at α = 0.05 level, based on Student’s t-method

Shoot dry matter

Grain yield

Iodine application

KI

(mg kg−1)

(g plant−1)

KIO3

0

0.93 c

0.1

1.08 abc

1.00 abc

1.04 AB

1.57 abc

1.56 abc

1.57 A

0.25

1.11 ab

1.17 a

1.14 A

1.53 abcd

1.51 abcd

1.52 A

1

1.10 abc

1.13 ab

1.12 AB

1.63 ab

1.49 abcde

1.56 A

2.5

1.10 abc

1.00 bc

1.05 AB

1.72 a

1.40 bcde

1.58 A

5

1.07 abc

1.05 abc

1.06 AB

1.57 abc

1.37 cde

1.47 A

10

1.02 abc

1.13 ab

1.08 AB

1.07 f

1.26 ef

1.16 B

20

0.38 d

1.02 abc

0.70 C

0.54 g

1.27 def

0.95 C

Mean

0.97 B

1.07 A

1.02

1.43

1.44

1.43

Mean

KI

KIO3

Mean

1.66 a

1.63 A

(g plant−1) 1.08 abc

1.00 B

LSD0.05 (I Form): 0.06

1.60 abc

LSD0.05 (I Form): -

LSD0.05 (I Application): 0.12


LSD0.05 (Interaction): 0.17


CV (%): 13.1

CV (%): 13.9

grain iodine concentrations. There were progressive increases in grain iodine concentration by increasing the rates of iodine in the spray solution

(Table 4). The highest grain iodine concentrations were obtained with the highest rates of iodine in the foliar spray, especially with KI.

Table 3 Effect of increasing rates of soil iodine applications in the form of KI and KIO3 on shoot iodine concentration at early stem elongation stage (i.e. 39-days-old plants) and grain iodine concentration at full maturity of bread wheat plants grown under

greenhouse conditions. Means sharing the same letter are not significantly different from one another at α = 0.05 level, based on Student’s t-method

Shoot iodine Iodine application

KI

(mg kg−1)

(mg kg−1)

Grain iodine KIO3

Mean

KI

KIO3

Mean

(μg kg−1)

0

0.2 f

0.4 f

0.3 D

22

25

23 C

0.1

0.3 f

0.3 f

0.3 D

25

35

30 C

0.25

0.3 f

0.4 f

0.4 D

24

30

27 C

1

0.7 f

1.4 f

1.1 D

28

27

28 C

2.5

7.0 fe

6.2 fe

6.6 D

28

32

30 C

5

35.1 d

18.8 ed

27.0 C

38

29

34 C

10

115.6 b

62.0 c

88.8 B

60

52

56 B

20

313.0 a

105.0 b

209.0 A

106

124

115 A

Mean

59.0 A

24.3 B

41.7

40

45

42

LSD0.05 (I Form): 5.8

LSD0.05 (I Form): -


LSD0.05 (I Application): 14


LSD0.05 (Interaction): -

CV (%): 31.2

CV (%): 38

Author's personal copy Plant Soil Table 4 Effect of increasing rates of foliar iodine applications in the form of KI and KIO3 on grain yield and grain iodine concentration of bread wheat plants at full maturity grown under greenhouse conditions. Iodine applications were conducted two times at

the booting and early milk stages. Means sharing the same letter are not significantly different from one another at α = 0.05 level, based on Student’s t-method

Grain yield Iodine spray sate KI (or KIO3) (%)

KI

Grain iodine KIO3

Mean

(g plant−1)

KI

KIO3

Mean

(μg kg−1)

0

1.62

1.66

1.64 A

14 ef

25 ef

20 E

0.005 (0.006)

1.58

1.57

1.58 A

28 ef

50 ef

39 DE

0.010 (0.013)

1.60

1.48

1.54 A

51 ef

44 ef

47 DE

0.025 (0.032)

1.50

1.82

1.66 A

97 e

104 de

100 D

0.050 (0.065)

1.51

1.65

1.58 A

170 de

247 d

208 C

0.100 (0.129)

1.23

1.30

1.26 B

445 b

396 c

420 B

0.250 (0.320)

1.10

1.44

1.27 B

1358 a

1009 b

1183 A

Mean

1.45

1.56

1.51

309 A

268 B

289

LSD0.05 (I Form): -

LSD0.05 (I Form): 35




LSD0.05 (Interaction): 92

CV (%): 16.9

CV (%): 25

In a third experiment, changes in grain iodine concentrations were studied as affected by the addition of 1% (w/ v) KNO3 and/or the non-ionic surfactant (0.05% v/v) into the foliar spray solutions with 0.05% (w/v) KI and 0.065% (w/v) KIO3. This rate of iodine was chosen as it was considered optimal based on the results of the previous experiments. Foliar spray with KI or KIO3 alone significantly increased grain iodine concentrations (Table 5) comparable to the previous experiment (Table 4). KIO3 resulted in higher concentration of grain iodine compared to the KI treatment, even though the means could not be statistically separated at the α chosen for analyses. Adding 1% (w/v) KNO3 or the surfactant significantly increased grain iodine concentrations as compared to the iodine treatments without any additive. The highest grain iodine concentrations were found when KNO3 and the surfactant were simultaneously added to the spray solutions containing KI or KIO3 (Table 5). An additional short-term experiment was conducted to study phloem transport of iodine in young wheat plants by immersion of the first leaves in an iodine containing solution with or without 1% KNO3 and/or the surfactant (0.02%, v/v). The results presented in Table 6 clearly show that iodine applied either in the form of KI or KIO3 is absorbed and transported from older into the younger leaves, resulting in a higher

concentration of iodine in the young – untreated - leaves compared to the control. Compared to the KIO3 treatment, the KI treatment resulted in a higher concentration of iodine in the young leaf at the same rate of iodine in the immersion solution (Table 6). Adding either KNO3 or the surfactant in the iodine containing immersion solution resulted in further enhanced absorption and translocation of iodine from older leaves. However, a combined addition of both KNO3 and surfactant in the immersion solution prepared with either KI or KIO3, did not cause any additive effect on iodine translocation from older leaves compared to the single additions (Table 6). Field experiments In the field experiments, rice and maize were included as well as wheat, to study the effect of the foliar spray of KI or KIO3 on grain iodine concentration in different cereal crops. Iodine concentrations of the brown rice from Thailand and Brazil (Table 7) and of wheat grain in Pakistan (Table 8) grown on the control plots without application of foliar iodine spray were very low: below 10 μg kg−1. A significant increase in grain iodine concentration of brown rice grown in Thailand and Brazil was found at increasing rates of both iodine forms in the foliar spray solution, applied twice (Table 7). In Thailand, the KI treatments caused

Author's personal copy Plant Soil Table 5 Changes in the grain iodine concentrations depending on different combined foliar application of KI or KIO3 together with a non-ionic surfactant (at the rate of 0,05% v/v), and 1% (w/v) KNO3. The treatments were realized twice once during the heading stage and the other one early milk stage. Means sharing the same letter are not significantly different from one another at α = 0.05 level, based on Student’s t-method) Treatments

Grain iodine (μg kg−1)

Control (no I-fertilization)

20.7 e

KI

92.7 d

KI + Surfactant

154.6 bc

KI + KNO3

159.1 bc

KI + Surfactant + KNO3

201.2 b

KIO3

126.3 cd

KIO3+ Surfactant

185.9 b

KIO3+ KNO3

185.2 b

KIO3+ Surfactant + KNO3

296.0 a

LSD0.05

48.6

CV (%)

21.7

higher increases in grain iodine than the KIO3 treatment, while in Brazil no significant effect of iodine form was found, nor was an interaction found between iodine form Table 6 Translocation of iodine from the first leaves immersed into KI- or KIO3-containing solution into the younger leaves of 18-day-old bread wheat plants. Besides KI or KIO3, the treatment solution contained also 1% KNO3 or a non-ionic surfactant (at the rate of 0.02% v/v). The leaf treatment with the iodine solution started when plants were 13-days old and was repeated twice per day over 4 days. Means sharing the same letter are not significantly different from one another at α = 0.05 level, based on Student’s tmethod) Treatments

Iodine in younger leaves (μg kg−1)

Control (no I-fertilization)

162 e

KI

364 cd

KI + Surfactant

582 a

KI + KNO3

402 c

KI + Surfactant + KNO3

506 b

KIO3

226 e

KIO3 + Surfactant

328 d

KIO3 + KNO3

314 d

KIO3 + Surfactant + KNO3

312 d

LSD0.05

69.5

CV (%)

13.8

and rate on grain iodine concentrations. The experiments in Brazil were conducted using rice cultivar Relampago. In the same trial period, 2 other rice cultivars (i.e., Calcula and Carevera) were tested, and the results obtained with these rice cultivars were very similar to the results obtained with the cultivar Relampago (data not shown). In Pakistan, the dose-dependent increase was also observed on grain iodine concentration of wheat after foliar application of KIO3 at increasing rates (Table 8). The highest grain iodine concentration (1524 μg kg−1) was obtained at the highest rate of iodine in the spray solution (0.13%, w/v KIO3). Under given experimental conditions, the iodine rates tested had no statistically significant effect on grain yield in rice in Brazil and Thailand (Table 9), while, in the field experiment conducted in Pakistan on wheat, the lowest spray rate of the KIO3 treatment (i.e., 0.01%) did increase grain yield significantly (Table 8). There was, however, a progressive decrease in wheat grain yield when foliar iodine spray rate was increased from 0.01% to 0.1% KIO3. In an additional field experiment conducted in Turkey, wheat, rice and maize plants were grown on large plots and treated 3 times with 0.05% (w/v) KIO3. Foliar spray of iodine had no distinct effect on grain yield in 3 cereal crops tested, but resulted in substantial increases in grain iodine concentrations (Fig. 2). In the control plots, grain iodine concentrations were 4 μg kg−1 for maize, 11 μg kg−1 for rice and 13 μg kg−1 for wheat. Among the cereal species tested, wheat and maize showed highest and lowest response to foliar iodine spray respectively, while rice (brown rice) showed an intermediate response (Fig. 2). Iodine in grain fractions By using various grain samples with different iodine concentrations obtained from several trials, distribution of iodine was studied in grain fractions of wheat including endosperm, bran and embryo. In all samples with elevated iodine content of the grain, a much higher concentration of iodine was found in the bran and embryo parts compared to the endosperm parts (Table 10). Generally, the bran part of the grains had the highest iodine concentration compared to the other fractions. In biofortified samples containing 87 μg I kg−1 grain, the endosperm concentration of iodine was approximately half of the concentration found in the whole grain. In the other samples with a higher iodine concentration in the grain, the endosperm iodine concentration was around one third that of the whole grain (Table 10). The higher iodine concentration in the

Author's personal copy Plant Soil Table 7 Effect of increasing rates of foliar iodine spray in forms of KI and KIO3 on grain iodine concentration of rice grown under field conditions in Thailand and in Brazil. As indicated below, same amount of iodine has been applied with KI and KIO3

treatments. Means sharing the same letter are not significantly different from one another at α = 0.05 level, based on Student’s t method

Grain iodine Brazil Iodine spray sate KI (or KIO3)

KI

(%)

(μg kg−1)

Thailand KIO3

Mean

KI

KIO3

Mean

(μg kg−1)

0

7

5

6e

9e

5e

7C

0.010 (0.013)

37

39

38 d

52 de

23 de

38 C

0.025 (0.032)

78

97

87 c

128 de

50 de

89 C

0.050 (0.065)

148

178

163 b

317 b

155 cd

236 B

0.100 (0.129)

332

346

339 a

551 a

261 bc

406 A

Mean

120

133

127

211 A

99 B

155

LSD0.05 (I Form): -

LSD0.05 (I Form): 60




LSD0.05 (Interaction): 133

CV (%): 23

CV (%): 61

endosperm compared to the whole grain found grains with iodine concentration of 18 μg kg−1 may be a consequence of iodine contamination from other grain fractions during the isolation of the endosperm part. The rice grains produced in Turkey from the experiment presented in Fig. 2 were used to analyze iodine concentration in the polished rice. Polishing rice from the control plots reduced iodine concentration from 11 μg kg−1 to 6 μg kg−1 (Fig. 3). A similar reduction in iodine was found in rice treated with iodine containing foliar spray. In this agronomic biofortified rice, Table 8 Effect of increasing rates of foliar iodine spray in the form of KIO3 on grain iodine concentration and grain yield of wheat grown under field conditions in Pakistan. Means sharing the same letter are not significantly different from one another at α = 0.05 level, based on Student’s t method Iodine rate

Grain iodine

Grain yield

(%)

(μg kg−1)

(ton ha−1)

0

8c

5.19 bc

0.01

76 c

5.74 a

0.025

202 c

5.42 ab

0.05

485 b

5.04 c

0.1

1524 a

4.55 d

LSD (0.05)

257

0.36

CV (%)

14.5

4.44

polished rice contained approximately half of the iodine found in brown rice (Fig. 3).

Discussion In the present study, soil or foliar application of KI or KIO3 at different rates on wheat, maize or rice did not show an increase in plant biomass or grain yield with the exception of the wheat experiment conducted in Pakistan. The lowest rate of the KIO3 spray to wheat (i.e., 0.01%) in Pakistan resulted in a significantly higher grain yield, in contrast to the results in Turkey, Thailand and Brazil. Iodine is not considered to be an essential element for higher plants (Marschner 2012). However, depending on the soil and growth conditions, iodine might be beneficial to plant growth. Positive effects of iodine applications on growth and development of various crops have been found after applications of relatively low doses of iodine (Medrano-Macias et al. 2016; Gonzali et al. 2017). Applied at very high rates (≥ 10 mg iodine per kg soil or applied foliar as ≥0.1% w/v KI or 0.125% w/v KIO3) iodine was depressive on growth of plants, especially when applied in the form of KI (Tables 2 and 4). This is comparable to previously published reports on adverse effects of iodine. For example, in pot trials using butterhead lettuce, soil applied iodine at concentrations more

Author's personal copy Plant Soil Table 9 Effect of increasing rates of foliar iodine spray in forms of KI and KIO3 on grain yield of rice grown under field conditions in Thailand and in Brazil. Means sharing the same letter are not

significantly different from one another at α = 0.05 level, based on Student’s t method

Grain yield Brazil Iodine rate

KI

(%)

(ton ha−1)

0

2.24

Thailand KIO3

Mean

KI

KIO3

Mean

2.09

2.16

4.34

4.24

4.29

0.01

2.30

1.96

2.13

4.43

4.03

4.23

0.025

1.85

1.98

1.91

4.82

4.10

4.46

0.05

1.97

2.24

2.10

5.20

3.80

4.50

0.1

2.84

2.28

2.56

3.89

4.10

3.99

Mean

2.24

2.11

2.17

4.53

4.05

4.29

LSD (0.05) (I Form)

n.s.

n.s.

LSD (0.05) (I Application)

n.s.

n.s.

LSD (0.05) (Interaction)

n.s.

n.s.

CV (%)

than 10 mg (L substrate)−1 caused negative effects on yield (Lawson et al. 2015). Higher sensitivity of plants to higher doses of soil-applied KI compared to soilapplied KIO3 was also found in rice (Kato et al. 2013), lettuce (Lawson et al. 2015) and strawberry (Li et al. 2017). The adverse effects of high iodine rates were more evident on grain yield than the vegetative growth (Table 2), indicating that development of generative organs may be more prone to suffer from excessive iodine applications than the vegetative growth. Additional experiments are needed for better understanding of the differential adverse effects of excessive iodine on generative and vegetative organs.

The iodide form (I−) of iodine is known to be more soluble in soils and better absorbable by the roots when compared to the iodate form (IO3−) (Hu et al. 2009; Kato et al. 2013; Medrano-Macias et al. 2016). The greater uptake of iodide compared to iodate was confirmed by the results given in Table 3, showing that at the soil applied iodine rates of KI and KIO3 which did not affect shoot dry matter (i.e., 5 and 10 mg kg−1), the iodine accumulation in the shoot was almost double when KI was applied compared with the KIO3 treatment. It is very likely that KI may show greater risk for toxicity to plants than KIO3, if applied at rates as shown in the present study. Based on these observations from the

18

Grain iodine (µg kg-1)

16

Y ie ld (t o n h a - 1 )

14 12 10 8 6 4 2

1800 1600 1400 1200 1000 800 600 400 200 15 0

0 Rice

Wheat

Maize

Rice

Wheat

Maize

Fig. 2 Grain yield and grain iodine concentration of rice, wheat and maize grown under field conditions on large plots (i.e., 150 m2). Bars show the standard deviation of the means for rice (n = 2), wheat (n = 4) and maize (n = 3)

Author's personal copy Plant Soil Table 10 Iodine concentration in various factions of seeds differing in iodine concentrations. The individual seed groups used in this study were obtained from field or greenhouse experiments.

Each value is the mean of 2 separate measurements (± standard deviation) per grain sample

Iodine concentration in seed fractions Production location

Cultivar

Grain iodine

Endosperm

(μg kg−1)

(μg kg−1)

Bran

Embryo

Sakarya-Field

Katea-1

18

23

±

0

48

±

1

27

±

2

Greenhouse

Tahirova 2000

87

44

±

2

381

±

9

143

±

27

Greenhouse

Tahirova 2000

184

69

±

1

607

±

16

283

±

0

Sakarya-Field

Katea-1

258

72

±

1

1359

±

126

533

±

35

Greenhouse

Tahirova 2000

284

103

±

5

791

±

26

360

±

48

Eskisehir-Field

Renan

1706

571

±

36

3190

±

55

3884

±

1011

present study and published results in literature it can be suggested that KIO3 is the preferred form of iodine for agronomic biofortification with iodine. The exact uptake mechanisms of iodide and iodate by the roots have not been clarified. It is known that plant roots exhibit iodate (IO3−) reduction capacity to iodide (I−), and the magnitude of this reducing activity differs markedly among the plant species (Kato et al. 2013). A future experiment studying capacity of wheat roots for iodate reduction activity may contribute better understanding of higher iodine accumulation in shoot of wheat from soil applied KI compared to KIO3. There was no distinct change in grain iodine concentrations among the soil-applied iodine treatments, at the 600

Iodine (mg kg-1)

500 400 300 200 10 0 Control

Foliar iodine treatment

Fig. 3 Iodine concentration in brown and polished rice. Grain samples used were from a rice production field in the Edirne locations where rice was biofortified with KIO3 on large plots with 200 m2 in 2 replicates. Bars show the standard deviation of the means of 2 replicates

rates below the highest 2 doses at which grain yield was depressed (Table 3). Similarly, the variation in the low soil iodine concentrations found in the locations of the field experiments (Table 1) was not related to the grain concentrations of the control (no I-treated) plants at the different locations (Fig. 2 and Tables 7, 8, 9). Grain iodine concentrations were significantly increased at the highest iodine application rates (i.e., 10 or 20 mg kg−1) (Table 3). At least part of the increases in grain iodine concentration at higher iodine rates might be associated with lower grain yields and thus Bconcentration effects^. Increase in nutrient concentrations as consequence of inhibited growth (i.e., Bconcentration effect^) is known under growthlimiting circumstances (Marschner 2012). However, there are net increases in grain iodine concentrations beyond these changes in grain yield. Since the grain yield is less affected by the KIO3 treatments (Table 2), a comparison can be made between shoot and grain concentrations of iodine for the plants treated with KIO3: The grain iodine concentration increased ~5 fold from 25 to 124 μg kg−1 grain (Table 3) by increasing application rates of iodine while shoot iodine concentrations of the same plants increased 262-fold from 400 to 105,000 μg kg−1 shoot dry weight during the stem elongation stage (Table 2). The huge difference in gradient between shoot and grain iodine may, at first thought, suggest that plants are not able to remobilize (translocate) the iodine, that is absorbed from roots and deposited in the vegetative organs, into the grain tissue. However, at high doses of KIO3 applied in the soil, it is clear that iodine can be transported from the vegetative tissue in the grain. The decrease in grain yield was around 30% at the highest soil-application rates of


KIO3 (i.e., 10 or 20 mg kg−1), while the increases in grain iodine concentrations were about 2-fold and 5-fold at the 10 mg kg−1 or 20 mg kg−1 soil iodine applications, respectively. From these results it is obvious that iodine has to be transported through the phloem. Phloem mobility of iodine in wheat plants is further confirmed by both the short-term and long-term experiments where iodine was applied on the foliage. Foliar absorbed iodine in wheat has been found to be remobilized to the grain, and this is clearly dose-dependent. Grain iodine concentrations increased significantly with increasing dose of iodine applied to shoots of plants growing under greenhouse conditions or under field conditions, as shown for wheat, rice and maize (Tables 4, 7 and 8; Fig. 2), suggesting a critical role for the fertilizer strategy in agronomic biofortification of cereal grains with iodine at sufficiently high levels for human nutrition. A part of the increase in grain iodine at the higher rate of foliar iodine treatments in the greenhouse trials could be ascribed to reductions in grain yield (‘concentration effect’), particularly in the case of the KI treatment. However, in case of the foliar KIO3 spray, the grain yield was not affected up to the rate of 0.065% KIO3, while at this rate of KIO3, grain iodine concentration still was increased by about 10-fold compared to the non-treated plants (Table 4). Adding 1% KNO3 or a surfactant in the spray solution promoted leaf penetration and remobilization of iodine to the grain (Table 5). Similar results were also found in a short term experiment conducted on young wheat plants. Iodine was able to translocate from the first (old) leaf into younger leaves after immersion of the old leaves in an iodine containing solution, and this translocation was further promoted when 1% w/v KNO3 or a surfactant was added in the immersion solution (Table 6). These results support the suggestion that iodine exhibits a phloem mobility in plants, at least in the tested cereal species of the present study, and that this is more pronounced when iodine is foliar applied compared to soil applications. The mechanism behind the positive effect of KNO3 on leaf absorption of iodine is yet to be revealed. Very little is known about iodine uptake and transport in plant cells, especially in the case of absorption of iodine in the leaf. Nitrate and iodate are known to be chemically similar and they may, therefore, compete during uptake and transport in biological systems. De la Cuesta and Manley (2009) indicated that nitrate and iodate possibly share the same transporter protein which may cause a competitive inhibition of iodate uptake in marine phytoplankton species if nitrate is high in the medium. By contrast, as

shown in this paper, NO3 supplied in form of KNO3 stimulated iodine uptake in leaf tissues. The positive effect of KNO3 could be ascribed to better leaf coverage of the spray solution. In a recently published paper, Lawson et al. (2016) showed that leaf absorption of iodate in butterhead lettuce plants is improved by inclusion of CaCl2 in the spray solution; but not affected by inclusion of Ca(NO3)2. The positive effect of CaCl2 on leaf absorption of iodate was ascribed to its humectant effect. Additional studies are needed for better characterization and understanding of the positive role of KNO3 in leaf absorption of iodine. KNO3 is one of the commonly applied foliar fertilizers with positive effects on yield and quality (Southwick et al. 1996; Howard et al. 1998; Shen et al. 2016; Morgan et al. 2016) and mitigation of biotic and abiotic stress factors as well (Bhuiyan et al. 2007; Zheng et al. 2010; Gimeno et al. 2014). Adding iodine into foliar KNO3 solutions, as shown in this paper, and possibly also other commonly foliar applied fertilizers or other agrochemicals, can be considered as an effective tool for agronomic biofortification of cereal grains with iodine. In the previous studies, considering substantially higher soil-to-plant transfer factors or tissue concentrations of iodine in leaves compared to seeds, it is often suggested that iodine has a very low phloem mobility in the plants (Muramatsu et al. 1995; Johnson 2003; Tsukada et al. 2008). By contrast, the results of more recent publications indicate that iodine is transported in phloem after its foliar spray to leaves. For example in lettuce plants, foliar-sprayed iodine was able to translocate in the roots (Smolen et al. 2014). In tomato plants, radio-labeled iodine (125I) was able to translocate from a treated leaf to the remaining shoots and fruits at significant amounts (Landini et al. 2011). Accumulation of iodine to concentrations high enough to be effective for agronomic biofortification in tomato fruits (Landini et al. 2011; Kiferle et al. 2013) and pepper fruits (Li et al. 2017) with application of iodine in the growth medium may indicate a contribution of phloem transport of iodine to fruit iodine concentrations. This cannot be attributed to xylem-transport only, because fruits have much lower transpiration capacity and xylem transport rates compared to the leaves (Hocking et al. 2016). Phloem transport of iodine from leaves into wheat grain was also found by Hurtevent et al. (2013) by applying radio-labelled iodine (125I) to leaves at low dosages comparable to atmospheric deposition of iodine. Compared to other elements, such as cesium, chloride and selenium, iodine had lower phloem mobility in wheat, and was classified as a medium phloem mobile element (Hurtevent et al. 2013).


The field trials conducted with 3 major staple-food cereals - wheat, rice and maize – showed a clear, differential response of these cereals to foliar iodine spray (Fig. 2). Grain accumulation of iodine following foliar spray of iodine was highest in wheat, while rice and maize showed an intermediate and lowest response, respectively. The reason for such differential response to foliar iodine spray among those cereal species is not known. It may be related to the differences in their leaf morphology (e.g., structure and composition of epidermis and cuticle) affecting the magnitude of the leaf absorption of iodine. Differences in leaf structure and especially in structure and composition of cuticular waxes among the plant species have important effects on leaf penetration of the sprayed nutrients (Fernández and Eichert 2009). The large differences in grain yield per hectare among the studied cereal species might be associated with possible Bdilution^ (due to higher yield, e.g., in maize) and Bconcentration^ (due to lower yield, e.g., in wheat) effects on grain iodine concentrations (Fig. 2) which might be a further contributing factor to the observed differences in grain iodine accumulation among those cereals. In addition, differential iodine accumulation among these 3 cereal species might be also related to the extent to which florets were directly fortified. The results with transport and seed accumulation of iodine seem to be similar to the Zn results obtained under the HarvestZinc project (Cakmak et al. 2010b; Zou et al. 2012). Foliar Zn applications are also highly effective in improving grain Zn, and by contrast, soil Zn applications result in only minimal increase of grain Zn. Probably, root absorbed iodine is deposited and immobilized in shoot tissues after its xylem transport, and not readily available for re-translocation through phloem during the reproductive growth stage. By contrast, spraying iodine during the late growth stage, when the extensive assimilate translocation through phloem into seeds takes place, creates a greater available pool of iodine in leaf tissue that is ready for an immediate loading and transportation in the phloem. Since in the current study, at least one of the spray applications is timed after anthesis, when the florets might be open, it is not impossible that grains were fortified directly with the iodine that was spray-applied. Thus, at least a part of measured iodine in the grains might be attributed to direct surface contamination from postanthesis foliar spray. However, the mentioned risk is minimized by washing the grains prior to analysis. Moreover, in case of maize, grain surface contamination with iodine will be minimal because maize grains are covered and wrapped very tightly by husk leaves.

More importantly, iodine delivered to seeds of the iodine treated plants was found in a significant concentration in the polished rice (Fig. 3) and also in the endosperm part of wheat (Table 10). In a survey study conducted in Japan, iodine concentration of polished rice collected from 20 different field sites varied between 1.4 to 18 μg kg−1 with an average value of 3.6 μg kg−1 (Tsukada et al. 2007). In the present study, iodine concentration of the polished rice was 6 μg kg−1 and this value had increased to about 220 μg kg−1 after foliar spray of iodine (Fig. 3). To our knowledge, there is no published information about the localization of iodine in wheat endosperm. Table 10 clearly shows that increases in iodine concentrations of whole grain of wheat by foliar iodine spray were well reflected in the endosperm part of the grains as well as in other seed fractions. The marked increases in iodine concentrations in whole grain of rice, maize and wheat as well as in polished rice and wheat endosperm reported in the present study highlight the great potential of agronomy (e.g., fertilizer strategy) for agronomic biofortification of cereal crops with iodine. Spraying KIO3 up to the rate of 0.05% w/v is recommended as the optimal form and rate to be used in agronomic biofortification of cereals with iodine. Uptake efficiency of iodine through the leaves could be further promoted by adding a surfactant or KNO3 to the foliar spray solution. Consuming cereal-based foods agronomically biofortified with iodine through a fertilizer strategy is expected to result in a significant contribution to iodine intake in human populations with high health impacts in the regions where iodine deficiency incidence is high and cereals are consumed regularly. Currently, experiments are investigating stability and concentration of iodine in processed and cooked cereal-based foods. Acknowledgements This study has been financially supported by SQM, Nestlé and International Fertilizer association (IFA) and benefited also from the activities of the on-going HarvestZinc Project (www.harvestzinc.org) that is supported by the HarvestPlus Program, SQM, ADOB, Bayer Cropscience, Mosaic Company, K + S Kali, International Fertilizer Industry (IFA), Valagro, ICL Fertilizers, ATP Nutrition, International Zinc Association (IZA), Aglukon and International Plant Nutrition Institute (IPNI).

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