Improving selenium status in plant nutrition and quality

M.L. Mora^1*, P. Durán¹, J. Acuña¹, P. Cartes¹, R. Demanet¹ and L. Gianfreda²

¹Center of Plant, Soil Interaction and Natural Resources Biotechnology, Scientific and Biotechnological Bioresource Nucleus, Universidad de La Frontera, Temuco, Chile. *Corresponding author: mariluz.mora@ufrontera.cl

²Università degli Studi di Napoli Federico II, Naples, Italy.

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Abstract

Selenium (Se) is an essential micronutrient for human health due to its antioxidant capabilities. The Se content around the world is highly variable from 0.005 mg kg^-1 in areas from China and Finland to 8,000 mg kg^-1 in seleniferous soils fromTuva-Russia. However, about one billion of people in the worldwide are Se deficient.

During the last decade, studies related with strategies for Se biofortification in food plants for human nutrition have significantly increased because this metalloid is incorporated into human metabolism mainly as a constituent of food plants. Similarly, Se biofortification is important in pastures for increasing the Se content in cattle to enrich meat and to prevent disease associated to Se deficiency as white muscle disease. In China, two endemic diseaseshave been relatedto Se deficiency: Keshan and Keshin–Beck diseases.

Agronomic biofortification by using inorganic Se sources is a current practice in countries as China, Finland, and USA. In Chile, fertilization by using chemical compounds with Se is an uncommon practice due the edaphoclimatic characteristics of Andisols, which represent around 60% of agricultural soils of southern Chile. Recent studies showed that microorganisms as bacteria and arbuscularmycorrhizal fungi play an important role in the transformations and Se availability, representing an interesting biotechnological alternative to Se biofortification.

This review is focalized to describing Se behavior in soil-plant system and the possible strategies to improving Se content, including the use of microorganisms as biotechnological tools for increasing plant nutrition and quality. Specific attention will be devoted to volcanic soils of Southern Chile, where different factors concur to enhance the Se-deficiency problem.

Keywords: Selenium, Andisols, selenobacteria, biofortification

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1. Introduction

Selenium (Se) is a naturally occurring chemical element found almost everywhere on earth. During several years, this metalloidhas been considered toxic and dangerous for human and animal health (O^,Toole and Raisbeck 1995). Only in the last decades, its physiological importance as a micronutrient fundamental to animal and human health has been assessed (Fernández-Martínez and Charlet, 2009). The essentiality of Se has been established when this metalloid has been recognized as a constituent of selenoenzymes such as glutathione peroxidase (GSH-Px), thioredoxin reductases (TR), and proteins with unknown functions that are involved in maintaining the cell redox potential (Rayman, 2000). Nowadays, 25 selenoproteins have been described in human metabolism, corresponding to selenoproteins in which Se is an enzymatic cofactor (Korlhe et al. 2000; Dodig et al., 2004; Rayman, 2000; Schomburg et al. 2004). Thus, Se deficiency increases the risk of oxidative damage and important associated pathologies, i.e. cancer and HIV (Facompre and El-Bayoumy, 2009; Méplan and Hesketh, 2012).

While selenoproteins have been discovered for bacteria and animals (and for many of them it has been identified their physiological role), experimental attempts made to recognize these proteins also in plants have so far failed and this question remains unresolved. However, studies realized by Pilon-Smits et al. (2009) showed that Se in plants improves the plant growth, increases the tolerance against biotic and abiotic stress (Hartikainen and Xue, 1999; Xue and Hartikainen, 2000; Pennanen et al. 2002) and improves other physiologic parameters (Xue et al, 2001; Djanaguiraman et al. 2004; Turakainen, 2006). This is interesting because the main source of Se nutrition for humans and animals is the soil-plant system, and a mineral imbalance can lead to Se-deficient food with consequences for human and animal nutrition (Govasmark and Salbu, 2011).

Plant foods are the major dietary sources of Se in most countries throughout the world (Rayman et al. 2008). The amount of Se available in soil, determines the amount of Se in the plant foods that are grown in this soil. The awareness that the intake of plants with desirable, non-toxic Se levels is the first step of Se entry in the food chain could explain why biofortification of this element has received great attention. For instance, field treatment with Se or Se rich-compounds can be considered a safe, low cost approach to achieve this target in Se deficient areas (de Souza, 1999; Ingh, 2005).

This review will address the complexity of Se behavior in plants and soils with particular reference to Se deficient soils and the possible strategies to improving Se content including the use of microorganisms capable to metabolize the inorganic Se for their use as seed inoculants for plant nutrition and quality. Specific attention will be devoted to volcanic soils of Southern Chile where different factors concur to enhance the Se-deficiency problem.

2. Quality of selenium supplemented plants

Plants are constantly and frequently exposed to the effects of reactive oxygen species. These free radicals are very reactive species producing oxidative stress, which results in internal H₂O₂ accumulation. Oxygen peroxide may act as signaling molecule and induce a sequence of reactions and/or provoke unspecific oxidation of proteins, membrane lipids or DNA injury. As well described in mammals and humans, and very likely in plants, several enzymatic (e.g. superoxide dismutase, SOD, glutathione peroxidase, GSH-Px, catalase, CAT, guayacol peroxidase, POD, ascorbate peroxidase, APX) and non-enzymatic (e.g. ascorbate, glutathione and vitamin E) actors participate to this mechanism (Miller et al. 1993).

Hartikainen et al. (1997) investigated the antioxidant effect of Se in lettuce (Lactuca sativa) and ryegrass (Lolium perenne) plants. The activity of GSH-Px increased in both plants at doses of Se increasing from 0 to 33 µg kg^-1. Later, Hartikainen et al. (2000) demonstrated the duality of Se effect depending on the applied Se doses. At low concentrations (0.1, 1.0, 10 mg Se kg^-1) Se behaved as antioxidant, whereas ad elevated concentration (30 mg Se kg^-1) it acted as pro-oxidant. It was hypothesized that while Se decreases the oxidative stress by increasing the activity of GSH-Px, which in turn reduces the levels of hydrogen peroxide, it can itself induce the spontaneous dismutation of the anion superoxide, thus diminishing the need of SOD action. This latter catalyzes the dismutation of the superoxide anion (that initiates a cascade of reaction of free radicals) to hydrogen peroxide (Mittler and Zilinskas, 1991). Sabeh et al. (1993) measured GSH-Px activity in purified Aloe vera extracts and proposed that this enzyme was a tetrameric protein with one atom of Se per subunit. In a study on the antioxidative properties of Se during photoxidative stress in potato, Seppänen et al. (2003) demonstrated the Se altered transcript accumulation of GSH-Px. Similarly, Cartes et al. (2005) evaluated the effect of selenite and selenateon the GSH-Px activity and lipid peroxidation. Thus, for both Se sources, there was a positive correlation between Se shoot concentration and GSH-Px, and lipid peroxidation measured by Thiobarbituric Acid Reactive Substances (TBARS), decreased at low level of Se shoot concentration. Similarly, Cartes et al. (2011), showed a reduction of oxidative damage in membrane ryegrass cv Aries and Nui by an activation of GSH-Px antioxidant enzyme, demonstrating the Se essentiality to plants due its significant role as antioxidant in plants.

Another important cause of oxidative stress is exposure to UV-B radiation, nowadays one of the major concerns to plant biologists. Several studies and contrasting results have been obtained on the combined effect of Se and UV-B radiation on plants. Hartikainen and Xue (1999) evaluated the Se effect on the growth and the membrane damage in plants exposed to UV-B radiation. It was also observed that the UV-B radiation diminished the toxic effect due to high doses of Se supplied. Similar study has been carry out in lettuce (Pennanen et al. 2002) and pumpkin (Germ et al. 2005). Heijari, (2006) observed that Se did not decrease the harmful effects of UV-B radiation in strawberry and only an increase of leave development occurred at low dose of Se (0.1 mg kg^-1).

Additionally, it is been also postulated that Se can contribute to decrease plant senescence and increase plant growth. One of the first beneficial effect of Se on plant growth was recorded by Singh et al. (1980), who observed that the application of 0.5 mg Se kg^-1, as selenite, stimulated the growth and the production of dry mass in Indian mustard and ryegrass, lettuce, potato and duckweed (Hartikainen, 2005). Thus, the main physiological benefits of Se in plants can be summarized as follow: enhancement of plant growth (Hartikainen et al. 1997; 2000); delay of plant senescence (Xue et al. 2001; Djanaguiraman et al. 2004); increase in carbohydrate accumulation (Turakainen, 2006); improved tolerance to photoxidative stress (Seppänen et al. 2003, Cartes et al. 2005; 2006; 2011); alleviation of abiotic and biotic stresses such as UV-B radiation (Hartikainen and Xue, 1999; Xue and Hartikainen, 2000; Pennanen et al. 2002) cadmium. (Filek et al. 2008; Pedrero et al. 2008) and fungal infection and herbivores (Hanson et al., 2003), phloem-feeding aphids (Hanson et al. 2004).

3. Selenium in soil-plant system

3.1. Selenium distribution in soil

The global Se distribution in soils varies greatly from 0.005 mg kg^-1 in Finland to 8,000 mg kg^-1 in Tuva-Russia (Chasteen and Bentley, 2002). Although Se concentrations are in normal range between 0.01–2.0 mg Se kg^-1 are considered as Se deficient soils; mean of world is about 0.4 mg Se kg^-1 and concentrations >1,200 mg Se kg^-1 can occur in seleniferous soils (Fordyce, 2005).

Selenium occurrence in agroecosystems is related to natural and anthropogenic process. In relation with natural process, Se is associated with volcanic eruptions, weathering and evaporation in the process of soil formation and alluvial deposition in soil (Haygarth, 1994). In fact, the persistence of Se in soil is associated with parent material, principal source of Se soil formation (Fernandez and Charlot, 2009). Igneous and volcanic rocks generally contain around 0.035 mg kg^-1, whereas sedimentary rocks present concentration between 0.05-0.06 mg kg^-1 (He et al. 2005). Sedimentary rocks represent the principal compounds of earth surface and their Se concentration is much higher than in igneous rocks (Fernandez and Charlot, 2009). This can be attributed to Se transfer to the atmosphere and hydrosphere during volcanic processes (Haug et al. 2007). On the other hand, early studies published by Lag and Steinnes, (1974) have reported that Se-supply from the sea via rain and snow and sulphuric acid-rich polluted rain was an important Se source. Thus, anthropogenic processes also generated inputs of Se in agroecosystems (He et al. 2005) as extraction and processing of various minerals, pharmaceutical manufactures, veterinary medicine, glassware manufacturing industry, electronics devices industry, lubricants manufacturers, etc. (Frankerberger and Benson, 1994; Wen and Carignan, 2007). It has been estimated that between 30% and 40% of total Se emissions to the atmosphere are due to human activities (Wen and Carignan, 2007), such as: extraction and processing of different elements (copper, zinc, uranium and phosphorus), the use of pesticides and the combustion of oil and coal. In the atmosphere, Se is transported associated to particulate matter (Bosco et al. 2005) and then it is deposited in the agroecosystems. In addition, the extensive uses of Se-containing fly ash as soil amendments (Dhillon and Dhillon, 2003) and the irrigation of cultivated soils with Se-contaminated waters (Lemly, 1998), have a major impact upon the selenium cycle. The selenium cycle in the agroecosystems is complex because Se has a broad range of oxidation states, from Se^2- (completely reduced) to Se⁶⁺ (completely oxidized), and can be transformed by both chemical and biological processes (Zhang et al. 2000).

3.2. Selenium deficient soil and plant nutrition

Selenium deficient soils are characterized by low Se-content and limited availability. In most of soils, Se content is found between 0.01 and 2.0 mg Se kg^-1 (Neal, 1995; Mayland 1994; Fordyce, 2007). Dhillon and Dhillon (2003) proposed that soils with concentrations below 0.1 mg Se kg^-1 of soil are described as Se deficient.

Selenium deficiency has been reported in a number of areas across the world, such as China, North America, New Zealand, Australia, Sweden and Finland (Gissel-Nielsen et al., 1984; Yläranta, 1985; Gupta and Gupta, 2000) which means that feed crops do not contain sufficient Se to meet animal requirements (Gupta and Watkinson, 1985). This Se deficiency has been related with important diseases as Keshan and Keshin–Beck, two endemic disease associated with a Se-deficiency, which are present in China and Siberia. Keschan disease is produced generally in children and woman of childbearing age and its symptoms are related with impairment of cardiac function, cardiac enlargement and arrhythmia (Xu et al. 1997). The diseases occurrence is involving Se and vitamin E deficiencies, and the presence of the Coxsackie B virus (Moreno-Reynes et al. 1998). Kaschin-Beck disease is an osteoarthropy, which manifests as enlarged joints, shortened fingers and toes, and in severe cases dwarfism and is attributable to Se and vitamin E deficiency (Coppinger and Diamond, 2001) and Iodine deficiency (Contempré et al. 1991). In addition, the low Se content in human diet also has been associated with different diseases such cardiovascular disease (Rayman, 2002), and dysfunction in immune systems (Bodoni et al., 2008).

3.3. Selenium in crop grown in Chilean Andisols

Se concentration in soil depends on geological source (parental rock) from which it derives and on biogeochemical processes. In volcanic soils, little is known about the specific interactions of Se oxyanions (predominant species of inorganic selenium in volcanic soils) and the mineralogical components specific to these soils (imogolite and allophane), which could be responsible of the low Se bioavailability (Fernandez-Martinez and Charlot, 2009). In addition, the organic matter has an important role in Se content in soil due to propensity for Se to be adsorbed to organic materials (Ander et al. 2010; Fordyce et al. 2010). According to a screening carried out by the Soil Service Laboratory of La Frontera University (Chile), in soils of the South of Chile amounts of Se range between 0.02 and 0.18 mg kg^-1soil. This is an important fact due that Andisols soils in southern Chile represent around 60% of agricultural soils. On the other hand, about 50% of the Chilean Andisols present a high soil acidity level, one of the main factors limiting agricultural production (Mora et al. 2005; 2006). Due to this fact, it is necessary to apply lime and gypsum and large amounts of P fertilizer (Mora et al. 1999). The application of lime to ameliorate soil acidity can be assumed to reduce Se sorption by soil (Ylaränta, 1983; 1990) and therefore, to increase Se uptake by plants. On the other hand, the use of fertilizers containing phosphate should be expected to raise selenite availability to plants, because in Andisols phosphate strongly competes with selenite by sorption sites (Barrow et al. 2005). Soil acidification can be accelerated over a few years under intensive agricultural systems, with the consequent increase of phytotoxic aluminum (Al³⁺) in the soil exchange complex, as demonstrated by the relationship between Al saturation percentage and pH for Chilean Andisols (Mora et al. 2005). Thus, inorganic Se forms as sodium selenite is bound to soil constituents as clays, oxy-hydroxides of aluminum (Al), iron (Fe) or manganese (Mn), remaining unavailable to plants (Cartes et al. 2005; Mora et al. 2008; Nakamaru and Altansuvd, 2014). As demonstrated in sorption experiments, selenite behaves like phosphate (Barrow et al. 2005) and therefore it is more stronger sorbed than selenate to the soil surfaces, thus becoming less bioavailable than selenate at equal rates of soil application (Cartes et al. 2005). However, sodium selenate could be leached at wet fall conditions. Competitive inhibition of selenate uptake and accumulation by sulfate has been extensively studied in solution or sand cultures (Bell et al. 1992; Kopsell and Randle, 1997; Zayed and Terry, 1992) and in soil experiments (Bañuelos and Meek, 1990; Yläranta, 1990). Two reasons drive research concerning the selenate-sulfate interactions in soils. First, selenate is more mobile than selenite in soil (Yläranta, 1991, Cartes et al. 2005). The other is related to the fact that sulfate competes with selenate for sorption sites since both of them might only form outer-sphere complexes with soil minerals (Zhang and Sparks, 1990; Jara et al. 2006).

3.4. Selenium in human nutrition

In relation to optimal Se status in human dietary, the Recommended Dietary Allowances (RDAs) indicates that 55 µg Se day^-1 is an adequate dose for adult men and women (RDAs, 2000). According to Rayman, (2004), based on the activity of the plasma selenoenzyme GSH-Px, several governments and international committees have recommended daily intakes varying from 30 to 85 µg day^-1 in human diets. The benefits of Se to human health are not only associated with its antioxidant function, but also with an improvement of the immune system, cancer risk and HIV (Rayman, 2002), specially at supranutritional levels up to 125 µg day^-1 in relation to the currently recommended intakes (Combs et al. 2001). Likewise, Se deficiencies in ruminant diets generatemetabolic disorders like the white muscle disease, alterations of both the immune responses and the reproductive performance, among others (Ceballos and Wittwer, 1999; Wittwer, 2002). According with NRC (2000), daily intakes between 100 and 300 µg kg^-1 of dry weight (DW) are adequate to achieve the requirements of beef and dairy cattle.Wittwer et al. (2002b) reported Se deficiencies in 83% of the forage samples of 12 dairy farms of Southern Chile.

The low Se concentration in forages of Bío-Bío, La Araucanía and Los Lagos Regions has been associated to a low GSH-Px activity in grazing cattle and horses (Ceballos et al. 1999; Laporte et al. 1998; Wittwer et al. 2002) and to the white muscle disease in Red Friesian calves (Contreras et al. 2005). For this reason, several efforts for enhancing the Se content in plants that are the bases to increase the Se content in human diet have been realized (Bañuelos et al. 2012; Chilimba et al. 2012; Acuña et al. 2013; Durán et al. 2013; Rahman et al. 2013).

4. Selenium biofortification

4.1. Agronomic Biofortification by using inorganic Se fertilizers

Agronomic biofortification, through the application of Se-fertilizers is widely applied in countries such as: Australia, USA, New Zealand, United Kingdom and Finland (Bañuelos et al. 2012). In this context, Finland government is pioneer in the application of inorganic Se fertilizer as a biofortification program (Broadley et al., 2006). In Africa, a national Farm Input Subsidy Program exists that includes the agronomic Sebiofortification with Se (Chilimba et al. 2012), where it has been tested successfully in maize Se supplemented. Similarly, Se biofortification has been tested in other several plant crop species Triticum aestivum, (Hawkesford and Zhao 2007; Govasmark and Salbu, 2011; Hart et al. 2011); Triticum durum (Poblaciones et al. 2014), Pisum sativum, (Poblaciones et al. 2013); rice (Premarathna et al. 2012); lentil (Rahman et al. 2013); mushrooms (Bhatia et al. 2013).

In Chile, selenium enrichment of fertilizer applications across coated seeds or by foliar applications in crops has been described as an effective strategy in order to increase Se availability to plants and concomitantly to enhance its content in pastures with strengthening the antioxidant system against aluminum toxicity (Mora et al. 2008). The benefits by Se on higher plants appear to be strictly dependent on the availability of both essential, as sulfur (S) and phosphorous (P), and toxic, as aluminum (Al), cadmium (Cd) and arsenic (As), elements in culture medium. Cartes et al. (2006) evaluated the effect of the simultaneous presence of selenite and S on the antioxidant Lolium perenne system. By increasing the levels of S, the GSH-Px activity decreased and the damage of the lipid membrane increased. As previously discussed, being Se transported inside the cell by S transporter and having this latter a higher affinity for S, higher amounts of S than Se were uptaken in the presence of higher concentration of sulfate. Mora et al. (2008) evaluated the impact of the level of soil acidity and P fertilization on the antioxidant responses of selenite-treated white clover plants, and they showed that the application of P and lime increased the antioxidant ability of plants by activating peroxidase (POD) and ascorbate peroxidase (APX) enzymes and by decreasing the lipid peroxidation at shoot Se concentration up to 0.2 mg Se kg^-1 DW. These results clearly indicate that different combinations of soil acidity and P fertilization may influence Se absorption by plants and be responsible of beneficial or unfavorable effects of Se on lipid peroxidation, activation of POD and APX in white clover.

Andisols from southern Chile have high amounts of interchangeable Al and low pH,properties that limit the Se-bioavailability in the soil solution (Mora et al. 1999; Mora and Demanet, 1999) and consequently the effectiveness of agronomic biofortification. Thus, inorganic selenite is bound to soil constituents, whereas selenate may be leached under wet fall conditions, limiting its availability to crops (Govasmark and Salbu, 2011; Hawkesford and Zhao, 2007). In addition, the Se transporters are similar to phosphorus or sulfur transporters, displayed in the low translocations rates to plants (Cartes et al. 2006; 2011). In fact, Inostroza-Blancheteau et al. (2012) showed that the high Se doses induce differentiation in wheat genotypes in terms of Se accumulation associated with highest expression level of sulfate transporters and lowest SOD activity.

4.2. Biofortification by using microorganisms

Microorganisms, mainly bacteria,play essential functions to plant growth, nutrition and disease control in agricultural systems (Nannipieri et al. 2003, Hawkes et al. 2007). Nowadays, studies have proposed the use of plant growth-promoting bacteria (PGPB) as alternative to enhance the uptake of micronutrients by plants (Tariq et al. 2007). Several reports showed the utilization of PGPB with abilities to mobilizing diverse micronutrients such as: Zinc (Zn), Iron (Fe), Manganese (Mn), Copper (Cu) and Selenium (Se) as biofortification strategies (Table 1).

Table 1. Diversity of PGPB used as a strategy of biofortification in plants

N.D; no indentified

Selenium in the agroecosystems is found in both inorganic and organic forms. The inorganic Se forms are present in four oxidation states, which are denoted as selenide (Se^2-), elemental Se (Se⁰), selenite (Se⁴⁺) and selenate (Se⁶⁺) (Fernandez-Martinez and Charlot, 2009). It is widely accepted that bacteria have capability to reduce oxidized and methylated Se oxyanions to some different Se-compounds. The reduction processes have been described as the ability of these microorganisms to convert the Se⁶⁺ and Se⁴⁺, to Se⁰ and finally generating Se methylated compounds (Losi and Frankerberger, 1996) and organic Se compounds as SeMet and SeMeSeCys (Duran 2015, under revision). Thus, biofortification by using microorganisms as alternatives to the chemical fertilization and the agronomic practices have gained great interest for researchers. Table 2 summarizes different studies related with Se biofortification.

Table 2. Selenium biofortification of crops

4.2.1. Rhizospheric bacteria for Se biofortification

Selenobacteria have been described as Se-respiring and Se-tolerant bacteria, associated to assimilation and metabolization mechanisms inside the cells (Kessi et al. 1999; Losi and Frankerberger, 1997; Stolz et al. 1999). Diverse studies showed that bacteria belonging to Firmicutes (Bacillus and Paenibacillus) and Proteobacteria (Pseudomonas and Enterobacter) stimulate Se transformation through oxidation, reduction and methylation from selenium contaminated soil called "selenifeorus soil" (Losi and Frankenberger 1997; Fordyce 2007; Fernandez-Martinez and Charlot, 2009).Studies by Acuña et al. (2013) showed that selenium deficient soils as Andisols possess a high diversity of culturable bacteria with the capabilities to metabolize and tolerate high Se doses. In Chile, there are about 5.1 million has of volcanic soils and about half of this area occurs under agricultural use, where the most important national milk, meat and cereal crops are produced (ODEPA, 2014).

Cereals, mainly wheat (Triticum aestivum L.), have been described as a good source for bioavailable Se, being SeMet its principal organic form in grain (Lyons et al., 2004). Broadley et al. (2006) have demonstrated the stronger link between the Se concentration of wheat grain and the optimal Se status in human diet (Broadley et al. 2006). Recent studies have demonstrated that the inoculation with Se-enriched selenobacteria increased the Se concentration in plant tissue of wheat (Acuña et al. 2013). Similarly, studies conducted by Duran et al., (2013) evaluated the synergism between selenobacteria and AMF in Se uptake and translocation in wheat plants. This report showed that plants co-inoculated with a mixture of selenobacteria strains and G. claroideum increased significantly the Se content in grain. Figure 1 shows the schematic representation of Se biofortification in Andisols (high Al saturation) of wheat plants by using bacteria, where seeds were coated with Se enriched bacteria and where the main Se forms found were elemental Se (Selenonanosphere), SeMet and SeMeSeCys. Wheat plants are able to improve the antioxidant activity and to enhance the Se content in shoots and grain to obtain enriched flour (Acuña et al. 2013; Durán et al. 2014, Durán data no shown)

Figure 1. Selenium biofortification by using microorganisms in Andisols. Selenobacteria reduce inorganic Se into Nanospheres and other organic Se forms. The microbial inocula can be coated in seed and wheat plant enhances the Se content in wheat grain for obtaining Se biofortified flour

4.2.2. Endophytic bacteria for Se biofortification

In a recent study, Durán et al. (2014) showed that plant growth promoting (PGP) endophytic bacteria Bacillus sp.E5 and Acinetobacter sp. E6.2 have ecological advantage over rhizospheric bacteria in terms of Se tolerance (Durán et al. 2014; Etesami et al. 2014). It should also be kept in mind that endophytes are better adapted to elevated Se concentration than rhizospheric bacteria. Indeed, rhizobacteria can only tolerate around 20 mM of sodium selenite (Acuña et al. 2013), whereas our endophytic strains showed tolerance to 60 - 180 mM of Se. Bactera strains were tagged with Green Fluorescent Protein and were observed by confocal microscopy inside the root plant (Figure 2 b,c). In this study also was evaluated the effect of arbuscularmycorrhizal fungi which enhance the Se content in plants and improve the antioxidant system in wheat (Figure 2d). Elevated Se tolerance by bacteria also was reported by Hunter and Manter (2009) for Pseudomonas sp. (150 mM Se). In addition, recently our research team showed that Bacillus sp.E5 and Acinetobacter sp.E6.2 exhibit mainly stable intracellular and extracellular NanoSe and important Se organic forms as seleno methyl selenocysteine (SeMeSeCys) and selenomethionine (SeMet), (Durán fata no shown), the three most important Se forms for cancer prevention. Similarly, in Lactuca sativa plants the enhancing of Se content in edible tissue and the performance of physiological parameters and scavenging reactive oxygen species for counteract the effect of drought stress were corroborated (Durán et al., 2015, submitted). Under field conditions similar benefit effects were found, as Se supplemented plants showed elevated SeMet in grain (dates in preparation).

Figure 2. Confocal laser scanning microscopy images of endophytic colonization: (A) control bacteria without Gfp tagged, (B) Gfp tagged bacteria, (C) root of wheat plants colonized by bacteria Gfp tagged and (F) optical microscopy image of root of wheat plants colonized by mycorrhiza

5. Conclusions and future perspectives

The use of selenium pelleted seeds appears to be an interesting alternative to ameliorate selenium deficiencies in grasslands systems. Similarly, the co-inoculation of selenobateria mixture and arbuscular mycorrhizal fungi represents a promising strategy for biofortification of wheat plants in order to produce Se enriched flour for supplementing foods for human consumption. Further studies are needed to confirm the effectiveness of biofertilization strategy under field conditions and to elucidate the main mechanisms of Se uptake by plants.

Acknowledgments

The authors acknowledged the important contribution of National Commission for scientific and technological Research (CONICYT) Program MEC 80130066 of Atraction an Integration of Advance Human Capital (Dr. Liliana Gianfreda) and the technical support of Scientific and Technological Bioresource Nucleus (BIOREN) from La Frontera University

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