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Recently, the daily consumption of functional foods for the prevention of some human diseases has become highly promoted by several health organizations in the world. In this context, the demand for blueberries (Vaccinium spp.) has progressively increased in some countries due to the high phenolic compounds concentration in their skins, which are characterized by a great antioxidant capacity (Cordes et al., 2016). Blueberry is a species evolved in rainy areas characterized by soils with an acidic pH from 4.5 to 5.5 (Retamales and Hancock, 2012), and when it is cultivated in lower rainfall zones, often characterized by alkaline or sub-alkaline soils, frequently manifest severe symptoms of iron (Fe) deficiency, drastically reducing plant growth and yield (Rombolà and Tagliavini, 2006).

Even though a few studies have assessed the effect of Fe deficiency on fruit chemical composition and quality in some fruit crops, knowledge regarding the impact of Fe chlorosis on blueberry quality, and healthiness is scarce. In peach fruits, some experiences indicate that Fe deficiency decreases fruit firmness and the red skin color, increasing organic anion concentrations (particularly succinate and quinate), vitamin C, and total phenolic compounds (Álvarez-Fernández et al., 2003). In grapevines, Fe deficiency has been shown to increase the phenolic compounds accumulation in berries (Bavaresco et al., 2010). In contrast, in the model plant Perilla frutescens, anthocyanin synthesis was negatively affected by Fe deficiency, likely associated to a reduced activity of the anthocyanidin synthase (ANS), an enzyme involved in the anthocyanidin synthesis (Saito et al., 1999; Turnbull et al., 2000; Nakajima et al., 2001), and whose activity requires a ferrous Fe center. The controversy on the impact of Fe deficiency on the secondary metabolites concentration in fruits might be due to a subtle effect of the environment on the biosynthetic regulatory signals, or a species dependent sensitivity. Even though blueberries are considered as a healthy nutritional fresh produce, precisely because of their fruit chemical composition, and also as a highly sensitive crop to Fe deficiency, rather scarce knowledge is available in this regard.

In blueberry orchards located in sub-alkaline areas, soil acidification by means of elemental sulfur (S) previous to planting or with sulfuric or phosphoric acid applications during the growing season through the irrigation system, is a common field practice worldwide. In the former case, soil pH varies after long periods of time since microorganisms mediate the transformation of S into inorganic acids. Moreover, depending on the initial soil pH values and the buffer capacity of the soil, extremely high S amounts should be applied to shift the pH down to optimal levels for blueberry (Horneck et al., 2004). As for sulfuric acid applications, soil pH is more quickly reduced, especially in weakly buffered soils. However, this material is hazardous and difficult for farm workers to use (Horneck et al., 2004), and repeated soil applications of sulfuric acid may have a detrimental effect on the microbial biomass and mycorrhizal fungi populations, and on carbon dioxide emissions to the atmosphere. On the other hand, the treatment for Fe chlorosis with Fe chelates is costly and not environmental nor human friendly (Rombolà and Tagliavini, 2006) due to the high risk of metals and chelating agents leaching into the deep soil layers and into the water table (Rombolà and Tagliavini, 2006).

The above mentioned evidences of the negative impact of traditional Fe corrective measures, have led to the use of natural Fe sources to control Fe deficiency. In this context, animal blood-derived compounds, composed of a relatively high Fe content (20–30 g Fe kg^-1) chelated by a heme group related to hemoglobin (López-Rayo et al., 2015), have been shown to be highly stable and capable of maintaining available Fe for plants in calcareous soils (Yunta et al., 2013). In controlled experiments conducted on grapevines cultivated in a calcareous soil, Fe-heme applications have increased the leaf chlorophyll concentration, plant growth, and potassium content in leaves (López-Rayo et al., 2015). On the other hand, some investigations have reported that intercropping systems with grasses are effective at preventing Fe chlorosis in co-cultivated crops (Cañasveras et al., 2014, Covarrubias et al., 2014, Granja and Covarrubias, 2018). The mechanism involved in improving Fe nutrition in associated plants is attributed to the ability of monocotyledoneous species to extrude mugineic acid family phytosiderophores (MAs), which are chelating compounds that form a soluble complex with Fe³⁺, increasing its availability also for the associated crops (Takagi, 1976; Ma et al., 2003; Xiong et al., 2013). For example, some authors have described that the main phytosiderophore secreted by Festuca rubra is 2′-deoxymugineic acid (DMA), whereas the graminaceous species Poa pratensis exudes DMA, avenic acid A (AVA) and 2′hydroxyavenic acid A (HAVA) (Ma et al., 2003; Ueno et al., 2007). Other studies have reported positive results from this sustainable management technique to prevent Fe chlorosis in fruit tree crops (Cañasveras et al., 2014, Covarrubias et al., 2014), however, to date there have been no reports published on a species highly susceptible to Fe deficiency like blueberry. In addition, some authors have described the possibility of the Fe³⁺-DMA complex being directly absorbed by intercropped peanut roots, since a functional Fe³⁺-DMA transporter, AhYSL1, was identified in this species (Xiong et al., 2013).

Even though investigations have been conducted on the effect of alternative and more sustainable management techniques for Fe correction in calcareous soils in blueberry productive and vegetative variables (Michel et al., 2019), the impact on the berry composition is lacking. Indeed, the use of animal blood-derived compounds and/or intercropping with graminaceous species implies the modification of soil nutrition properties related to nitrogen (N) availability, eventual phytoalexins, or simply modification of the competition for light, water and nutrients with eventual impacts on the fruit yield and metabolism (Michel et al., 2019). Accordingly, the present study is focused to evaluate the effect of Fe-heme applications or intercropping with graminaceous species on productive and berry quality variables in blueberry cv. Emerald, cultivated in a sub-alkaline soil.

Data collected in our experiment indicate that those treatments resulting in higher leaf chlorophyll concentrations at harvest (see section “Materials and Methods”), such as intercropping with F. rubra and P. pratensis, together with Fe-EDDHA applications, were effective in inducing changes in some productive and berry morphological characteristics compared to the control and Fe-heme plants. Indeed, these treatments increased the fruit load and, for plants intercropped with F. rubra, also an increase in the yield per plant was evident. In addition, the same treatments decreased the skin/flesh ratio (Figure 1). The skin to flesh ratio is affected by the berry volume, as it might be the case for the Fe-EDDHA treated plants, with the highest fruit average weight as well as the lowest skin/flesh relationship (Figure 1C,D), but may be also due to a more thick epidermis tissue. The lower crop load recorded in plants with a lower leaf chlorophyll concentration, that is control and Fe-heme, could be related to a lower fruit set or a higher early abscission of berries in these plants, considering that Fe chlorosis promotes the expression of genes involved in the synthesis and signaling of ethylene, resulting in the abscission of young fruits (Iqbal et al., 2013). Also, blueberries are regarded as a highly sensitive species to sink/source relationship (Jorquera-Fontena et al., 2018; Petridis et al., 2018), where the non-structural carbon reserves status in shoots and storage organs are determinant in the extent of the post-fruit set fall (Buwalda and Smith, 1990; Mehouachi et al., 1995). In fact, it is well known that the Fe status of plants is closely linked to the photosynthetic capacity of leaves (Briat et al., 2007), and in citrus, Fe deficiency induced reductions in the carbon status of plants, increasing the early fruit fall (Gómez-Cadenas et al., 2000). In our study, a lower leaf net photosynthesis during the season was registered, precisely, in control and Fe-heme plants in comparison with those treated with Fe-EDDHA and intercropping with grasses (Michel et al., 2019). Likewise, the effect of Fe nutrition on the carbon availability within the plant could contribute to explain the higher pulp fraction in relation to the skin in berries collected from plants with a better Fe status, such as those treated with Fe-EDDHA and intercropped with graminaceous, as well as the higher yield in plants associated with F. rubra than the control plants.

The ripening process, on the other hand, regarding SS, pH, acidity and SS acidity ratio were not altered by any of the Fe-correcting strategies (Table 1). This result, suggests that any eventual impact of the treatments on yield was rather the result of a sink to source balance, with no impact on the primary metabolism of the berries. Similar results from Fe nutrition studies have been reported for peach, citrus and pear (Álvarez-Fernández et al., 2003, 2011; Bañuls et al., 2003). However, as for berry firmness, a relevant trait for post-harvest market considerations, differences between the treatments assessed in the present study were recorded (Table 1). Firmness is associated to a combination of ripening stages, water content and cell wall properties in berries. In blueberry, the berry firmness varies according the ripeness status, being maximum before berry veraison and progressively decreasing after it, and the SS to acidity ratio has been reported as a very reliable indicator of the ripening stage and fruit firmness (Moggia et al., 2017). As for our results, no significant differences in the SS/acidity ratio was observed between treatments, even though differences of up to a 13% between the minimum -Fe-EDDHA- and maximum –intercropping with P. pratensis- were detected, both treatments with significant differences in the berry firmness. This might suggest that the primary metabolites and their ratio, suggested as a proxy for ripening state in fruits, are highly variable as compared to firmness. Alternatively, a more subtle effect of the treatments might be indirectly affecting the water status and physical skin properties of the berries, such as plant vigor and light interception and temperature on fruits, but those were not evident from our observations.

Data concerning anthocyanin content in ‘Emerald’ blueberries from our study, revealed the presence of 9 different compounds in the skins (Table 3), while 13 were reported in Italy (Giovanelli and Buratti, 2009), 15 in France (Kader et al., 1996), 25 in Canada (Gao and Mazza, 1994), 15 in United States (Ballington et al., 1987; Mazza and Miniati, 1993), and 10 in other countries and other varieties (Sapers et al., 1984). The anthocyanin compounds identified in berry skins were galactosides, glucosides and arabinosides of delphinidin, cyanidin, petunidin and malvidin, similar to previous studies (Kader et al., 1996; Taruscio et al., 2004; Giovanelli and Buratti, 2009). However, and in contrast with other investigations, peonidin was not detected (Kader et al., 1996; Tomás-Barberán and Espín, 2001; Lohachoompol et al., 2008; Giovanelli and Buratti, 2009). In addition, the major anthocyanins concentration corresponded to delphinidin 3-galactoside and malvidin 3-galactoside, followed by cyanidin 3-galactoside and delphinidin 3-arabinoside. Also, and consistent with determinations from different Vaccinium corymbosum varieties, anthocyanin compounds reached 30–35% of total phenols (Moyer et al., 2002; Sellapan et al., 2002; Lee et al., 2004; Giovanelli and Buratti, 2009).

As for low molecular weight phenols, the hydroxycinnamic acids were the largest group contained in skins. Within that group, the highly antioxidant molecule chlorogenic acid predominated, while ferulic acid was present in lower concentrations (Table 4), similar to previous reports (Häkkinen and Törrönen, 2000; Taruscio et al., 2004). As for the hydroxybenzoic acids group, the highest compound identified was vanillinic acid, followed by gallic and protocatechuic acids (Table 4). In the flavanols group, epicatechin predominated over catechin (Table 5), as seen in nine different Vaccinium species (Taruscio et al., 2004). Between di-OH flavanols and flavonols, astilbin showed a higher concentration than quercetin, and no myricetin or kaempferol was detected (Table 5), as reported by Taruscio et al. (2004). In this sense, Kader et al. (1996) and Sellapan et al. (2002) found kaempferol, although in a smaller amount than quercetin, a molecule regarded as the more abundant flavonol in blueberry (Kader et al., 1996, Häkkinen et al., 1998). Up to our knowledge, this is the first report on the phenolic composition in “Emerald” blueberries grown in Chile. Thus, although the phenolic composition and antioxidant capacity of blueberry is more influenced by the genotype than by the growing season, it is advisable to evaluate the same species for several producing seasons in order to precisely determine whether the genotype maintains its phenolic composition over environmental variations.

It has been widely reported that changes in berry weight usually leads to changes in phenol concentration in berries, as these compounds are affected by the berry size due to a dilution factor (Ojeda et al., 2002; Roby et al., 2004). In contrast, our results did not reveal significant differences between treatments in total phenol, total anthocyanin and total tannin concentrations, when are expressed as mg g^-1 berry (data not reported), despite the effect of treatments on the skin/flesh ratio. This suggest, that the phenol content in berries were not necessarily the result of berry size variations but to an actual positive differential synthesis vs. degradation activities in skins. Indeed, our results are expressed as for skin weight basis (Tables 2–5). Data related to total phenols and total anthocyanins (Table 2) as well as the anthocyanins and low molecular weight phenols profiles (Tables 3–5) revealed a higher accumulation of these compounds in skins from plants treated with Fe-EDDHA and in intercropped plants than the control and Fe-heme. These results suggest that by better counteracting the Fe deficiency of blueberry plants, the Fe-chelate as well as the intercropping treatments favored the berry fruits chemical composition. Fe is a cofactor of the ANS enzyme, which is involved in the anthocyanin synthesis pathway (Saito et al., 1999; Turnbull et al., 2000), the activity of which requires a ferrous Fe center to catalyze the reaction that converts dihydroflavones (flavan-3,4-diol) into anthocyanidins (Turnbull et al., 2004). Similarly, for the low molecular weight phenols, Fe is also involved in its synthesis pathway as a cofactor of flavanone-3β-hydroxylase (F3H) and flavonol synthase (FLS) enzymes, which catalyze the consecutive conversion of flavanones to flavanonols, and of flavanonols to flavonols, respectively. In this sense, the oxidation reactions involving Fe-dependent enzymes play a crucial role in flavonoid biosynthesis (Saito and Yamazaki, 2002; Springob et al., 2003), and clearly, corrective and effective field practices regarding Fe nutrition have a positive impact on berry quality in field grown blueberries.

On the other hand, data related to both cinnamic and benzoic acids suggest that Fe might also be participating in the non-flavonoid compounds concentration in blueberry skins, but further studies are needed to clarify this issue. The synthesis and accumulation of phenolic compounds are strongly influenced by the photosynthetic capacity of plants (Smart and Robinson, 1991) which, in our study, was higher in plants treated with Fe-EDDHA and intercropped with the graminaceous species (data not shown). In fact, the synthesis of the shikimate pathway precursors come from hexoses derived from photosynthesis, ending in the formation of polyphenols such as flavonols, flavanols, anthocyanins, or tannins. Whether the higher benzoic and cinnamic acid concentrations results purely from a more favorable carbon supply condition in the Fe chelate and intercropping with grasses treatments, or a more subtle effect are involved, as it could be the need for antioxidant defenses and related systemic acquired resistance in Fe-deficient plants, depleting benzoic acids intermediates, is not clear and needs further investigations.

In view of the strong and positive relationship among the antioxidant capacity and the total phenol and anthocyanin content (Ehlenfeldt and Prior, 2001; Moyer et al., 2002; Sellapan et al., 2002), the study of sustainable management techniques, such as intercropping with grasses, that affect the crop and influence the phenolic accumulation of the fruits, will be useful to providing the consumer with a better product.

The datasets generated for this study are available on request to the corresponding author.

JC conceived the work and wrote the manuscript. LM conducted the experiment and measured the field. ÁP performed the chemical analysis in berries. CP, PB, and AR contributed to the manuscript writing.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.