Acidic soils (pH ≤ 5.5) are widely distributed, representing about 30% of the total world’s land area and, approximately 50% of the world’s potential arable lands. However, soil acidity is an important limitation for crop productivity. This is because, various restrictive factors for agricultural production usually coincide in these soils, such as toxic levels of aluminum (Al) and deficiencies in essential minerals for robust plant nutrition, such as phosphorus (P). Although Al is the third most abundant element in the earth’s crust, it lacks any discernible role in known biological processes (Tolrà et al., 2011). Under acidic conditions, the Al is released from soil particles into the soil solution as the trivalent ionic form (Al³⁺), which is particularly toxic for plants (Kochian et al., 2015). Aluminum rapidly hinders root growth, limiting both root expansion, and consequently constraining the absorption of water and essential nutrients vital for the plant’s fitness. Aluminum exists not only in the Al³⁺ ionic form but also is found as several insoluble forms including aluminosilicates, Al-humus complexes and other precipitated forms. Both the soluble and insoluble forms of Al can react and strongly retain minerals with negative charge, such as phosphate (H₂PO₄ ^-, HPO₄ ^2-), decreasing its availability for plant nutrition (Kochian et al., 2004). Thus, both Al toxicity and P deficiency converge to exert a deep impact on plant growth and crop yields on acidic soils (Kochian et al., 2004; Chen and Liao, 2016).

In Southern Chile, soils from volcanic origin represent about 50-60% of arable land and support most of the agricultural production and forestry activities of the country (Borie and Rubio, 2003). The two main soil orders found in the southern-central Chile are Andisols and Ultisols, which inherently predisposed to natural acidity, harbor substantial amounts of active Al³⁺ and strong phosphate retention (Mora et al., 2006, 2017). Therefore, these soils are characterized by having high amounts of total P although with a very low P availability (Borie and Rubio, 2003). Consequently, traditional agricultural practices on acidic soils requires continuous application of agrochemicals, such as P fertilizers (a non-renewable resource) to increase P availability, and lime application (calcium carbonate) to increase soil pH and reduce Al phytotoxicity (Mora et al., 1999). However, these agricultural practices continued over time are unsustainable, since often they present negative environmental and socio-economic impact, and are not economically viable or physically manageable for many farmers worldwide (Samac and Tesfaye, 2003). Consequently, there is an increasing interest to develop environmentally friendly strategies to diminish our agrochemical dependency, and contribute to more sustainable agriculture.

Intercropping systems have proven to be an effective and sustainable alternative to counteract nutrient deficiency, along with increasing the use efficiency of fertilizers in the soil. This achievement can be attributed to the phenomenon of interspecific facilitation, encompassing both above- and belowground interactions (Michel et al., 2019; Schoebitz et al., 2020; Xu et al., 2020). Within belowground interactions, there are several studies evidencing that nutrient mobilization by the roots of some species plays an important role in the facilitation process, enhancing the nutrient uptake and growth of neighboring non-mobilizing species (Li et al., 2014; Lambers et al., 2018; Shen et al., 2023). In general, the nutrient mobilization is via root exudation of organic compounds, including phosphatases, organic acids, phenolic compounds, which increases the mineralization and solubilization of nutrients in the soil. The interspecific facilitation of nutrient acquisition by association of two or more plant species having different abilities to mobilize nutrient, have involved numerous species, including species bearing specialized root structures, such as cluster roots (Muler et al., 2013; Teste et al., 2014; Shen et al., 2023).

Cluster roots are described as ephemeral rootlets and root hairs around the central root forming structures type “brush” or “raceme” that actively exude organic compounds which play a pivotal role in efficiently mobilizing essential nutrients (Lambers et al., 2006; Lambers et al., 2015a). Many species have this type of root adaptation, including species that belong to Proteaceae family, which have been widely described as plants with remarkable ability to both acquire and utilize P efficiently (Lambers et al., 2015a). The nutrient-acquisition strategy of Proteaceae involves chemical modification of its rhizosphere through the exudates from their cluster roots, which release nutrients from the soil (Delgado et al., 2015; Lambers et al., 2015a). The primary exudates consist of organic acids which carry negative charges, enabling them to displace phosphate bound in the soil through interactions with cations such as Al³⁺, Fe³⁺ and Ca²⁺ (Ryan et al., 2001), making the previously bound phosphate readily available for plant uptake. Additionally, the organic acids chelate Al³⁺ to form stable and non-toxic complexes and, therefore, it is also involved in the detoxification of this metal (Kochian et al., 2015; Chen and Liao, 2016).

Certain cluster root-bearing species, such as several members of Proteaceae family have been classified as Al hyperaccumulator species. This means that they are able to accumulate high concentrations of Al within their leaves (≥ 1000 mg kg⁻¹ dry weight) without manifesting any signs of stress or toxicity (Jansen et al., 2002). This intriguing trait suggests that the roots of these Al hyperaccumulator plants might absorb Al from their rhizosphere, potentially leading to a reduction of soil Al levels through continuous Al accumulation in leaves, bark and wood, as was early proposed by Webb (1954). Conversely, there are studies indicating that Al-hyperaccumulator species might acidify the rhizosphere, thereby increasing Al availability, as observed in Camellia sinensis L (Ruan et al., 2004; Chen et al., 2006). Despite these insights, our current understanding remains lacking in providing a comprehensive clarification regarding the influence of Al-hyperaccumulator species on soil Al availability, particularly within the context of species possessing cluster roots. Furthermore, the potential facilitative effect of an Al hyperaccumulator species, coupled with its ability to solubilize P, on a plant species sensitive to both Al toxicity and P deficiency, remains largely unexplored. In light of this, our study aimed to evaluate the effects of an Al hyperaccumulator species bearing cluster roots, Gevuina avellana Mol (Delgado et al., 2019), on the growth and performance of a neighboring species. For this investigation, we used Vaccinium corymbosum L., an economically important crop species, as a model species sensitive to both Al toxicity (Cárcamo-Fincheira et al., 2023) and P deficiency (Pinochet et al., 2014). Consequently, we posit that species with the dual ability to accumulate Al and solubilize P, may be suitable for the establishment and growth of neighboring plants in soils with high Al content and low P availability, such as volcanic soils in Southern Chile.

Contrary to initial expectations, there was no evidence of the facilitating effect of G. avellana on the V. corymbosum seedlings growth ( Figure 1 ). Rather surprisingly, G. avellana exhibited an enhancement in its own growth when grown alongside with V. corymbosum ( Figure 1 ; Supplementary Table S1 ). These findings align with the observations made by Fajardo and Piper (2019), who reported that Nothofagus species did not experience an improvement in their survival and growth when they were planted next to G. avellana. Notably, however, G. avellana did exhibit growth enhancement when co-cultivated with Nothofagus or some conspecifics species. These authors concluded that Proteaceae species have a competitive advantage over non-cluster root-bearing species at the seedling stage, especially on nutrient-rich substrates. In our study, it was observed that V. corymbosum showed lower roots P concentration when it grows in association with G. avellana ( Table 3 ). This suggests that the competitive advantage of G. avellana may be attributed to the significant exudation of carboxylates such as oxalate and succinate from its large cluster roots (Delgado et al., 2021; Zúñiga-Feest et al., 2021). These exudates likely contributed to the higher leaf P concentration compared with V. corymbosum ( Tables 3 and 4 ). While there are currently no studies specifically addressing P mobilization around the cluster roots of G. avellana, research conducted on other Proteaceae species, such as Embothrium coccineum (Delgado et al., 2015; Renderos et al., 2022), have shown similar levels in the labile P fraction in the rhizosphere of mature cluster roots compared to non-cluster roots and bulk soil, hinting at the possibility of localized P mobilization and accelerated P uptake by plants with cluster roots.

Based on the N:P ratio thresholds established by Koerselman and Meuleman (1996), our findings indicate that G. avellana consistently experiences co-limitation of both N and P across all treatments. In contrast, the growth of V. corymbosum appeared to be predominantly constrained by P, when plants were grown under control soil conditions ( Figure 3 ). This observation reaffirms the high P demands of the V. corymbosum species, as previously described Pinochet et al. (2014) (≥ 16 mg P kg^-1 soil). Interestingly, in the Al-supplemented soil, V. corymbosum plants were co-limited by N and P. This was attributed to a decrease in foliar N concentration, particularly evident in conspecific plant pairs. The decrease in foliar N concentration due to Al toxicity has been previously reported in V. corymbosum (cv. Brigitta) (Qilong et al., 2020). This phenomenon is primarily associated with reduced root growth, resulting in a diminished nutrient uptake ability. In our study, a reduction in roots growth of V. corymbosum ( Supplementary Figure S3 ) was not observed. It can be speculated that a nutrient imbalance, rather than direct Al toxicity, was most likely the underlying reason for the negative effects of soil Al₂(SO₄) supplementation on the foliar N concentration.

Although it was expected to observe a facilitating effect on P acquisition, as in the case of other Proteaceae species inhabiting in extremely P-poor environments (Teste et al., 2020; Shen et al., 2023), a facilitating effect was found for G. avellana on V. corymbosum in foliar chlorophyll concentration and photosynthesis rate ( Figure 2 , Table 2 ), when both species growth together in Al supplemented soil. Since N is a fundamental element for photosynthetic pigments and consequently for the photosynthesis process (Lambers and Oliveira, 2019), the facilitating effect of G. avellana can be attributed to the fact that this species induced a higher N uptake rate in V. corymbosum when it grew in Al supplemented soil, presenting values similar to those when it grew in the control soil ( Table 4 ). In this context, it is relevant to mention that there is some evidence showing that, unlike other species of Proteaceae species inhabiting in extremely P-impoverished soil (e.g. South Africa and Australia), the southern South American Proteaceae Embothrium coccineum induced cluster roots formation mainly under poor N soils, suggesting that this roots structures could promote N–acquisition (Piper et al., 2013). In Hakea actites (Proteaceae), the cluster roots formation and expression of peptide transporter were up-regulated in response to N starvation, probably to increase N uptake under conditions of low N availability (Paungfoo-Lonhienne et al., 2009). From these insights, it is proposed that cluster roots of G. avellana could indeed possess mechanisms which can facilitate N availability to neighboring plant species. In this regard, one plausible mechanism could involve organic acid release, which stimulate microbial activity in the rhizosphere. Microbial degradation of organic matter can release N, which may subsequently contribute to higher levels of available N for plants. While this hypothesis provides a compelling direction, additional studies are required to reveal the actual role of cluster roots of G. avellana in N acquisition and its possible mechanisms underlying this process.

A significantly higher photosynthetic rate was observed in G. avellana plants when they were grown in Al supplemented soil, regardless of the plant combinations involved. This intriguing finding is congruent with the growing body of evidence which underscores the benefits associated with the presence of Al in the soil. This is especially pertinent for plant species that are naturally adapted to acidic environments and particularly pronounced in Al hyperaccumulator species (Watanabe and Osaki, 2002; Bojórquez-Quintal et al., 2017; Muhammad et al., 2018; Sun et al., 2020). Similarly, studies have reported higher photosynthetic rates in Al hyperaccumulator plants, such as Camelia sinensis and C. japonica when were supplied with Al compared to control conditions (Mukhopadyay et al., 2012; Hajiboland et al., 2013; Liu et al., 2020). In Qualea grandiflora, another Al hyperaccumulator plant species, a significant increase in photosynthetic pigment concentrations, encompassing chlorophyll a, b, and carotenes, was observed in plants supplied with Al (Cury et al., 2019). Our findings support the contention that Al may exert a positive impact on the photosynthetic rate in the Al-hyperaccumulator plant. In our study, the increase in the photosynthetic rate in G. avellana is probably due to the increased stomatal conductance ( Table 2 ) and increased proportion of chlorophyll a ( Figure 2C ), the primary chlorophyll pigment for photochemisty (Björn et al., 2009). Furthermore, the increase in leaf Mg concentration ( Table 3 ), a central element within the chlorophyll molecule, may also contribute to this effect (Farhat et al., 2016).

In addition to Mg, the concentration of other essential mineral nutrients such as K and Mn significantly increased in both leaves and roots of both species when grown in the Al supplemented soil. This effect held true regardless of whether the neighboring species is interspecific or conspecific. Furthermore, in the Al hyperaccumulator species, G. avellana, there was a significant rise in Na levels in leaves; and a concurrent increase in Fe, Cu and Zn in the roots was observed. The beneficial effects of Al on plant nutrient uptake have been observed in other species. For instance, Osaki et al. (1997) reported increased content of N, P, Mg and K in different organs of several plant species upon Al treatment. Similarly, the stimulation of growth by Al in the Al hyperaccumulator C. sinensis was attributed to the increased absorption of vital plant nutrients, such as Ca, Mg, K and Mn (Fung et al., 2008), as well as N and P (Konishi et al., 1985). Bojórquez-Quintal et al. (2017) proposed that Al could induce the expression or activity of transport proteins (channels and transporters) and an alteration in the membrane potential and proton (H+) flux. This, in turn, could facilitate nutrients uptake by plant roots.

Carotenoids are known to play important roles in plants as antioxidants, and during environmental stress, plants usually respond by increasing their carotenoid concentrations (Havaux, 2014). Intriguingly, our observations revealed significantly lower concentrations in leaf carotenoids of G. avellana plants cultivated in Al-supplemented soil compared to control ( Figure 2E ), suggesting that this species may not have been experiencing stress during Al exposure. Indeed, the lipid peroxidation of G. avellana leaves was lower in plants that were cultivated in the Al supplemented soil. Similar results were found in the roots of C. sinensis, where it was observed that Al reduced the lipid peroxidation in root tips, which was related to the fact that Al-induced an increase in the activities of antioxidant enzymes (e.g. superoxide dismutase, catalase, and ascorbate peroxidase (Ghanati et al., 2005). Thus, the results suggest a positive effects of Al on the membrane integrity of G. avellana plants. On the contrary, higher lipid peroxidation was observed in the leaves of V. corymbosum plants that were cultivated in the Al supplemented soil, supporting previous reports indicating membrane damage induced by Al in this species (Ulloa-Inostroza et al., 2016; González-Villagra et al., 2021). Interestingly, V. corymbosum accompanied by G. avellana reduced significantly the lipid peroxidation in leaves when grew in the Al supplemented soil, clearly showing a facilitating effect of G. avellana towards V. corymbosum. It is proposed that, by increasing the N concentration of V. corymbosum when it grew with G. avellana ( Table 4 ), V. corymbosum could have increased the activities of antioxidant enzymes, thus decreasing lipid oxidation.

In both species, leaf Al concentration was similar among treatments, suggesting a high self-regulation in Al uptake. This adaptive mechanism is highlighted by Delgado et al. (2019), who found that G. avellana growing in natural conditions showed similar leaf Al concentrations, independent of Al saturation percentage and soil pH. These authors reported ranges from 3,959 to 6,256 mg Al kg^-1 for mature leaves and 7,150 to 11,040 mg Al kg^-1 for senescent leaves of adult plants. In contrast, the current study used of 2-year-old plants, with an average of 1,505 mg Al kg^-1. It is most probable that the capacity for Al hyperaccumulation in this species, increases with the age of the plant. With respect to roots Al concentration, G. avellana plants exhibit significantly higher values when grown alongside V. corymbosum, compared to cultivation alongside conspecific species in soil without Al supplementation ( Table 3 ). It was found that in this soil condition, leaves of V. corymbosum were limited by P ( Figure 3 ) and therefore, it is likely that V. corymbosum activated mechanisms such as roots carboxylate exudation to mobilize P from the soil. Such responses are common among plants facing P deficiency (Chen and Liao, 2016). Beyond liberating P from the soil, it also serves as a formidable defense against Al toxicity, particularly in plants that possess the inherent ability to exclude the entry of Al into their tissues. This is achieved through the formation of harmless Al-organic complexes within the rhizosphere, reducing the potential harm posed by Al (Kochian et al., 2004; Chen and Liao, 2016). Thus, it is proposed that the greater Al uptake of G. avellana was due to the fact that it took up the Al excluded by V. corymbosum. Interestingly, V. corymbosum presented higher Al concentrations in the roots than in leaves (36 to 69 times more), a pattern observed in Al-sensitive plants (Kochian et al., 2004; Alarcón-Poblete et al., 2020; Cárcamo-Fincheira et al., 2023). This trend underscores a limited translocation of Al, thus, confirming the low Al tolerance in the aerial part of the V. corymbosum (Star cultivar) plant.

When plants were grown in Al supplemented soil, both species increased their Mn uptake. This could be due to Al supplementation decreasing the soil pH from 6.1 to 4.7 ( Table 1 ) thereby increasing the solubility of Mn with decreasing soil pH (Lambers and Oliveira, 2019). On the other hand, several authors have reported that leaf Mn concentration is a good proxy for rhizosphere carboxylate concentrations (Shane and Lambers, 2005; Lambers et al., 2015b; Pang et al., 2018; Lambers et al., 2021). This is because carboxylates exuded by roots simultaneously mobilize P and other nutrients from the rhizosphere, especially Mn, which enters roots via transporters with limited substrate specificity (Lambers et al., 2021). Indeed, the leaf Mn concentration in 100 chickpea genotypes has been positively correlated with carboxylate amount in rhizosheath (Pang et al., 2018). In the current study, there was a 2-fold increase in foliar Mn concentration in G. avellana when grown in Al supplemented soil, while in V. corymbosum this increase was 7-fold, which was independent of the neighboring species. These results suggest that V. corymbosum presented high stress in the Al-supplemented soil and it is likely that this species may have exuded large amounts of carboxylates under these conditions.

The S values were exceptionally high in the soil supplemented with Al₂(SO₄)₃ compared with those values found in the soil of control treatment ( Table 1 ). Consequently, leaf S concentrations of G. avellana and V. corymbosum plants supplemented with Al₂(SO₄)₃ were up to 12 and 15 times more than control plants ( Tables 3 , 4 ), with these values being considered in the high ranges for plants (~ 10 mg kg^-1 DW) (Lambers and Oliveira, 2019). Sulfur is integral to numerous physiological processes in plants, including protein synthesis, enzyme activation, chlorophyll formation, defense mechanisms, and secondary metabolite production (Kopriva et al., 2019). It has been reported that S can alleviate the toxicity of metals such as cadmium (Ferri et al., 2017; Lyčka et al., 2023) and Al (Guo et al., 2017), because some S-containing compounds, such as glutathione and phytochelatins, play vital roles in the complexation of metals and their subsequent sequestration into vacuoles. Additionally, these S-containing compounds contribute to the alleviation of oxidative stress by functioning as antioxidant molecules. Our results showed that the addition of Al₂(SO₄)₃ seems to benefit G. avellana, due to increased proportion of chlorophyll a ( Figure 2C ), photosynthetic rate ( Table 2 ), leaf Mg concentration ( Table 3 ) and decreased lipid peroxidation ( Figure 4 ). On the contrary, V. corymbosum plants worsens their condition under the soil supplemented with Al₂(SO₄)₃, raising the possibility that S supplementation may not have been sufficient to alleviate Al toxicity or that the S could have generated a toxic effect on the plant. Most studies have focused on the interactions occurring between S and other macro and micro-nutrients when plants are subjected to S deficiency (Courbet et al., 2019), however, the excess of S and its effect on the plant has been scarcely explored. We recognize that future studies are necessary to elucidate the effect of Al and S separately on these plants. In any case, it is important to highlight and report that G. avellana facilitate to V. corymbosum when both species grown in the Al₂(SO₄)₃ supplemented soil. Additionally, it is interesting to mention that unlike G. avellana, another Proteaceae species, Hakea prostrata, tightly controls its S acquisition (Prodhan et al., 2017). The ability to modulate S and N uptake in some species, has been linked to match its low protein concentration and low demand for rRNA, and its consequently low P requirements (Prodhan et al., 2017), as an adaptation of plants that have evolved in extremely P-impoverished habitats [values of total soil P ranging from 6.6 to 20.3 mg P kg⁻¹ (Hayes et al., 2014)]. Thus, it is probable that G. avellana, which has evolved on relatively nutrient-rich soils (values of total soil P ranging from 63.1 to 951.6 mg P kg⁻¹ (Delgado et al., 2018)), has not evolved the ability to regulate S uptake.

Contrary to expectations, it was rather surprisingly found that G. avellana did not contribute to higher growth rates either P acquisition in V. corymbosum. On the contrary, G. avellana shows better growth rates when grown associated with V. corymbosum. In addition, V. corymbosum had a decrease in root P concentrations, when grown in association with G. avellana, indicating a highly competitive capacity of this latter species for P acquisition. On the other hand, G. avellana did not assist in the decrease of Al uptake of V. corymbosum, although it contributed to an increase in N acquisition, and consequently higher chlorophyll concentrations and photosynthesis rates. Besides, V. corymbosum decreased the lipid peroxidation in leaves when grown in soil with Al supplementation and accompanied by G. avellana. Overall, our results suggest a facilitating effect of G. avellana to V. corymbosum and vice versa. The benefit of facilitation of G. avellana to V. corymbosum is only observed when both species grown in the Al supplemented soil. The information provided in this manuscript is relevant to know some nutritional and physiological aspects of these species. Future research is required to reveal the possible role of cluster roots of G. avellana in the N acquisition. Finally, this study has opened avenues for future research, suggesting the need for additional facilitation tests employing soil with more pronounced contrasts of the studied element (e.g. N, P or Al, and maintaining other soil properties constant) in order to find a greater impact of the nutrient under study on growth of the plant.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

MD: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. PB: Conceptualization, Funding acquisition, Investigation, Methodology, Supervision, Writing – review & editing. GB: Funding acquisition, Methodology, Writing – review & editing, Formal analysis. ML: Investigation, Methodology, Resources, Writing – review & editing. PD: Investigation, Methodology, Resources, Writing – review & editing. AV: Conceptualization, Supervision, Visualization, Writing – review & editing. MR-D: Investigation, Methodology, Resources, Writing – review & editing.