Genotypic variation in cadmium uptake and accumulation among fine-aroma cacao genotypes from northern Peru: a model hydroponic culture study

ABSTRACT

The regulation established by the European Union regarding cadmium (Cd) is hindering the global trade of cocoa and cocoa products. The objective was to investigate the variation in Cd uptake and accumulation in plants of five genotypes of ‘fine-aroma’ cocoa grown under hydroponic conditions with the addition of 20 µmol of CdCl₂. Our results showed that Cd uptake, translocation, and accumulation at leaf and root tissue levels varied significantly among genotypes. Cd accumulation in the root system was manifested after 24 h of cultivation exposed to Cd, with concentrations ranging from 72.40 mg kg⁻¹ and 112.24 mg kg⁻¹. Polynomial analysis showed a clear relationship between Cd accumulation and plant exposure time. It was observed the INDES 38 genotype is the most promising for future breeding programs because despite high Cd accumulation in its roots (156.75 ± 0.90 mg kg⁻¹), the amount of metal transferred to leaves was low.

KEYWORDS:

• Biodiversity
• foodchain
• genotypic variation
• mitigation strategies
• Theobroma cacao

Introducction

Cadmium (Cd), a heavy metal widely used in various industries, has been extensively studied in plants due to its toxic effects [1]. Its existence within ecosystems has a natural origin, but it’s various anthropogenic processes have led to a continuous increase in the level of this element [2].

The risk of Cd contamination derives not only from its high level of toxicity and mobility but also from its capacity to bioaccumulate in different living beings, making it alarming that, even at low concentrations, its availability is prolonged over time [3]. Dietary intake as the main source of exposure and accumulation in humans has been made public through important agricultural crops such as cocoa [4,5], potato [6,7], blueberry [8], rice [9,10], wheat [11], maize [12] and different root, fruit and leaf vegetables [13–15]. However, despite their known detrimental health effects of this metal, exposure to it continues and is increasing in the population, particularly in developing countries [16,17].

Cocoa (Theobroma cacao L.) is currently one of the most important crops in the world because its seeds are the main raw material for the chocolate industry [18]. Furthermore, it is arguably one of the crops that should be prioritized to boost social development programs, as it economically benefits many of the world’s poorest sectors [19,20]. In Peru, for example, cocoa is the second most economically important crop after coffee [21]. However, the current interest in cocoa is not only because of its high economic value, but also because of the bioaccumulation of Cd in the beans. In a recent study, it was found that in some regions of Peru, such as Tumbes, Piura, Amazonas, Loreto, Huanuco and San Martin, the Cd content in cocoa exceeds regulatory limits, which may have an impact on more than 20% of producers nationwide [5]. In other words, this scenario generates significant costs for the economy, fostering to date great concern for the cocoa sector, since according to European Union (EU) regulations, any derived product containing at least 50% cocoa must have Cd levels below 0.8 mg kg⁻¹ [22].

The regulation that came into force at the beginning of 2019 requires extensive research in laboratory, greenhouse, and field conditions to find solutions to the problem posed by Cd enrichment of cocoa beans. According to the review of the current state of research on Cd in cocoa, the use of genotypes with low Cd accumulation/translocation as rootstocks could be an alternative in the short term to reduce Cd bioaccumulation [21,23,24]. However, this requires a prior understanding of the mechanisms by which Cd accumulates in cocoa plants. As suggested by Barati et al. (2023), understanding the process of mineral uptake and translocation can provide crucial information on the physiological mechanisms necessary for plant growth and development. On the other hand, the study of within-species diversity can improve our understanding of the evolutionary history of the crop and provide valuable information to develop conservation and breeding programs [25]. In this particular case, this could allow the identification of cocoa genotypes that absorb and accumulate reduced concentrations of Cd in different tissues and organs [26]. Therefore, there is a case for an independent study of new tolerant genotypes, which could have novel mechanisms to combat Cd bioaccumulation.

In the present study, Cd uptake and accumulation in juvenile cacao plants grown under hydroponic conditions were characterized. For this purpose, leaves and roots of five fine aroma cacao genotypes were analyzed to (i) study the dynamics of Cd absorption and accumulation and (ii) select which genotypes show low-affinity for Cd translocation. This research will serve as a basis to promote new research using biotechnological tools to develop plants with a lower affinity for Cd transport and to ensure the commercialization and sustainability of cocoa production in the long term.

2. Materials and methods

The study was conducted in the northern Peruvian province of Chachapoyas at the Universidad Nacional Toribio Rodríguez de Mendoza (6° 24′ 58.1″ S, 77° 24′ 47.1″ W; 2276 m s.n.m.). The trials were conducted under greenhouse conditions with an average day/night temperature of 25/14°C and a relative humidity of 70%.

2.1. Identification of cocoa genotypes

The plant material was identified in plots in the district of Copallin in the province of Bagua, Amazonas region – Peru (Figure 1), based on studies of fine aroma cacao diversity carried out in northern Peru [27–29]. The initial screening required calculating the Cd levels accumulated in the leaves of 13 ‘native fine aroma’ cacao genotypes, as well as analyzing soil samples collected in the vicinity of the identified plants. The results obtained made it possible to determine the Cd absorption ratio (_{Cdleaf/Cdsoil}) for each genotype and finally to select 5 genotypes with an absorption ratio between 1.40 and 4.79.

Figure 1. Distribution map of the five fine-aroma cocoa genotypes collected for the Cd sensitivity test.

2.2. Cadmium sensitivity test setup

The seeds of each genotype were washed with water to remove mucilage before planting in sawdust for germination. Subsequently, at the age of sixty days, seedlings were transferred to a hydroponic system containing Hoagland’s solution (pH 5.2) as a nutrient source [30]. Each plastic tray contained thirty seedlings and 10 L of nutrient solution (replaced once a week and continuously aerated by air pumps). All the products for preparing the nutrient solution were obtained from Sigma‒Aldrich (99.99% trace metals basis). Ninety days after planting, the plants were stressed with 20 µmol Cd (addition of CdCl₂; 99.99% trace metals basis, Sigma‒Aldrich) for six days (Figure 2).

Figure 2. Scheme of assembly of the floating root hydroponic system for Cd sensitivity testing in young plants of five genotypes of fine-aroma cocoa.

2.3. Quantification of cadmium uptake

After Cd application, sample collection (roots and leaves; Figure 3) was performed at different time intervals (0, 24, 48, 96, 120, and 144 hours; Figure 2). Leaves and roots were washed with plenty of deionized water and then dried at 70°C for 48 h in an oven (Binder FD 115, Binder GmbH, Germany). To remove the apoplastically bound Cd, roots were immersed for 15 min in 20 mmol L⁻¹ EDTA-Na₂ (ACS reagent, Sigma‒Aldrich) [31].

Figure 3. Sample collection scheme and analysis process for the quantification of Cd accumulation in young plants of five fine-aroma cocoa genotypes.

Mineralization of leaf and root samples was carried out by the dry method (Carter & Gregorich, 2007). For this purpose, 1000 mg of the sample was calcined at 480°C for 8 h in a muffle furnace (Thermolyne, Thermo Scientific, U.S.A.). Next, digestion was performed using 2 mL of concentrated HCl (ACS reagent, Sigma‒Aldrich) and 2 mL of deionized water in a fume hood (Protector Basic 47, Labconco Corp., U.S.A.). The digested sample was filtered and then diluted with deionized water to a volume of 25 mL. The total cadmium concentration in the samples was determined spectrophotometrically using a microwave plasma atomic emission spectrophotometer (4100 MP-AES, Agilent Technologies, U.S.A.) (Figure 3).

To ensure the quality of the analysis, calibration standards were prepared from 1000 mg L⁻¹ of Cd (traceable to SRM from NIST Cd(NO₃)₂ in HNO₃ 0.5 mol L⁻¹ 1000 mg L⁻¹ Cd Certipur®, Supelco). The seven-point calibration curve for Cd 228.802 nm (six standard references and a reagent blank) showed excellent linearity with a calibration coefficient greater than 0.999. A measurement precision of < 4% relative standard deviation (RSD) and recovery > 97% were achieved.

2.4. Determination of accumulation capacity and translocation factor

The efficiency of the plant in accumulating Cd in its tissues from the surrounding environment is calculated as follows [32]:

$\begin{array}{rl}& \mathrm{B}\mathrm{i}\mathrm{o}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{F}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{o}\mathrm{r}\left(\mathrm{B}\mathrm{C}\mathrm{F}\right)=\\ & \mathrm{C}{\mathrm{d}}_{\mathrm{p}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{t}}/\mathrm{C}{\mathrm{d}}_{\mathrm{h}\mathrm{i}\mathrm{d}\mathrm{r}\mathrm{o}\mathrm{p}\mathrm{o}\mathrm{n}\mathrm{i}\mathrm{c}\mathrm{s}\mathrm{o}\mathrm{l}\mathrm{u}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}}\end{array}$

The efficiency of the plant to translocate Cd from the root to the leaves was calculated as follows [33]:

$\mathrm{T}\mathrm{r}\mathrm{a}\mathrm{n}\mathrm{s}\mathrm{l}\mathrm{o}\mathrm{c}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{f}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{o}\mathrm{r}\left(\mathrm{T}\mathrm{F}\right)=\mathrm{C}{\mathrm{d}}_{\mathrm{l}\mathrm{e}\mathrm{a}\mathrm{f}}/\mathrm{C}{\mathrm{d}}_{\mathrm{r}\mathrm{o}\mathrm{o}\mathrm{t}}$

2.5. Relative chlorophyll content

One leaf from each plant was selected and labeled to determine the relative chlorophyll content. A portable measuring device (SPAD-502 Plus, Konica Minolta, Japan) was used for the reading. Measurements were taken under indirect sunlight on the fourth leaf (outside its midrib) (Figure 7a), and the average of three readings per leaf was recorded.

2.6. Experimental design and data analysis

The experiment was arranged as a completely randomized design, with two treatments: control (0 µmol CdCl₂) and Cd stress (20 µmol CdCl₂) and three replicates per genotype (i.e. JSM 2, JMB 1, INDES 39, INDES 38 and JSM 3) for each treatment. The experimental unit consisted of a 10 L tray, each containing 30 seedlings of each genotype. For data analysis, net Cd accumulation was estimated, which was derived from the difference between the Cd concentrations recorded in the stress-tested seedlings and the control seedlings.

The assumptions of normality (Shapiro-Wilk) and homogeneity of variances (Levene) were verified. Data were subjected to a one-way analysis of variance (ANOVA). Means that were statistically significant were compared using Tukey’s test (p < 0.05). Additionally, a polynomial regression analysis was performed (p < 0.05). All analyses were performed with Minitab 21.4 statistical software.

3. Results

3.1. Accumulation of cd in the different genotypes

The analysis of variance showed that there were significant differences (p < 0.05) in the levels of Cd accumulation in the leaves and roots of the five genotypes of fine-aroma native cocoa (Figure 4).

Figure 4. Evolution of Cd accumulation levels in leaves (a) and roots (b) of five fine aroma cocoa genotypes. P values less than 0.05 (*) and less than 0.001 (****), according to Tukey’s test at the 95% confidence level. Mean values and vertical line indicate ± standard deviation (SD).

Cd accumulation levels in leaves during the first 48 hours of exposure (CdCl₂) were not significantly different between genotypes, although genotype JSM 2 was found to have somewhat higher levels (up to 0.31 ± 0.04 mg kg⁻¹) (Figure 4a). Cumulative Cd levels showed a significant increase between 96 and 144 hours of exposure (CdCl₂), especially in genotypes JSM 2 and JMB 1, which had increases of 125% (0.93 mg kg⁻¹) and 1906% (1.27 mg kg⁻¹), respectively (Figure 4a). On the other hand, although at 144 hours, Cd accumulation levels in genotypes INDES 39 (0.76 ± 0.10 mg kg⁻¹) and JSM 3 (0.63 ± 0.07 mg kg⁻¹) were significantly low (with respect to genotypes JSM 2 and JMB 1), there was an increase in Cd levels between 145 and 190%. Finally, it is important to note that genotype INDES 38 presented the lowest amount of Cd accumulation, with a level below 0.50 mg kg⁻¹ (Figure 4a).

At the root system level, Cd accumulation in all genotypes was evident after 24 hours of exposure to CdCl₂, with values ranging from a minimum of 72.40 mg kg⁻¹ to a maximum of 112.24 mg kg⁻¹ (Figure 4b). Genotypes INDES 38 (156.75 ± 0.90 mg kg⁻¹) and INDES 39 (141.72 ± 0.59 mg kg⁻¹) recorded their maximum Cd accumulation 48 hours after being established in the hydroponic system treated with CdCl₂, averaging an increase of approximately 45% within 24 hours (Figure 4b). For genotype JMB 1, there was a significant increase in Cd levels between 24 and 96 hours (time of maximum accumulation), going from 112.24 ± 0.40 mg kg⁻¹ to 143.44 ± 0.43 mg kg⁻¹, which represented an increase of 27.80%. On the other hand, it was observed that in the JSM 2 genotype, Cd accumulation increased considerably during the first 24 hours of exposure (CdCl₂) before reaching a stable phase during the following 96 hours (Figure 4b).

3.2. Polynomial regression analysis of cd accumulation in the different genotypes

To better understand the influence of time on Cd accumulation in the leaves and roots of cocoa genotypes with different fine aromas, a polynomial regression analysis was performed (Figure 5). Two of the five genotypes studied (INDES 39 and JMB 1) had a good fit to the nonlinear regression model (R² >0.75) at the leaf tissue level, while JSM 3 had a poor fit to the relationship between Cd accumulation and time of exposure to CdCl₂ (Figure 5a).

Figure 5. Polynomial regression curves for cadmium accumulation in leaves (a) and roots (b) of five genotypes of fine-aroma cocoa grown under exposure to CdCl₂ for different times.

For root tissue, a good polynomial fit was observed, indicating a clear relationship between Cd accumulation and CdCl² exposure time with high R² values (R² >0.90) (Figure 5b). The analysis demonstrates a clear trend toward an increase in tissue Cd levels during the first 72 hours of exposure for four genotypes (INDES 38, JSM 2, INDES 39, and JMB 1) and a subsequent decreasing trend. On the other hand, polynomial fitting suggests that Cd accumulation increases beyond 144 hours after CdCl₂ exposure for genotype JSM 3 (Figure 5b).

3.3. Distribution and translocation factor of cd in the different genotypes

The distribution of Cd for each genotype is presented as a percentage of the average amount of metal accumulated in the plant throughout the study (excluding the initial evaluation at 0 hours). In all genotypes, Cd accumulation was found mainly in the roots, representing between 99.24% and 99.84% of the total plant Cd content (Figure 6a). On the other hand, as expected, Cd bioaccumulation was high since hydroponic systems allow easy availability of the metal, with factors higher than 30 for all genotypes (Figure 6a).

Figure 6. (a) Cd distribution in leaves (green color) and roots (brown color) of five fine aroma cacao genotypes. (b) polynomial regression fitting curves for the root-leaf Cd translocation factor in five cacao genotypes. TF: translocation factor; BF: bioconcentration factor.

The translocation factor results clearly show an increasing trend of Cd accumulation with time (Figure 6b). It is observed that JSM 2 exhibits clearly higher values than INDES 38, indicating that while the latter absorbs more Cd from the medium (a genotype with a high bioconcentration factor), little metal is released from the root to the various aerial structures of the plant. The translocation factor was fitted to a polynomial trend line with a fit greater than 91% for all genotypes.

3.4. Relative chlorophyll content

The methodological procedure and the results of the SPAD index for each cocoa genotype are shown in Figure 7. The results show that genotype JSM 3 had a significantly lower SPAD value (23.56 ± 2.71) compared to the other genotypes. At the general level, it was observed that genotypes INDES 39 (29.61 ± 2.71) and JSM 2 (28.03 ± 5.22) were distinguished by recording a relative chlorophyll content slightly higher than INDES 38 (26.94 ± 2.36) and JMB 1 (27.39 ± 2.07) but not significantly different from the latter two (Figure 7b).

Figure 7. (a) schematic diagram for SPAD measurements and (b) relative chlorophyll content in five fine-flavored cocoa genotypes. P value less than 0.001 (****).

4. Discussion

4.1. Plant growth and cadmium uptake

Although Cd is present in the earth’s crust, its level has steadily increased due to a variety of events caused by mankind [2]. For example, the hydrocarbon industry is a process related to the origin and/or increase of heavy metals (such as Cd and Pb) in the soil and river sources of the Peruvian Amazon because of accidental spills or habitual discharges of formation waters extracted from the oil fields of the Northern Peruvian Pipeline [34,35]. Therefore, the flora and fauna of the region, as well as the local population, are at risk due to this contamination.

Due to its harmful effects on humans, animals, and plants, Cd is a heavy metal that has caused much concern. Although it does not serve an essential function in plants, it can be readily taken up by roots and concentrated in the apoplasm and root cells [36,37]. The accumulation of this metal can have a negative impact on processes such as photosynthesis [38,39] and nutrient uptake [40,41] and, consequently, alter normal plant growth and development [42–44]. Cocoa cultivation represents a significant threat due to its negative impact on physiological processes but also because of its accumulation in beans. Therefore, it is crucial to develop research to understand the mechanisms of Cd uptake and accumulation in plants [45,46].

4.2. Kinetics of cadmium accumulation and translocation in cocoa

In the current study, it was found that the concentration of Cd in the roots was higher than that in the leaves. This implies that only a small amount of Cd entering the root system was transferred to the aerial part of the plant. Several species, such as Lactuca sativa [47], Arabidopsis thaliana [48], Miscanthus sacchariflorus [45], and Theobroma cacao [49], have this common feature. Therefore, the retention mechanism that the cells of the root system exert on the metal (through the cell wall and/or vacuoles), which limits its entry into the xylem (pathway to transfer Cd: root → shoot-leaves), could explain the root>shoot-leaves distribution pattern [50–54]. In general, Cd content in plants decreases in the order of roots > stems > leaves > fruits > seeds [42]. That said, Cd sequestration in roots is possibly a defense mechanism against its toxic effects that can damage the aerial organs of the plant [55].

Cocoa plants in the present study showed that Cd accumulation in leaves increased in relation to the time they remained in the hydroponic system. Previous studies also observed similar responses in M. sacchariflorus [45] and Celosia argentea [56]. These results show a logical behavior since Cd uptake is mediated by the uptake of essential elements (such as Ca, Fe, mg, Cu, and Zn) for plant development [54,57]. Therefore, for entry into the plant, Cd may compete with nutrients because they share physiological mechanisms for uptake, although it is possible that they use different membrane transporters for translocation from root to aerial organs [58]. On the other hand, as observed in the study, the tendency to increase the levels of absorbed Cd may be mainly related to the use of juvenile cacao plants, since these had small biomass to distribute the metal. Conversely, due to the larger biomass of an adult plant, the dilution effect may reduce the concentration of Cd in leaves [59,60]. However, increased leaf biomass could mean higher Cd uptake and accumulation [56].

In the root system, it was observed that after reaching a peak concentration, Cd accumulation levels decreased. This behavior can be explained by events such as the increase in Cd translocation to the aerial organs of the plant and the reduction in the fraction of Cd available in the medium [61,62]. Interestingly, in this study, it was observed that the increase in leaf Cd coincided with the decrease in root Cd levels; this implies that Cd mobilization is mainly unidirectional. This result also indicates that Cd continues to accumulate even in older leaves and is not redistributed with the onset of senescence [31]. However, it is important to note that the average Cd accumulation in the roots of each of the five cocoa genotypes exceeded 90 mg kg⁻¹. The high level of Cd accumulated in the root system indicates that defense mechanisms are activated, such as Cd sequestration in root vacuoles, to minimize toxic effects [43,63]. Nishizono et al. [64] pointed out that the ability of root cell walls to trap Cd is due to the number of available functional groups (such as hydroxyl, carboxyl) that can form complexes with trace metals. However, it is important to note that Cd accumulation in roots varies depending on the type of plant and the concentration of available Cd [65]. Here, our results suggest that some of the fine aroma cacao genotypes can be considered Cd-exclusive plants attributed to low-affinity transport. This is because absorbed Cd is sequestered or immobilized in roots, which limits its translocation to shoots and leaves [63,66].

4.3. Role of genetic diversity in cd uptake in cocoa

According to the findings of this study, there are variations in Cd uptake, accumulation, and translocation among cocoa genotypes. In this regard, Xin et al. [67] reported that subcellular accumulation of Cd in plants varies significantly among plant species, between cultivars and genotypes of the same species. In this context, of particular interest is the INDES 38 genotype, as it is characterized by a low propensity for Cd translocation. Overall, these results suggest that the search for long-term methods/strategies to reduce Cd concentrations in cocoa beans requires genetic approaches. In that context, many studies in recent years have reported on genotypic variability and its relevance to counteract heavy metal accumulation [4,26,68]. For example, the results from a study examining 100 cocoa accessions revealed that Cd accumulation exhibits significant differences among accessions [26], revealing possible genetic differences in Cd uptake and translocation due to the expression of specific transporter proteins [31]. Therefore, it is crucial to continue developing studies that help to understand the mechanisms of Cd adsorption in cocoa.

The root is the main organ of entry and accumulation of heavy metals in plants because not all tissues or organs have the same capacity to accumulate heavy metals [69]. After metal uptake into the root symplasm, three processes control their movement from the root into the xylem: sequestration of metals within the root cells, symplastic transport into the stele, and release into the xylem [70]. Inside the root, after it is released into the xylem, Cd moves into the aerial organs of the plant via apoplastic and symplastic pathways [70,71]. Recently, by stable isotope pulse chasing (¹⁰⁸Cd), a study revealed that in cocoa beans, Cd is only incorporated through the phloem from a stationary reservoir in the tree and not by direct xylem transfer from the roots [72]. In addition, it has been suggested that there are different genes/proteins that are responsible for Cd uptake, transport, and accumulation in plants. These include the calcium transporter LCT1 [73], IRT1, which belongs to the Zn and Fe transporter family [74], YSL (Yellow Stripe-Like), which belongs to the oligopeptide transporter family [57], and NRAMP, a macrophage gene associated with natural resistance in T. cacao [31,75].

Taken together, the results of this study show promise for identifying genotypes with low affinity for Cd uptake and transport. A recent study in northeastern Peru found moderate genetic diversity; however, they noted that internal and external correspondence analysis revealed that the cocoa samples collected were generally distinct and unique [27]. The combination of genes during a crossing of plant material ensures a high degree of variability [76,77], and consequently, genotypes with desired agronomic traits are likely to be found [77]. In this regard, Bustamante et al. [27] highlighted that in the Amazon region, the use of crosses between trees for genetic improvement is possible due to their low or null fixation indexes (indicative of the absence of inbreeding). This scenario opens a gap for the development of breeding programs that can contribute to the selection of genotypes that regulate the accumulation of Cd in their organs.

5. Conclusion

The study confirms that cocoa genetic diversity can help to comply with the new European Union regulations on Cd limits for cocoa marketing. The Cd uptake and accumulation of the five genotypes varied significantly, but in all cases, Cd accumulation at the root system level represents more than 99% of the total Cd accumulated by the plant. The results confirm that cocoa plants show different intragenotypic responses, which may be related to defense mechanisms that decrease the transfer of Cd from the roots to the aerial tissues and organs. In this study, the ability to absorb, accumulate and translocate Cd allows the INDES 38 genotype to be potentially useful for breeding programs due to its low-affinity transport trait.

Overall, the study demonstrates that the identification of genotypes with a low-affinity for Cd transport together with agronomic practices such as the use of grafting can be a viable alternative to grow cocoa in a safe and sustainable manner with less health risks to consumers of cocoa products. However, to achieve this, it will require a deeper understanding of the complexity of the rootstock-scion relationship.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The research has received financial support from the SNIP project (312252)/CUI (2252878) “Creación del Servicio de un Laboratorio de Fisiología y Biotecnología Vegetal de la Universidad Nacional Toribio Rodríguez de Mendoza de Amazonas” (Creation of a Plant Physiology and Biotechnology Laboratory Service of the National University Toribio Rodríguez de Mendoza de Amazonas), and the project “High-Altitude Agriculture: Sustainable Crop Production Under Platic Tunnels in the Andes”, contract N° 100K-CAFSMPRPE-9, funded by PARTNERS OF THE AMERICAS of the Program The 100,000 STRONG in the Americas, executed by the Research Institute for the Sustainable Development of Ceja de Selva.