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by soil salinity, and more than 77 million ha from the arable area are affected by high salt contents [2,3].

Nevertheless, the main problem is that the principal crops are using plant species adapted to "old climatic conditions". Hence, it is necessary to look for alternative crops or "new crops" to face the "new climatic conditions". In this sense, it is crucial to consider some species that grew during millennia in mountain regions under extreme environmental conditions. Mountain plants, especially those adapted and cultivated in different altitudinal levels, can be crucial due to the gene pool that allowed these adaptations. In this scenario, quinoa (*Chenopodium quinoa* Willd.), which has grown throughout the Andes in South America for 5000 to 7000 years [4,5], can be considered a good option. During a long period of cultivation by the Aymaras and Inca populations, this crop was grown in different ecological zones, from sea level, in Chilean varieties [6], to 2000 to 4000 m above sea level (a.s.l.) along the Andes. Quinoa presents a C3 photosynthetic pathway according to anatomic and carbon isotope discrimination studies [6], with high photosynthetic assimilation and an intrinsic water use efficiency (iWUE) [7,8].

Several studies confirmed quinoa as an important source of nutritional components such as essential amino acids, fatty acids, minerals, soluble sugars, and bioactive components [9–11]. Furthermore, numerous reports in the field or lab conditions showed that quinoa is a species with high resilience to abiotic stress, including salinity, drought, high temperature, and ultraviolet B (UV-B) radiation [12–14]. Quinoa can tolerate very high salinity concentrations, producing a complete life cycle even at water salinities of 500–750 mM NaCl [15–17]. Thus, it can be grown in very marginal environments, for example, in North Africa, where soil salinization and drought are serious issues. The high nutritional value maintenance under different stresses makes quinoa an excellent crop to grow in the aforementioned marginal environments and face climate change. This quinoa tolerance to edaphic and harsh climatic conditions is related to this crop's high diversity along the Andes. In effect, there are more than 16,000 quinoa accessions stored in different seed banks in 30 countries, most of which are concentrated in Bolivia and Peru [18–20]. These accessions include the five ecotypes classified by Tapia (2015) [21]: (i) Valley quinoa; (ii) Altiplano quinoa; (iii) Salar quinoa; (iv) Sea level quinoa; and (v) Subtropical quinoa. Quinoa accessions of the different ecotypes are considered multipurpose plants: the seeds and leaves can be used as food, the biomass can be used as animal feed or as a cover crop, the colorants and the saponin content can be used in pharmaceutical and agroindustry, and plantings can serve as a phytoremediation tool for environmental cleanup [22–24].

Tapia's classification accepts an implicit fact: each ecotype can thrive in the environment in which it was adapted. However, quinoa has been introduced at higher latitudes as a complementary crop with good adaptation [25]. Currently, quinoa is cultivated and experimented on in almost 130 countries [19], including The United States [26,27], India [28], Italy [29], and Egypt [16], among others. Quinoa adaptation's success is due to its high plasticity to reach places that differ from its original location managing the sowing dates, taking advantage of the environmental offer (basically temperature and light). One of the perfect examples of quinoa plasticity is the CICA-17 variety. It was selected at 3800 m a.s.l. in Cuzco-Peru from the local variety Amarilla de Maranganí at Centro de Investigaciones de Cultivos Andinos (CICA, Universidad Nacional de San Antonio Abad del Cusco, Peru). CICA-17 belongs to the Altiplano ecotype, and it is tolerant to cold temperatures, low precipitation, and high salinity conditions. CICA-17 quinoa was introduced in northwestern Argentina in 1996–1998 from the American and European Test of Quinoa conducted by FAO-CIP [30]. Nowadays, CICA-17 is the variety most used by small producers in Northwest Argentina and especially in arid high mountain valleys (above 2000 m a.s.l.) where the climate is desert type. This variety has been cultivated in Egypt for ten years because of its good adaptation to its marginal places. Egypt has a considerable extension in arid, semi-arid, and marginal lands that constrain classical crop productivity. In this scenario, quinoa is becoming a complementary crop of high nutritional value. CICA-17 has a notable yield (near 2000 kg ha−1 ) either in mountain valleys at 2000 m a.s.l. as in lowlands

at 200 m a.s.l. in northwestern Argentina and Egypt [7,31,32]. Eisa et al. (2018) [33] and Ebrahim et al. [34] showed that the CICA-17 yield varied between 2000 and 3000 kg ha−1 in a marginal land at El-Fayoum oasis (Egypt).

CICA-17 quinoa can provide a new complementary crop for dry-saline lands. Still, it makes it necessary to study the mineral concentrations in different grain and seed organs, especially if different soil salinities influence these elements. Often, the mineral study in quinoa was focused on Na and K because of their relationship with the osmotic adjustment mechanisms that halophytes exhibit [35] or on the presence of a few minerals in different quinoa seed organs, and the abrasion effect on the Ca and K pericarp content [36]. All these approaches are essential to understand the physiological behavior of this promising species. It is also relevant to understand how these mineral contents can vary in different soils and climatic conditions in field conditions if quinoa is used as food in marginal lands. Nowadays, it is known that quinoa's seeds and leaves are a significant source of major minerals (calcium, magnesium, potassium, phosphorus, sulfur, and sodium), trace elements (iron, cobalt, zinc, copper, and manganese), and ultratrace elements (chromium, lithium, arsenic, nickel, molybdenum, selenium, tin, and vanadium) and that their content varies according to the genotype and the place where the cultivation is carried out [33,36–38]. However, the detailed mineral composition in different quinoa ecotypes is still scarce and even more so is their spatial distribution. For example, Prado et al. [37] reported 18 minerals present in quinoa grains, while Konishi et al. [36] mentioned only six minerals. Hence, we investigated the grain yield and size, the occurrence and content of minerals and their spatial distribution in the different grain tissues, in the CICA-17 quinoa crop grown in field conditions on soils with different salinity in the marginal lands of the Egypt.

#### **2. Results**

#### *2.1. Soil and Irrigation Water Analysis*

Electrical conductivity (EC) and organic matter, Cl, Na, Mg, K, Ca, SO4, and Fe contents are higher in saline soil (Table 1). These parameters increase 43, 24, 17, 9, 2.6, 4, and 1.3%, respectively, compared to the non-saline soil. Table 2 summarizes the water irrigation analysis for both cases.

**Table 1.** Physical and chemical properties of soil samples collected in two saline and non-saline locations of Egypt.

1 Sp: Saturation percentage; 2 EC: electrical conductivity; 3 DB: bulk density; 4 OM: Organic matter. Each value is the mean of n = 6 samples.

**Table 2.** Physical and chemical properties of irrigation water of saline and non-saline locations.

#### *2.2. Grain Yield* The weight of 1000 seeds of CICA-17 quinoa decreased by 13% in saline conditions *2.2. Grain Yield*

*2.3. Grain Weight and Sizes*

*2.2. Grain Yield*

1).

Soil salinity negatively affected grain yield. A reduction of close to 47% concerning the grain yield was obtained in saline conditions compared to the non-saline one (Figure 1). compared to non-saline conditions (Figure 2). On the other hand, the grain size distribution (A: <1.41 – >1.0 mm; B: <1.68 – >1.41 mm; C: <2.00 – >1.68 mm; D: ≥2 mm) showed that the two largest grain sizes were unfavored by salinity conditions (Figure 3). Soil salinity negatively affected grain yield. A reduction of close to 47 % concerning the grain yield was obtained in saline conditions compared to the non-saline one (Figure 1).

Soil salinity negatively affected grain yield. A reduction of close to 47 % concerning the grain yield was obtained in saline conditions compared to the non-saline one (Figure

**−2 SO4**

**−2 SO4**

**−2**

**−2**

*Plants* **2021**, *10*, x FOR PEER REVIEW 4 of 14

**Table 2.** Physical and chemical properties of irrigation water of saline and non-saline locations.

*Plants* **2021**, *10*, x FOR PEER REVIEW 4 of 14

**Table 2.** Physical and chemical properties of irrigation water of saline and non-saline locations.

Non-saline 0.43 7.16 1.04 0.21 1.40 1.22 1.02 0.62 0.00 1.29 Saline 1.70 7.23 6.37 0.35 3.40 4.50 8.50 4.60 0.00 6.12

Saline 1.70 7.23 6.37 0.35 3.40 4.50 8.50 4.60 0.00 6.12

**Location EC pH Na+ K+ Ca2+ Mg2+ Cl- HCO−3 CO3**

**Location EC pH Na+ K+ Ca2+ Mg2+ Cl- HCO−3 CO3**

**dSm−1 meq L−1**

**dSm−1 meq L−1**

#### *2.3. Grain Weight and Sizes*

The weight of 1000 seeds of CICA-17 quinoa decreased by 13% in saline conditions compared to non-saline conditions (Figure 2). On the other hand, the grain size distribution (A: <1.41 – >1.0 mm; B: <1.68 – >1.41 mm; C: <2.00 – >1.68 mm; D: ≥2 mm) showed that the two largest grain sizes were unfavored by salinity conditions (Figure 3). **Figure 1.** Effect of non-saline and saline soils on grain yield (mean ± SE) on *C. quinoa* cv. CICA-17. Different letters above columns indicate significant differences between means at *p* < 0.05, according to the t-test.

**Figure 2.** Effects of non-saline and saline soils on the weight (mean ± SE) of 1000-seeds of *C. quinoa* cv. CICA-17 plants. Significant differences between means (*p* ≤ 0.05) are indicated by different letters above columns according to the t-test. **Figure 2.** Effects of non-saline and saline soils on the weight (mean ± SE) of 1000-seeds of *C. quinoa* cv. CICA-17 plants. Significant differences between means (*p* ≤ 0.05) are indicated by different letters above columns according to the *t*-test.

#### *2.4. Mineral Content in Quinoa Grains*

The most important mineral elements with biological activities showed two trends. While phosphorus (P), magnesium (Mg), and sodium (Na) increased their content under saline conditions, silicon (Si), potassium (K), calcium (Ca), and iron (Fe) contents decreased (Table 3). The same increase or decrease were detected for the other elements except for Mn, Cu, and Co, which exhibited the same behavior under non-saline and saline conditions.

**Figure 3.** Seeds diameter distribution of *C. quinoa* cv. CICA-17 obtained in non-saline and saline soils. All the differences were significant (*p* ≤ 0.05) according to the t-test. A: <1.41 – >1.0 mm; B: <1.68 – >1.41 mm; C: <2.00 – >1.68 mm; D: ≥2 mm. **Figure 3.** Seeds diameter distribution of *C. quinoa* cv. CICA-17 obtained in non-saline and saline soils. All the differences were significant (*p* ≤ 0.05) according to the *t*-test. A: <1.41 – >1.0 mm; B: <1.68 – >1.41 mm; C: <2.00 – >1.68 mm; D: ≥2 mm.

Cu 6.70 a 6.34 a 5.3 Li 0.04 b 0.05 a 19.1 Co 0.06 a 0.06 a 2.8 Nb\* 0.04 a 0.03 b 34.2 *\** Element detected for the first time in quinoa.

#### **Ultratrace elements** Y\* 0.04 a 0.02 b 39.0 *2.5.  Quinoa seed has different tissues (Figure 4). Using SEM-EDX analysis, we found that only the pericarp accumulated sodium in both non-saline and saline soils. Grains developed under saline conditions showed 6.3 times greater sodium content than those detected under non-saline conditions (Table 4, Figure 5). Chlorine, P, and Br also increased significantly in pericarp in saline conditions. By contrast, soil salinity led to a decrease in Ca, Al, Fe, Cu, and Si. Meanwhile, potassium was located mainly in the pericarp without significant differences due to the soil salinity condition. In the endosperm and the perisperm, the K content increased in saline soil (2.5 and 2.2 times, respectively) while its concentration was more significant (2.7 times) in the embryo in non-saline soil. The other elements studied did not change significantly.

*2.5. Mineral Spatial Distribution on Quinoa Grains*

ements studied did not change significantly.

Quinoa seed has different tissues (Figure 4). Using SEM-EDX analysis, we found that only the pericarp accumulated sodium in both non-saline and saline soils. Grains developed under saline conditions showed 6.3 times greater sodium content than those detected under non-saline conditions (Table 4, Figure 5). Chlorine, P, and Br also increased significantly in pericarp in saline conditions. By contrast, soil salinity led to a decrease in Ca, Al, Fe, Cu, and Si. Meanwhile, potassium was located mainly in the pericarp without significant differences due to the soil salinity condition. In the endosperm and the perisperm, the K content increased in saline soil (2.5 and 2.2 times, respectively) while its concentration was more significant (2.7 times) in the embryo in non-saline soil. The other el-

**Figure 4.** SEM photographs of *C. quinoa* cv. CICA-17. (**A**) grain external view; (**B**) seed with manually removed epicarp; and (**C**) longitudinal medial grain section. Pe, pericarp; Pe r, pericarp partially removed; Ep, episperm; En, endosperm; P, perisperm; Emb, embryo with a radicle-hypocotyl axis (r) and cotyledon (co). **Figure 4.** SEM photographs of *C. quinoa* cv. CICA-17. (**A**) grain external view; (**B**) seed with manually removed epicarp; and (**C**) longitudinal medial grain section. Pe, pericarp; Pe r, pericarp partially removed; Ep, episperm; En, endosperm; P, perisperm; Emb, embryo with a radicle-hypocotyl axis (r) and cotyledon (co).

**Table 4.** SEM-EDX comparative mineral relative percentages on quinoa grain tissues cultivated under non-saline and saline conditions (mean ± SD; n.d., not detected). Significant differences between means (*p* ≤ 0.05) are indicated by different letters behind the values according to the *t*-test. Comparisons were made between identical tissues under the two salinity conditions.

**Figure 5.** Spatial distribution of elements analyzed by SEM-EDX in grain longitudinal medial sections of *C. quinoa* cv. CICA-17, cultivated under non-saline (A–K) and saline (L–V) conditions. Ep, episperm; En, endosperm; P, perisperm; Pe, pericarp; Emb, embryo with the radicle-hypocotyl axis (r) and cotyledon (co). Arrowhead indicates the greater abundance of the mapped element. **Figure 5.** Spatial distribution of elements analyzed by SEM-EDX in grain longitudinal medial sections of *C. quinoa* cv. CICA-17, cultivated under non-saline (**A**–**K**) and saline (**L**–**V**) conditions. Ep, episperm; En, endosperm; P, perisperm; Pe, pericarp; Emb, embryo with the radicle-hypocotyl axis (r) and cotyledon (co). Arrowhead indicates the greater abundance of the mapped element.

The Mg content increased significantly in the endosperm under saline soil, while Cl content increased in embryo, and perisperm and decreased in the endosperm. Sulfur content differed between soil salinities. It was stored mainly in the embryo in non-saline conditions, meanwhile, it accumulated more and perisperm in saline soil (6.3 and 3 times, respectively). Nitrogen was detected only in the cotyledons, endosperm, and perisperm, reducing their content in the last two tissues under salinity conditions. Silicon, Fe, Br, Al, and Cu were below the detection limit for embryo, endosperm, and perisperm as for Na, they are detected only in pericarp.

#### **3. Discussion**

The high salinity soils of Sah El Tina have not produced harmful effects on the quinoa crop cultivation (cv. CICA-17). As a halophyte, the plant displayed a series of physiological and morphological adaptations that allowed it to complete its cycle [35–39]. Saline soils affect grain yield and seed size, according to previous research [40,41]. In our case, we detected a decrease near 47% compared to the yield get in non-saline conditions. The weight of 1000 seeds decreased by 13% in saline soil, and only smaller grains (< 1.68 mm) were favored. From a commercial point of view, this finding is also essential because markets prefer large grains instead of small ones.

Regardless of the soil's saline conditions where quinoa was grown, the most abundant minerals in the quinoa grains were Si, K, P, Mg, Ca, Na, Mn, Fe, Cu, Al, and Zn. However, an increase in P, Na, and Mg contents was observed due to soil salinity. Sodium, an essential mineral in the cell ionic balance, was detected only in the pericarp and not in other tissues (embryo, endosperm, and perisperm). It is evident that the pericarp structure (with different cell layers) is a shield to prevent the entry of sodium to the underlying tissues, but not for the chlorides that increased in saline conditions. Contradictorily, the mineral content of Mg and Mn was reduced by salinity in saline-sodic soils in Greece [42]. However, the salinity conditions of that field experiment are low (6.5 dS m−1 ) in comparison to our experiment (26 dS m−1 ) (Table 1). It is necessary to consider that 26 dS/m is the starting value of the saline soil's electrical conductivity (EC). It probably increases during the life cycle because of the soil and water quality used (with high EC). So we can assume that EC is further increased during cultivation, and the seeds were produced under more significant saline stress conditions than the starting one. This hypothesis must be verified in future studies in the field.

Regarding the occurrence of certain minerals in quinoa, it is necessary to consider whether the analyses are performed on grains (pericarp + embryo) or only in seeds (without pericarp). The desaponification process removes the pericarp and probably all the elements specifically present in this tissue (Table 4) This feature is mentioned in many cases where the analysis was performed on flour, but it is not clear whether the grains used contained the pericarp or not. Our results showed that Na was only in the pericarp, Mg was present in the pericarp, embryo, endosperm, and perisperm. While S was found in all the tissues, P was only absent in the perisperm. All these features should be considered to prepare quinoa-based foods since the product will not have the same mineral composition based on whether or not it is desaponified. It is important to point out that saponins are present in different quinoa organs (leaves, flowers, fruits, and seeds), especially in seed coats (pericarp). Saponins must be removed by different methods (physical or chemical) to avoid conferring a bitter flavor to the quinoa products. In general, saponin concentration ranges from 0.01 to 5% on a dry weight basis [43,44]. There are no data concerning saponin concentration in the quinoa crop of Egypt. However, González et al. [45] observed that saponin content in CICA -17, grown in desert climatic conditions in a high valley in Northwest Argentina, varied from 2.3 to 6.9% according to different nitrogen treatments. Considering that saponins can increase their concentration under saline condition [29], we can conclude that desaponification must be a necessary process before consuming quinoa either as grain or as flour because the maximum acceptable level of saponin in quinoa for human consumption varies from 0.06 to 0.12% [46,47]. Besides, desaponification removes sodium from the grain, avoiding its potential negative effects when consumed.

Except for Sn, Ni, and As, already found by Prado et al. (2014), the other ultratrace mineral elements (Rb, Sn, Th, Nd, Pr, Nb, Sm, Ni, Y, La, Ce, As, Ti, Ge, V, Zr, Ga, Zn, Ba, and

Pb) were first detected in quinoa in both non-saline and saline conditions. The presence of Cs was reported [48], but it was observed in aboveground parts of plants (stem and leaves). For many trace and ultratrace minerals detected in quinoa, the cellular level function is unknown, and their presence is only a passive accumulation from the soil and irrigation water. The role of Cr, Li, Si, Ni in human metabolism was already demonstrated [49,50]. Chromium participates in protein transport and improves diabetes [51], while Li is an essential element for regulating the central nervous system [52]. Following our results, Li has also been found in quinoa and amaranth (*Amaranthus caudatus*) consumed as food in the Northwest of Argentina [53]. Silicon is essential for Ca assimilation, the formation of new cells, and tissue nutrition [54], and Ni is necessary for the proper functioning of the pancreas [55]. Regarding arsenic, which is dangerous in high concentrations, several studies suggest too that it probably plays a physiological role in the metabolism of methionine, acting as an effector of acid metabolism amino sulfur [56]. Aluminum is typically considered a toxic element, but some studies in vitro suggest that this element plays an essential role in different biological systems (e.g., DNA synthesis stimulation or bone formation) [57].

In summary, quinoa can be considered a source of minerals as Cr, Li, Si, Ni, As, and Al concerning nutritious food and health. Besides, considering that quinoa foliage can accumulate some minerals such Ni, Cr, Cu, and Cd [24] and the hyperaccumulation of heavy metals in roots [58], we hypothesize that this species may be a good alternative for the remediation of contaminated soils.

#### **4. Materials and Methods**

#### *4.1. Plant Material, Site Description, and Experimental Design*

Grains of CICA-17 quinoa cultivar were selected for this study. Field experiments were conducted in 2015/2016 (mid-November to end-March) in two places in Egypt. One was in the Sahl El-Tina plain (named saline in this work), located in the northwestern coast of Sinai Peninsula (31◦ 020 N, 32◦ 350 E), and other in the Experimental Station (named non-saline) of Ain Shams University, Cairo (30◦ 030 N, 31◦ 140 E). Both places are arid, with an average annual precipitation of 60 mm yr−1 (Sahl El-Tina) and 20 mm yr−1 (Cairo), with rainfall concentrated between October and April. The average monthly temperature was 20.5, 12.8, 16.7 ◦C and 21.7, 12.1, 16.9 ◦C for maximum, minimum, and mean temperature in saline and non-saline places during the crop cycle, respectively. Representative soil samples in both locations were collected in the center of each plot at 0.60 m depth. Soil samples (6 in total) were obtained with a soil borer. Samples were mixed in the lab, and physical and chemical analyses were performed according to the standard methods published by Page et al. (1982) [59]. Results are listed in Table 1.

Experimental soils were prepared, including the construction of ridges. Compost with a rate of 8 t ha−1 and phosphorus at a 120 kg P2O5 ha−1 was added during the land preparation. Nitrogen was added as side-dressing at 160 kg N ha−1 in two equal rates after 30 and 51 days from the sowing date. Potassium was added at a rate of 140 kg K2O ha−1 at the flowering stage. Seeds of quinoa were sterilized with sodium hypochlorite solution (5% active chloride) for 10 min and then washed with distilled water several times and dried with tissue paper before planting. We sowed about ten seeds per hill to ensure germination. A complete randomized block design with six replicates (experimental plots) was used, with an average of 18 m2 for each plot (6 ridges with 5 m length and 0.6 m width). After four weeks from the sowing date, the seedlings were thinned to two or three seedlings per hill. Local sources provided irrigation water in both places, and Table 2 lists their chemical properties. A detailed description of the procedure is in Eisa et al. (2017) [16].

#### *4.2. Yield Components*

Ten quinoa plants in each experimental plot were cut and air-dried for 7–10 days at the harvesting stage. The dried panicles were threshed by hand. Subsequently, we determined the grain yield (kg ha−1 ), the seed sizes, and the weight of 1000 seeds (g). The percentage of grain sizes was determined with sieves of different meshes.

#### *4.3. Mineral Content*

The grain samples were dried in an oven at 65 ◦C to constant weight. The dried samples were ground into a fine powder, passed through a 60-mesh sieve, and ashed (electric oven at 575 ◦C for 16 h). After cooling, ash subsamples (0.1 g) were digested with 10 mL of HF/HClO4/HNO3 (5.0/2.5/2.5, *v*/*v*) mixture in a closed teflon vessel (Savillex, Canada) at 90 ◦C for at least 12 h. Once digestion finished, the remaining acid was evaporated on a hotplate to incipient dryness. Next, 1 mL of HNO3 was added twice and was evaporated again to incipient dryness. Residual sediment was dissolved in HNO3 (1 mL) and transferred into a 100 mL volumetric flask. The digestion vessel was rinsed with deionized water several times, and washing water was also transferred to the volumetric flask. Flask volume was made up with deionized water and then used for chemical element analysis. A total of 49 elements, including major (e.g., Ca, K, Mg, Na, and P), minor (e.g., Zn, Fe, Cu. Mn, Co, and Na), and ultratrace mineral elements (e.g., Cr, Li, As, Ni. Mo, Se, Sn, and V) were quantitatively determined by high resolution inductively coupled plasma mass spectrometry (Element XR HR-ICP-MS, Thermo Scientific, Germany) at the labGEOTOP of institute Geosciences Barcelona (Spanish Research Council, CSIC, Barcelona, Spain) [37]. Quality control of element determinations was carried out using internal standards and control samples of known composition for each analyzed element (BDH Chemical, England). Of the 49 elements analyzed, only 34 were listed because the rest (15 elements) were lower than the equipment's detection limit. Element concentration (mean of 3 repetitions) was expressed on a dry weight (dw) basis (Table 1). The accuracy and precision of analytical determinations by HR-ICP-MS were lower than 10%.

#### *4.4. Mineral Spatial Distribution*

To determine the spatial distributions and relative abundances of C, O, N, Na, K, Ca, Mg, P, Cl, Cu, Br, Al, Fe, Si, and S, three samples of achenes per saline and non-saline locations were analyzed by scanning electron microscopy (SEM, Supra55VP) coupled to an energy dispersive X-ray analyzer (EDX). SEM-EDX conditions were −10 ◦C, sample chamber pressure of −50 Pa, accelerating voltage of 20 kV). These analyses were conducted at CIME facilities (Integral Center of Electronic Microscopy of CONICET-UNT, Tucumán, Argentina).

Complete grains (to study pericarps), seeds with manually removed pericarp (to expose the seed coat), and median longitudinal sections of whole grains (cut with a blade) were mounted and fixed with a commercial adhesive on sample plates. Maps of mineral distribution were made in the median longitudinal sections. Semi-quantitative analyses of mineral abundance were obtained from equivalent areas of different tissues or organs (pericarp, episperm, endosperm, perisperm, embryo, and cotyledon mesophyll).

#### *4.5. Statistical Analysis*

The mean values between treatments per tissue/organs were compared using the *t*-test at *p* ≤ 0.05 level of probability (statistical package SPSS Inc., version 11.0, Chicago, IL, USA).

#### **5. Conclusions**

This study demonstrated the high resilience of the CICA-17 variety of quinoa to two contrasting edaphic situations in Egypt. It was clear that mineral concentration can change when interacting with different soil types, irrigation waters, and other environmental conditions. Soil salinity induces substantial changes in the distribution of minerals in the different grain tissues. Although quinoa is a significant source of major minerals such as K, P, Mg, Ca, and Na, the contents of Si, Ni, Cr, and Li may be important for their healthy behavior in human metabolism. The detection for the first time of 17 elements (Rb, Th, Nd,

Pr, Nb, Sm, Y, La, Ce, Ti, Ge, V, Zr, Ga, Zn, Ba, and Pb) in quinoa seed add a new value to quinoa crops as a potential application for phytoremediation processes. From our results, we can conclude that cv. CICA-17 may be a complementary crop in the marginal lands of high salinity in Egypt and the Mediterranean region, showing high potential concerning healthy food and environmental issues as phytoremediation.

**Author Contributions:** Conceptualization, J.A.G., S.E., and L.H.; methodology, M.I.M., G.I.P., D.A.G., M.R., M.E.A.E., and J.-L.F.-T.; formal analysis an investigation, J.A.G., S.E., D.B. and S.H.; writingoriginal draft preparation, J.A.G., S.E.; E.H.A.E.-S.; writing-review and editing, J.A.G., L.H., and D.B.; project administration, E.H.A.E.-S., and A.A.-A.; funding acquisition, J.A.G., and S.E. All authors have read and agreed to the published version of the manuscript.

**Funding:** The Ministry of Foreign Affairs, International Trade and Worship, Buenos Aires, Argentina, partially supported this research through the South-South cooperation project "Development of quinoa crop in marginal areas of Egypt", FO.AR-6429, and Fundación Miguel Lillo (Miguel Lillo 251, T4000, Tucumán, Argentina) under the Project B-0004-1 ("Ecophysiology of selected varieties of quinoa and others species with nutritional values in lab and field").

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available in this article.

**Acknowledgments:** Authors are thankful to Lic. Sebastián Buedo (Fundación Miguel Lillo) for providing laboratory facilities. We acknowledge the analytical assistance of labGEOTOP Geochemistry Laboratory (infrastructure co-funded by ERDF–EU Ref. CSIC08–4E–001).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

## *Article* **Development of Quinoa Value Chain to Improve Food and Nutritional Security in Rural Communities in Rehamna, Morocco: Lessons Learned and Perspectives**

**Abdelaziz Hirich 1,\*, Sifeddine Rafik 2,3, Mohamed Rahmani 3 , Amira Fetouab 2 , Fatima Azaykou 2 , Kaoutar Filali 4 , Hayatullah Ahmadzai 5 , Younes Jnaoui 2 , Aziz Soulaimani 2 , Mariam Moussafir 2 , Mohamed El Gharous 2 , Salwa Karboune 6 , Abdelaziz Sbai 4 and Redouane Choukr-Allah 4**

**Abstract:** Agricultural production in the Rehamna region, Morocco is limited with various challenges including drought and salinity. Introduction of climate resilient and rustic crops such as quinoa was an optimal solution to increase farmer's income and improve food security. This study summarizes results obtained from a research project aiming to develop quinoa value chain in Morocco. The study tackled several aspects including agronomic traits (yield and growth), transformation, quality (nutritional and antinutritional traits) and economic analysis and, finally, a strength– weaknesses–opportunities–threats analysis, lessons learned and development perspectives were presented. From an agronomic point of view, introduced new quinoa cultivars showed higher performance than locally cultivated seeds and, furthermore, the use of irrigation and organic amendment has tremendously improved seed yield by double and three times, respectively, compared to rainfed conditions. Nutritional analysis revealed that protein and phosphorus content remained stable after seed pearling while most of the micronutrients content decreased after seed pearling. However, saponins content was reduced by 68% using mechanical pearling compared to 57% using both traditional abrasion and washing. The economic analysis showed that production cost of quinoa seeds could be further decreased using mechanized intensive tools along with irrigation and organic amendment supply. This study revealed several lessons learned from the field experience and proposed several development actions for each value chain component that can be implemented within a national quinoa program.

**Keywords:** production cost; pearling; yield; irrigation; mechanization; harvest

### **1. Introduction**

Today, more than 120 countries around the world cultivate quinoa (*Chenopodium quinoa* Willd.) or try to adapt it to their environmental conditions. The continued expansion of its cultivation in all continents challenges the prejudices of that quinoa is a species, which can

**Citation:** Hirich, A.; Rafik, S.; Rahmani, M.; Fetouab, A.; Azaykou, F.; Filali, K.; Ahmadzai, H.; Jnaoui, Y.; Soulaimani, A.; Moussafir, M.; et al. Development of Quinoa Value Chain to Improve Food and Nutritional Security in Rural Communities in Rehamna, Morocco: Lessons Learned and Perspectives. *Plants* **2021**, *10*, 301. https://doi.org/10.3390/plants 10020301

Academic Editors: Cataldo Pulvento Received: 31 December 2020 Accepted: 13 January 2021 Published: 5 February 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

only grow in the high plains of the Andes on the shores of Lake Titicaca. After a first boom in quinoa cultivation in the 1990s mainly linked to the demand of vegetarians for products rich in vegetable proteins from organic farming, a second boom in the 2000s was based on the values of fair trade, and, today, we are facing a third quinoa boom at the global level with the production of quinoa in new countries that were not even importing quinoa [1]. Morocco falls into the last category of the country having initiated its cultivation before importing it for its own consumption. These changes on a global scale are such that great transformations in progress in the way quinoa is produced, the networks related to its distribution and in the ways of considering it and incorporating it into various local diets.

The year 2013 has been declared the International Year of Quinoa (IYQ) by the United Nations. This made it possible to recognize the importance of the biodiversity of quinoa and the high nutritional value of its seeds [2]. Within this dynamic, quinoa has been introduced in Morocco since the 1999/2000 season and was considered as an important alternative to traditional crops such as cereals, which are strongly subjected to climate change effects and soil degradation due to salinization making quinoa a judicious solution and potential crop that may contribute to national food security [3]. In this Moroccan context, quinoa is proving to be an interesting solution to limit the risk of agricultural production failure associated with the yield losses observed on traditional cereals cropping systems, which sometimes contribute to soil degradation because of the monoculture practiced in several regions. The fact that quinoa is considered a rustic crop resistant to various abiotic stress makes it a resilient and climate smart crop that could be used for climate change adaptation [4].

In Morocco, quinoa was subjected to several field trials evaluating the performance of introduced cultivars and the effect of various cropping practices on its productivity. First a collection of quinoa accessions was tested for the adaptation goal in the Khenifra region in the year of 2000 resulting in a selection of 14 accessions, which were believed to be tolerant to drought. Then, experiments on quinoa in Morocco were intensified within the SWUP-MED EU funded project (sustainable water use securing food production in dry areas of the Mediterranean region) where quinoa was introduced and tested in several regions including Rehamna, Rabat and Agadir [5]. Secondly, research activities were focusing on testing the effect of several practices on quinoa such as irrigation with saline water [6,7], deficit irrigation [8,9], organic amendment [10], sowing dates [11], use of wastewater for irrigation [12], etc.

At the nutritional level, for some people, quinoa is a new and nutritious food that has recently been found in supermarkets and restaurants and can replace many common grains. Certainly, in many regions of the world, this vision corresponds to reality but it should be known that quinoa was one of the main food crops of the pre-Columbian civilizations of Latin America and remains an important food for the Quechuas and Aymaras settled in rural areas of the Andes, South America. In the Quechua language, quinoa is called chisiya, which means mother grain [13]. Quinoa provides as much energy as foods used in a similar way, such as beans, corn, rice or wheat. It is also an important source of quality protein, dietary fiber, polyunsaturated fatty acids and minerals [14]. Protein content in of quinoa seeds varies between 12% and 20%; however, it is reported as 16% on average [15].

One of the obstacles for quinoa seed valorization is its content in terms of saponins because of their bitter taste and toxic effects, which necessitates their elimination. Several pearling techniques and methods are used to eliminate saponins from the quinoa seeds; the wet technique remains the most used one especially in Morocco combined with preliminary manual abrasion [16].

In this study we provided an evaluation of the agronomic performances of introduced quinoa cultivars grown under different production scenarios and the effect of seed pearling on nutritional and saponin contents. The study also presents a technical and economic analysis of the quinoa production and transformation. Through the conducted investigations the strengths, weaknesses, opportunities, and threats related to the existing quinoa value

chain in Morocco were revealed and the lessons learned. Finally, we proposed development perspectives for each value chain component.

#### **2. Results**

#### *2.1. New High Yielding Chenopodium Quinoa Cultivars Introduction*

Table 1 presents obtained results in terms of plant productivity and growth. The data clearly indicate that introduced ICBA (International Center for Biosaline Agriculture) cultivars performed better than other tested varieties in terms of yield while locally produced bulk seeds showed the lowest performances. Irrigation supply and organic amendment have greatly increased the seed yield for all tested cultivars. Under farm conditions, yield was doubled under irrigation supply and tripled under combined irrigation and amendment application. More or less the same effect has been noticed for plant height. Quinoa dry matter responded very well to the amendment application in the case of ICBA-Q3 cultivar while no significant changes were noticed for local bulk seeds. Results obtained for 1000 seed weight indicate that Titicaca cultivar had the highest seed weight and size while Puno cultivar had the lowest. Organic amendment had a notable effect on 1000 seed weight in the case of local bulk seed while no significant difference was obtained for ICBA-Q3 cultivar.

**Table 1.** Seed yield, plant height, dry matter and 1000 seed weight of quinoa tested in the Rehamna region under several experimental conditions. Different letters (a, b, ab, c) indicate a significant difference according to the Tukey test (*p* < 0.05).

**Table 1.** *Cont.*

### *2.2. Quinoa Seed Processing*

Winnower

Winnower

Sheller (pearling machine)

Sheller (pearling machine)

2.2.1. Harvest and Postharvest Machines

Several mechanized tools were locally developed or adapted to be used for quinoa harvest and post-harvest operations by a private entrepreneur (BenRim farm) as presented in Table 2. Supporting local private entrepreneurs to manufacture those tools was one of the key outcomes of this project. *Plants* **2021**, *10*, x FOR PEER REVIEW 5 of 28 *Plants* **2021**, *10*, x FOR PEER REVIEW 5 of 28

> 150 kg.hr−1

150 kg.hr−1

200 kg.hr−1

200 kg.hr−1

Couscous is a famous Mediterranean dish and widely consumed in Morocco. It is now produced out of quinoa by several women cooperatives in the Rehamna region using both quinoa flour and semolina. The pathway for quinoa-based products processing is described in Figure 1. Quinoa based products described in this study were produced traditionally by women following several steps such as seed pearling, washing and drying

Couscous is a famous Mediterranean dish and widely consumed in Morocco. It is now produced out of quinoa by several women cooperatives in the Rehamna region using both quinoa flour and semolina. The pathway for quinoa-based products processing is described in Figure 1. Quinoa based products described in this study were produced traditionally by women following several steps such as seed pearling, washing and drying

2.2.2. Quinoa Transformation Pathway

2.2.2. Quinoa Transformation Pathway

Threshing quinoa panicles results in a mixture of grains, small residues and chaffs. The mechanical winnowing consists of using a winnowing fan that

Threshing quinoa panicles results in a mixture of grains, small residues and chaffs. The mechanical winnowing consists of using a winnowing fan that creates wind that blows away the lighter chaff, while

It is equipped with two motors, the first one is designed to turn a drum with a rotation speed of 750 rpm. The second one is more powerful (3000 rpm) and designed to extract the fine dust produced during the pearling process. The rotating drum is made of 80 cm long perforated inox steel and has 6 baffles distributed throughout the drum.

It is equipped with two motors, the first one is designed to turn a drum with a rotation speed of 750 rpm. The second one is more powerful (3000 rpm) and designed to extract the fine dust produced during the pearling process. The rotating drum is made of 80 cm long perforated inox steel and has 6 baffles distributed throughout the drum.

**Table 2.** *Cont.*

**Machines Description Capacity**

**Machines Description Capacity**

The cereal's combined harvester was used to harvest quinoa with few adaptations at sieves levels to match quinoa seed size

The cereal's combined harvester was used to harvest quinoa with few adaptations at sieves levels to match quinoa seed size

Thresher The machine can be powered by an electric or a diesel

Thresher The machine can be powered by an electric or a diesel

engine. It adopts axial-flow roller

engine. It adopts axial-flow roller

Combined harvester

Combined harvester

*Plants* **2021**, *10*, x FOR PEER REVIEW 5 of 28

*Plants* **2021**, *10*, x FOR PEER REVIEW 5 of 28

**Table 2.** Harvest and post-harvest machines locally developed.

**Table 2.** Harvest and post-harvest machines locally developed.

1.5 ha.hr−1

1.5 ha.hr−1

200 kg.hr−1

200 kg.hr−1

#### 2.2.2. Quinoa Transformation Pathway 2.2.2. Quinoa Transformation Pathway Couscous is a famous Mediterranean dish and widely consumed in Morocco. It is 2.2.2. Quinoa Transformation Pathway

Couscous is a famous Mediterranean dish and widely consumed in Morocco. It is now produced out of quinoa by several women cooperatives in the Rehamna region using both quinoa flour and semolina. The pathway for quinoa-based products processing is described in Figure 1. Quinoa based products described in this study were produced traditionally by women following several steps such as seed pearling, washing and drying now produced out of quinoa by several women cooperatives in the Rehamna region using both quinoa flour and semolina. The pathway for quinoa-based products processing is described in Figure 1. Quinoa based products described in this study were produced traditionally by women following several steps such as seed pearling, washing and drying Couscous is a famous Mediterranean dish and widely consumed in Morocco. It is now produced out of quinoa by several women cooperatives in the Rehamna region using both quinoa flour and semolina. The pathway for quinoa-based products processing is described in Figure 1. Quinoa based products described in this study were produced traditionally by women following several steps such as seed pearling, washing and drying to produce processed seeds and milling, grinding and sieving to have quinoa flour and semolina. The key steps of couscous production are manual rolling, forced sieving, precooking and drying. However, 27 steps from raw material reception to final product shipping are required to produce traditional couscous.

**Figure 1.** Processing diagram for quinoa based products adapted from the "3rd Millennium" cooperative.

#### 2.2.3. Shelling Impacts on the Nutritional and Antinutritional Aspect of Quinoa Seeds

Table 3 shows the nutrients content of raw, processed seeds and quinoa bran for Puno, Titicaca and ICBA-Q5 cultivars. Data clearly indicate that nutrient content varies from cultivar to another. Titicaca processed seeds presents the highest content in terms of proteins, Mg, P, Ca, Zn and Cu, for Puno cultivar the highest content was observed for C and Fe, while ICBA-Q5 processed seeds presents the highest content in terms of K, Na, Mn and Ash.

**Table 3.** Nutrients content of raw, processed seeds and quinoa bran for Puno, Titicaca and ICBA-Q5 cultivars.

Figure 2 presents the saponin content in two different seeds polished using two different methods, bulk seeds polished manually by the women cooperative and Puno seeds polished mechanically using a locally manufactured pearling machine. Obtained results indicate clearly that bulk seeds accumulate more saponins compared to Puno seeds. Pearling using the mechanized tool was shown to be more efficient than manual abrasion as the saponin content was reduced by 68% in the case of Puno and 57% in the case of bulk seeds.

**Figure 2.** Saponin content (%) of raw and processed bulk and Puno seeds. Bulk seeds were pearled manually and Puno seeds mechanically using quinoa sheller.

#### *2.3. Quinoa Import*

Import of quinoa in Morocco has known a great evolution since 2015 (first year of record) to reach 84 tons in 2019 with a total value of 2.4 million MAD (Figure 3). The average price per kilogram was greatly decreased. In 2015 the average import price was equal to 80 MAD.kg−1 while in 2019 it was equal to 29 MAD·kg−1 following the worldwide trend in quinoa price as per the Statista database [17].

**Figure 3.** Evolution of quinoa import in Morocco in terms of quantity and value. 1 USD = 9 MAD.

#### *2.4. Economic Analysis of Quinoa Seed Production*

#### 2.4.1. Quinoa vs. Traditional Cereals: Production Cost and Net Margin

Data presented in Table 4 clearly indicate that for quinoa cultivation, production cost per kilogram is higher under the rainfed and manual production mode compared to the scenario where irrigation is supplied along with fertilizers and organic amendments. Thus, production cost per kilogram decreased from 27 to 11 MAD and this can be explained by the improved yield and reduced cost attributed mainly to irrigation and fertilization and the use of the mechanized tool for quinoa cultivation. Consequently, the net margin was improved significantly due to input supply and the mechanized tools adoption. The presented data also indicate that quinoa is more remunerating than cultivated cereals as it generates five times and twice the net margin generated by barley and wheat, respectively, grown under the rainfed and mechanized scenario.

**Table 4.** Production cost and net margin of processed quinoa seeds compared to traditional cereals cultivated in the Rehamna region. 1 USD = 9 MAD.

#### 2.4.2. Quinoa Production Cost Breakdown

Cost breakdown for quinoa cultivation operations is presented in Table 5. Harvest and post-harvest operations account for the largest part in the overall production cost in the case of rainfed and manual cultivation contributing with 63% in the total production cost. While this percentage was decreased to 50% when mechanized tools for harvest and post-harvest operations were adopted. In the case of the optimized production mode using irrigation and fertilization the part of harvest and post-harvest operations in the total cost was greatly decreased due to high cost attributed to irrigation system depreciation, pumping and fertilizers and was equal to 48 and 26% under manual and mechanized production mode, respectively.

**Table 5.** Cost breakdown for quinoa cultivation and seed processing operations. 1 USD= 9 MAD.

2.4.3. Sensitivity Analysis of Net Margin vs. Production Cost, Grain Yield and Sale Price

Figure 4 presents the sensitivity analysis elucidating the impacts of +25 and −25% changes of total cost, grain yield and sale price on net profit. Results indicate clearly that net profit is affected greatly by sale price as a reduction or increase of 25% in sale price is likely to have the largest impact on net profit either negatively or positively but with a more pronounced effect on the net profit in the case of price reduction. Holding all other variables at their base-value, a 25% reduction in the output price will reduce net profit by 94.2, 82.8, 74.2 and 67.7 percent under scenario I, II, III and IV, respectively.

**Figure 4.** *Cont*.

**Figure 4.** Changes in net profit as responses to ±25% variation in sale price, grain yield and total cost under tested production scenarios. 1 USD = 9 MAD.

#### 2.4.4. Monte Carlo Simulation Analysis (10,000 Iterations)

Monte Carlo simulation performs risk analysis by building models of possible results by substituting a range of values—a probability distribution—for any factor that has inherent uncertainty. It then calculates results over and over, each time using a different set of random values from the probability functions. Depending upon the number of uncertainties and the ranges specified for them, a Monte Carlo simulation could involve thousands or tens of thousands of recalculations before it is complete. In our case the number of iterations made is 10,000 simulating the net profit as affected by changes in market price and yield.

The Monte Carlo simulations presented in Figure 5 as frequencies derived after 10,000 iteration of simulation show that the risk of having a financial loss when producing quinoa is about 2.55%, 0.55%, 0.05% and 0% respectively for scenario I, II, III and IV assuming that changes in yield may occur from 200 to 800 kg·ha−1 for scenario I and II and from 1000 to 3000 kg·ha−1 for scenario III and IV, and allowing for simultaneous variation in price from a lower bond of 30 MAD·kg−1 to an upper bond of 120 MAD·kg−1 .

We also carried out a break-even analysis for each of the four cases to identify values of the key parameters that make revenues equal to the cost of production, and as a result the net gains were zero (Table 6). The estimated break-even points were lower than the lower bonds assumed in the simulations (200 kg·ha−1 of yield for scenario I and II, 2000 kg·ha−1 yield under scenario III and IV and 30 MAD for the price) indicating higher confidence for the main results presented in Table 5 to remain unaffected.

**Figure 5.** Monte Carlo simulation frequency of net profit as affected by a change in market price and yield. 1 USD = 9 MAD.

#### 2.4.5. Cost Breakdown of Quinoa Based Products

Table 7 presents cost breakdown for quinoa products such as quinoa couscous, processed seeds and quinoa flour in Morocco. Raw material has the largest contribution to the total production cost for all products with almost 80%, 87% and 85% for quinoa couscous, flour and processed seeds, respectively. Labor cost consisting of women working on different steps

of production is also important as it represents more than 13% in the case of couscous while it represents only 1.88 and 3.64% of the total production cost for quinoa flour and processed seeds, respectively. Consequently, among all quinoa products, quinoa couscous remains very expensive compared to other cereals couscous (max market price is equal to 20 MAD·kg−1 ). Obtained data indicate that the women cooperatives producing quinoa products should reduce raw material cost through their own production of quinoa seeds, better planning for quinoa stocks and elaborating the sale contract with quinoa producers for a low price.

**Table 7.** Cost breakdown for quinoa couscous, flour and processed seeds production. 1 USD= 9 MAD.

#### *2.5. SWOT Analysis of the Quinoa Value Chain in Morocco*

Table 8 presents analysis of the quinoa value chain in Morocco including its strength. weaknesses. opportunities and threats at different levels.

#### *2.6. Lessons Learned*

Several lessons were revealed by the present study as presented in Table 9.

#### *2.7. Perspectives of Development*

With several challenges (drought and salinity) facing staple crops such as cereals in many marginal areas in Morocco. quinoa could be a judicious solution to improve food security and increase farmer's income. A national program of quinoa in Morocco is becoming necessary and needs to tackle all value chain components. We suggest several actions to improve the quinoa value chain in Morocco summarized in Table 10.

**Table 8.** SWOT (strength. weaknesses. opportunities and threats) analysis of the quinoa value chain in Morocco.

**Table 9.** Lessons learned from the quinoa value chain in Morocco.

**Table 9.** *Cont.*

**Table 10.** Proposed development actions and involved actors for each value chain components.

#### **3. Discussion**

Since its introduction to Morocco in the 2000s. quinoa was seen as a rustic and stress tolerant crop with several potentialities to replace cereals and other traditional crops in the marginal environment of Morocco. Therefore. it was subjected to various trials at the field and pot level in several regions to evaluate its productivity and responses to various stresses. For instance. the finding of this study indicates that quinoa yield was tripled for most of the tested cultivars under full irrigation conditions compared to rainfed. which support the results obtained by Fghire et al. [8] who found that the yield of Puno cultivar (one of the tested cultivars in the present study) conducted in the same study area (Tnin bouchane) was increased by 236% under full irrigation (100% ETp) compared to rainfed irrigation. Our results are in agreement with Geerts et al. [20] who reported that full irrigation increased quinoa yield with 27% compared to rainfed conditions. Contrarily to our case. this low increase percentage is mainly explained by the high amount of rain received (330 mm) for rainfed treatment compared to irrigated treatment (245 mm irrigation

+ 330 mm rainfall). While in the present study quinoa under farm conditions received only 43 mm of rain during its growing season while the irrigated treatment received 200 mm of irrigation. which obviously explain the tremendous increase of yield and other growth parameters as a response to irrigation.

It is well known that organic matter amendment has a positive effect on crop growth and productivity. In the case of quinoa. very few studies are available evaluating the effect of organic amendment on yield; nevertheless. other crops were cultivated under the organic amendment and showed a positive response [21–23]. Our results suggest that the ICBA-Q3 cultivar grown under controlled experimental conditions significantly (*p* < 0.01) responded to the organic amendment only after applying 40 T/ha of manure while no significant difference was observed under a lower dose. While under farm conditions. quinoa yield and plant height were significantly improved under organic amendment application. Our results are in agreement with Hirich et al. [10] who found that that organic amendment of 10 t ha−1 and 5 t ha−1 significantly increased seed yield by 13% and 3%. respectively. under full irrigation. The yield improvement under organic amendment is explained by a soil content increase in terms of nutrients after mineralization of the organic matter; therefore. the nutrients uptake will be increased. which will result in high plant growth and productivity [24].

Our finding in terms of seed nutrient content indicates that most of the micronutrients content was reduced in the processed seeds due to seed pearling. which means that they are mostly concentrated in the pericarp (bran) as suggested by Konishi et al. [25] and D'Amico et al. [26] who reported that minerals are accumulated in the pericarp (seed out layer) and proteins are mostly accumulated in the endosperm tissues. In terms of mineral content. Konishi et al. [25] reported that phosphorus is mainly localized in the embryonic tissues. which explain why P content in the present study for all tested varieties was higher in the processed seeds and lower in bran. However. in terms of magnesium and potassium our finding indicates high content in the seed bran compared to processed seeds. which disagrees with Konishi et al. [25] who reported that both magnesium and potassium are located in the embryonic tissues.

One of the limiting factors for quinoa valorization and transformation is its content in terms of saponins. which are mainly concentrated in the pericarp or bran and need to be removed before use [27]. The cultivars we tested in this study are classified as bitter [28]. and the saponins removal level by either mechanical abrasion or manual polishing was not enough to classify the quinoa as sweet. since the saponin content threshold for human consumption is equal to 0.12% according to CODEX [18]. Our results indicate that Puno seeds pearling using mechanical abrasion resulted in 68% reduction in terms of saponin content. which confirm the finding of Hirano and Konishi [29] who reported that quinoa seed pericarps contain 67.6% of the total saponin content in the whole grain while the rest is remaining in the seed endosperm and other internal layer. Our results are in agreement with Mhada et al. [30] who reported that mechanical abrasion allowed the reduction of saponins level from 1.4 to 0.51% for the Puno cultivar. a reduction of 64% of the initial saponin level. Our findings are also in line with Gómez-Caravaca et al. [31] who reported that an abrasion degree of 20% allowed reducing the saponin levels in pearled quinoa (129.8 mg/100 g d.w.) more than 50% comparing with the initial saponin content in whole quinoa (244.3 mg/100 g d.w.).

In Morocco the quinoa market is very limited and still a niche market. In order for the market to expand. huge effort is required to promote for quinoa and a rise in awareness among consumers about its health benefits. Like other countries. such as Turkey. quinoa consumption is limited to those with knowledge of health foods for specific health benefits. including its gluten free status. Quinoa is not a product "consumed by the masses". but rather one "discovered" by educated. health-conscious consumers [32]. The economic analysis showed that under rainfed conditions production cost at harvest per hectare varies from 6650 to 7900 MAD (739–878 USD) for the mechanized and manual production mode. respectively. While under irrigated conditions the production cost

increased to 18.445 and 19.695 MAD·ha−1 (2049 and 2188 USD·ha−1 ) for the mechanized and manual production mode. respectively. due to depreciation of the irrigation system. energy and fertilizers input costs. The same trend was reported by Yazar et al. [32] in Turkey who found that the production cost of quinoa was equal to 728 and 1650 USD·ha−1 under rainfed and irrigated conditions. respectively. Our results are also in agreement with Mercado and Ubillus [33] who reported that the production cost of quinoa in Peru varies from 676 to 2604 USD·ha−1 for traditional rainfed and conventional production system. respectively. with a profitability that varies from 100 to 200% and market price varies from 1.7 to 2 USD·kg−1 . However. in Morocco the profitability could vary from 150 to 500% due to high quinoa price and the use of intensive production systems (irrigation and mechanized tools).

This study presents a SWOT analysis of the quinoa value chain in Morocco. which revealed that one of the main weaknesses limiting quinoa market expansion in Morocco is the traditional production and valorization and the lack of using intensive production tools. Thus. quinoa price in Morocco remains relatively high above middle class consumer's purchasing power even at farmgate and only rich people can afford it. This way the local quinoa products with a high price and relatively lower quality could never compete with an imported one. which have usually good quality. The trendy nature of the market for quinoa in Morocco has had both positive and negative aspects. Certainly. growers have benefited from the rising prices that the crop commands. though the various intermediaries may reap more of the profits than the small growers [34]. Another bottleneck in the quinoa value chain is a lack of promotion around quinoa benefits using public channels and social media. which is considered a key point for any new product development [19].

#### **4. Materials and Methods**

#### *4.1. Study Area*

The province of Rehamna is geographically located between Marrakech (South). Settat and El Jadida (North). El Kelaa des Sraghna (East) and Sidi Bennour. Youssoufia to the west (Figure 6). This region is characterized by an average rainfall of 177 mm with an intra and interannual variation (Figure 7). Temperatures are relatively homogenous throughout the zone. with temperatures ranging from 4 to 46 ◦C. Prevailing winds are from the North-East in winter and from the West in summer. Warm winds (Chergui) are frequent and blow from the East and South. The total agricultural area of the region of Rehamna is 591.125 Ha with an arable land area of 342.500 Ha (35.425 Ha of irrigated area and 307.075 Ha of rainfed area) (DPA Rehamna 2018).

**Figure 6.** Rehamna province localization (study area).

**Figure 7.** Rehamna climatic conditions taken from the UM6P Experimental Farm meteorological station.

#### *4.2. Soil and Water Analysis*

Table 11 presents the physicochemical analysis of soils at the UM6P experimental station and farm level. Soil texture is clay loam at the UM6P experimental station and sandy loam at the farm level. Both soils are considered poor in terms of organic matter and rich in terms of potassium.

**Table 11.** Physicochemical analysis of soil at the UM6P experimental station and farm level.

According to Table 12. both irrigation waters were slightly saline with more salinity and mineral content obtained for irrigation water at the UM6P experimental station.

#### *4.3. Tested Cultivars*

In this study ICBA (International Center for Biosaline Agriculture) quinoa cultivars were introduced due to their high adaptation to MENA and Morocco conditions and resistance to drought and salinity [4]. The origins of those cultivars are: low land. Bolivia for ICBA-Q1. Q2 and Q3 and coast. Chile for ICBA-Q4 and Q5. Those cultivars were already introduced and tested in the south of Morocco (Laayoune area) within a previous R&D project and showed high performance under salinity conditions with an average seed yield exceeding 2 t/ha. In addition to ICBA cultivars. two Danish public varieties were tested. Titicaca and Puno. two short cycle varieties widely cultivated in Morocco and showed a good adaptation with higher yield compared to other quinoa accessions [4,5,30,35].

**Table 12.** Physicochemical analysis of irrigation water at the UM6P experimental station and farm level.

#### *4.4. Trial Installation*

#### 4.4.1. At the Farm Level

An on-farm trial was conducted at the farm level (Tnin Bouchane. 32◦14.62670 N. 8 ◦19.81810 W. 280 m + MSL (mean sea level)) testing ICBA cultivars (ICBA Q1-Q5) compared to locally cultivated bulk seeds (mixture of L119 and L143 accession) under rainfed. irrigation and irrigation with cow manure amendment (40 T/ha) conditions in a split plot design with 4 replications (plot size was equal to 10 m2 ). Organic amendment was applied along with soil preparation before sowing. Irrigation practices were performed according to farmer usual practices with an irrigation supply of about 200 mm (2000 m3/ha) for the whole cropping period. using drip irrigation following evapotranspiration demand according to Allen et al. [36]. Quinoa seeds were sown using a plant density of 8 plants/m2 (50 cm between lines and 25 cm between plants). The trials were carried out between 18 February and 30 June 2018.

#### 4.4.2. At the UM6P Experimental Farm

Another trial was carried out in the UM6P experimental farm (Ben Guerir. 32◦13.08" N. 7◦53.230 W. 468 m + MSL (mean sea level)) to investigate the performance of six quinoa cultivars including ICBA-Q1. ICBA-Q2. ICBA-Q5. Titicaca. Puno and locally cultivated bulk seeds under Rehamna conditions. The objective of this trial was to assess the productivity of tested cultivars and their adaptation to Rehamna agroclimatic conditions. The trial was conducted in a completely randomized block design with four replications. Plot size was equal to 100 m2 . Irrigation was applied following the evapotranspiration method according to Allen et al. [36] using parameters from the existing weather station. Irrigation volume supplied was equal to 300 mm (3000 m3/ha). Trials were carried out between 21 February and 25 June 2018.

#### *4.5. Agronomic Practices and Seed Yield Determination*

All trials were subjected to commercial agronomic practices such as soil preparation. preirrigation. weeding (3 times during the growing period). phytosanitary treatments (application of insecticide treatment against caterpillar in the seedling stage) and plant thinning (keeping only one or two plants per sowing hole). Quinoa seeds were sown using a plant density of 8 plants/m2 (50 cm between lines and 25 cm between plants).

Seed yield for all trials was determined after maturity. Quinoa panicles were harvested first and dried in open air. Seeds were extracted using manual threshing followed by seed polishing and cleaning.

#### *4.6. Seed Pearling*

The saponin elimination process remains as a critical operation in seed processing. Recently. many appropriate technologies have been developed to remove saponins to an acceptable threshold without affecting the nutritional properties of the seed.

Puno seeds processed mechanically by Benrim farm were polished using a pearling machine that was locally manufactured for a duration of two minutes. The machine operates on a semi-industrial scale with a transformation capacity of 120 kg·hr−1 . it is equipped with two motors. the first one is designed to turn a drum with a rotation speed of 750 rpm. The second one is more powerful (3000 rpm) and designed to extract the fine dust produced during the pearling process. The rotating drum is made of 80 cm long perforated stainless steel and has 6 baffles distributed throughout the drum. During seed processing. the speed of rotation and friction (seed-seed. seed-drum and seed-baffles) gradually increased the temperature of the seeds to 35 ◦C and decreased the moisture from 13 to 10%.

At the level of the women's cooperatives in Morocco. saponins elimination from quinoa seeds was performed manually with traditional equipment. The majority of valorization units (women's cooperatives. startups. etc.) used combined operations starting with a dry method (manual abrasion) and finishing by a wet one (washing using water). First. a manual abrasion using a glove against a rough surface (e.g., rubber. sieve) is carried out to eliminate the external coat of the episperm (bran). This manual abrasion operation is time consuming and requires effort; it takes one hour to dehull 6 kg of quinoa seeds. Women's cooperatives are using a partial manual abrasion in order to avoid losing the embryo and preserve the seed morphological aspect. However, the residual saponin still remains above the CODEX [36] threshold (0.12% of dry matter) and the bitterness perception is still present. Hence. they add a washing step as a supplementary operation to totally eliminate the bitterness. For the washing operation. seeds soaked with water allowing saponins to dissolve. They use a water quantity of 20 L per 5 kg of quinoa seeds for 10–15 min. This quantity is used three times soaking a total of 15 kg of polished quinoa seeds. Finally, the processed quinoa is dried for 5 h.

#### *4.7. Chemical Analysis*

#### 4.7.1. Nutrient Analysis

Raw and mechanically processed seeds and resulted bran of the Puno variety were used for nutrient content determination following the steps below:

#### 4.7.2. Saponin Analysis

In addition to Puno seeds (raw and mechanically processed seeds) used for nutrient content determination. the extraction and quantification of saponin content was performed on locally produced and manually processed by the Bouchane cooperative.

Saponin extraction was performed grinding one gram of dried sample to a fine powder and dissolving in 20 mL of 20% isopropanol. The blend was heated to 86 ◦C for 20 min for saponin extraction by a microwave-assisted method and filtered (Whatman filter paper) for further quantification. Saponin concentrations were measured by spectrophotometric methods as described by Gianna et al. [37] with minor modifications. The Liebermann-Burchards (LB) reagent was used to quantify saponins. as it is capable of producing a light brown coloration if these compounds are present in a sample. The LB reagent was a 1:5 mixture of acetic acid and sulphuric acid. respectively. Following mixing 1 mL sample solution with 3.5 mL LB reagent. the absorbance at wavelength 580 nm was measured in all samples after 10 min. A calibration curve based on pure quinoa saponins was used for determining the final saponin concentration (mg/mL) in each solution on the basis of absorbance measurements (absorbance = 4.5725 × saponin concentration + 0.0164). The percentage of saponin content was determined on the basis of fresh weight [38]. For nutrient content and saponin determination. three replications have been analyzed. Pictures of grains have been taken using optical microscopy Nikon Eclipse Lv100nd-motorized microscope (Nikon, France) with episcopic/diascopic illumination that enables control of objectives and light intensity from the camera control unit and automatically detects the observation method.

#### *4.8. Farmer's Survey*

Quinoa field production cost was determined using face-to-face interviews with farmers cultivating quinoa bulk seeds under different cropping systems (rainfed. irrigated. organic amendment and mechanized). Three farmers from each cropping system were selected and interviewed. The following questions related to production cost breakdown were included in the survey:

#### *4.9. Sensitivity Analysis and Monte Carlo Simulations*

Quinoa is not yet a well-established crop in the local production systems. A lack of farmers' experience and the possibility of a shortage of inputs. especially planting materials. could possibly result in greater yield variability among farmers. The economic performance of quinoa will depend critically on the actual yield performance of different varieties and households' characteristics and their management practices. which greatly varies among farmers. Beyond production. the markets for quinoa are not well developed. Imperfect

and non-competitive markets may fail to clear at competitively determined prices. Poorly functioning markets may therefore pose price risks to the local producers. Hence. such variabilities in yield and potential price volatility may alter the results presented under the base-case scenarios (Table 4). We run simulations of the base-case results to factor in for potential production and price uncertainty.

We first conduct deterministic sensitivity analysis by changing a single parameter. whilst holding all other parameters of the model at their baseline values. In our case. the deterministic analysis was carried out by allowing for a 25% change in yields. prices and total costs to model and assess the sensitivity of net gains for each scenario. While the conventional one-way sensitivity helps determine the scale of impact of a single parameter and its limitation is that it does not proved insights into the probability of such a change (e.g., it does not explain how likely it is for the parameter of interest to take a specific value). Moreover. the deterministic approach fails to take into account the correlation between the values taken by the parameter of interest and other parameters in the model that are held constant [39].

Unlike the deterministic case. simulations allow for stochastic and simultaneous variations and shocks in multiple parameters using the principals of inferential statistics. To evaluate the contemporaneous impact of variations in yields and prices. we then construct a dynamic variant of the model to estimate all possible outcomes given a probabilistic distribution in yields and prices. Using a Monte-Carlo simulation method. we assign multiple values to yields and prices by generating random numbers that follow a symmetric triangular distribution and uniform distribution with lower and upper bonds. respectively. Note that the symmetric triangular distribution is a probability distribution with a probability density function (PDF) shaped like a triangle allowing for central tendency towards the "most-likely or the base-case value". It therefore gives due weightage to the mean value in the yield with frequent outcomes clustered around the most-likely value. Uniform or triangular distribution assumed for the price variable. on the other hand, allow for the randomly generated number to take any value between the specified upper and lower bonds based on a constant probability. Hence. any value in the specified interval is just as likely and probable [40].

#### *4.10. Valorization Cooperative's Survey*

A technical and financial assessment was carried out conducting a diagnostic of the 3rd Millennium" cooperative (a quinoa valorization unit in Rehamna region) in order to assess the technical pathway of quinoa transformation and determine production costs of processed products. The cooperative used Puno variety. which is the most common used by cooperatives and it is provided by the Benrim farm in the Berrechid area where the quinoa price is the most affordable.

#### *4.11. Quinoa Import Data*

Quinoa import data in terms of quantity and value have been extracted from the change office database [41]. Data were first downloaded searching for quinoa as a keyword and processed using Excel software.

#### *4.12. Statistical Analysis*

Differences in response variables to applied treatments were assessed using a general linear model with StatSoft STATISTICA 8.0.550 software (StatSoft Inc. Tulsa, OK, USA). Statistical differences were all significant at α = 0.05 or less. The means comparison was based on a one-way ANOVA analysis.

#### **5. Conclusions and Recommendations**

In the light of obtained results quinoa was shown to be a potential and resilient crop that could be an alternative to traditional cereals in the marginal area such as the Rehamna region where traditional cereals are not performing well at both the agronomic

and economic level. Furthermore. quinoa offers better remuneration and yield under both rainfed and irrigated cultivation. It is also recommended for farmers to adopt mechanized tools for quinoa cultivation and seed processing to reduce production cost and improve their income. The access to those mechanized tools will be easier if farmers are gathered in cooperatives or associations. Quinoa price structuration remains a bottleneck in its value chain in Morocco as quinoa still have a niche market and demand on quinoa products still does not meet the producer's expectations. Furthermore. production cost of the quinoabased product such as couscous remains very high due to a high cost of raw material and involved labor force. Therefore. it is recommended for women's cooperatives valorizing quinoa to have their own quinoa production.

Several scenarios of cost–benefit analysis were conducted to assess the economic viability of quinoa production in Morocco. The results across multiple scenarios consistently indicated that quinoa is highly profitable. yielding a net margin ranging from 21.100 to 111.555 MAD depending on the scenario (e.g., irrigated vs. rainfed and manual vs. mechanized systems). Further sensitivity analysis and simulations were undertaken to analyze the potential impacts of uncertainty in key variables and assumptions. particularly taking into account variability in yield performance. market prices and production costs. The sensitivity analysis showed that output price has the largest and significant impact on the quinoa profitability. However. as is indicated by the results of the simulation. the likelihood of net profit to be negative is neglected with a probability ranging from 0.5 to 2.55% depending on the scenario.

This study revealed several lessons learned from the field experience and proposed several development actions for each value chain component that can be implemented within a national quinoa program. which may be funded within the new Moroccan agricultural development plan called the "green generation".

The Rehamna Quinoa upscaling project has identified the suitable varieties. and the best production and management practices to maximize yields. In addition. the nutritional analysis of the genotypes with the highest potential was carried out. Seed multiplication of the most promising genotypes has been developed with a private local company to ensure enough seeds are available in the market for the scaling up production. On a global scale. the impact of the project on small farmers and the gender issues has been positive in general. as it secures a minimum revenue for the farmers even in dry years. Therefore. it will be judicious to pursue implementing cutting edge research to collect. screen and identify the quinoa genotypes that have the best potential for wide scale adoption in different agroecological zones and marginal environments.

We recommend then to set up support for all players in the sector through coordination of the quinoa interprofession in Morocco supported by the structures of the Department of Agriculture. Additionally. we need much better organization of the quinoa sector. in order to have more visibility to the consumers. This starts with focusing on more development of quinoa on public awareness and promotion-marketing. More research is needed in reducing saponin content in the different quinoa product. which represent one of the current weak points in the sector and it is necessary to remove this bottleneck as it does affect. today. the quality of the product and the entire value chain. Morocco's situation within the Maghreb region places it in an advance position in the development of quinoa value chain. This should trigger a promoting cooperation within the Maghreb countries. It is also possible to create a Mediterranean or African network on quinoa to extend ideas and research results.

**Author Contributions:** A.H. supervised agronomic trials, conducted economic assessment, analyzed the data and wrote the manuscript; S.R. conducted chemical analysis and conducted valorization and transformation assessment; M.R. analyzed quinoa transformation pathway; A.F. carried out agronomic trial at the UM6P experimental station about organic amendments; F.A. carried out agronomic trial at the UM6P experimental station about new cultivars performance; K.F. carried out trials at farm levels; H.A. conducted the cost analysis; Y.J. contributed to field trials running; A.S.(Aziz Soulaimani) conducted chemical analysis; M.M. supervised agronomic trial at the UM6P

experimental station; M.E.G. supervised all agronomic trials; S.K. conducted saponin analysis; A.S.(Abdelaziz Sbai) and R.C.-A. elaborated SWOT analysis, lessons learned and development perspectives. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was carried out within an IDRC (International Development Research Center-Canada) funded project entitled: Scaling up quinoa value chain to improve food and nutritional security in poor rural communities of Morocco under the agreement number 108637-001.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data generated within this work are open access and available to be shared with interested persons according to signed agreement with IDRC.

**Acknowledgments:** First. we thank our sponsor the International Development Research Center (IDRC)—Canada for providing fund to conduct the project activities. Besides. we appreciate the efforts deployed by the technical team of UM6P- Agricultural Innovation and Technology Transfer Center (AITTC) and the technical support of the International Center of Biosaline Agriculture (ICBA). Special thanks to members of the 3rd Millennium Cooperative for couscous production and Benrim Farm (Haj Mohamed Ibrahimi) for their cooperation. Our gratitude goes to our local partners and facilitators (farmers. women cooperatives. startups. OCP group. DRA Marrakech-Safi. DPA Rehamna. ONCA Rehamna. ONSSA Rehamna. etc.).

**Conflicts of Interest:** The authors declare that they have no conflicts of interest.

#### **References**

### *Review* **Quinoa in Ecuador: Recent Advances under Global Expansion**

**Leonardo Hinojosa 1,\* , Alex Leguizamo 2 , Carlos Carpio 3 , Diego Muñoz 4 , Camilo Mestanza 5 , José Ochoa 6,7 , Carmen Castillo 6 , Angel Murillo 6 , Elena Villacréz 6 , Carlos Monar 8 , Nicolas Pichazaca 9 and Kevin Murphy 10**

**Abstract:** Quinoa is a highly diverse crop domesticated in the Andean region of South America with broad adaptation to a wide range of marginal environments. Quinoa has garnered interest worldwide due to its nutritional and health benefits. Over the last decade, quinoa production has expanded outside of the Andean region, prompting multiple studies investigating the potential for quinoa cultivation in novel environments. Currently, quinoa is grown in countries spanning five continents, including North America, Europe, Asia, Africa, and Oceania. Here, we update the advances of quinoa research in Ecuador across different topics, including (a) current quinoa production situation with a focus on breeding progress, (b) traditional seed production, and (c) the impact of the work of the nongovernment organization "European Committee for Training and Agriculture" with quinoa farmers in Chimborazo province. Additionally, we discuss genetic diversity, primary pests and diseases, actions for adapting quinoa to tropical areas, and recent innovations in quinoa processing in Ecuador. Finally, we report a case study describing a participatory breeding project between Washington State University and the Association of Andean Seed and Nutritional Food Producers Mushuk Yuyay in the province of Cañar.

**Keywords:** *Chenopodium quinoa*; quinoa production; participatory plant breeding; quinoa processing

### **1. Introduction**

Quinoa (*Chenopodium quinoa* Willd.) is an annual crop from South America. Originally thought to be domesticated in the highland region of the Andes Mountains, a recent report suggests that quinoa domestication from its wild relative *Chenopodium hircinum*

**Citation:** Hinojosa, L.; Leguizamo, A.; Carpio, C.; Muñoz, D.; Mestanza, C.; Ochoa, J.; Castillo, C.; Murillo, A.; Villacréz, E.; Monar, C.; et al. Quinoa in Ecuador: Recent Advances under Global Expansion. *Plants* **2021**, *10*, 298. https://doi.org/10.3390/ plants10020298

Academic Editors: Cataldo Pulvento and Veronica De Micco Received: 1 January 2021 Accepted: 1 February 2021 Published: 4 February 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Schard. occurred independently in the Andean highlands and on the Chilean coast [1]. Five ecotypes have been used to classify quinoas: (1) valley = grown at 2000 to 3500 m above sea level (m a.s.l.); (2) altiplano = grown at high altitudes of more than 3500 m a.s.l.; (3) salares = grown in the salt flats of Bolivia and Chile; (4) sea-level = grown at low altitude; (5) subtropical or yungas = grown in humid valleys of Bolivia [2].

Quinoa is adapted to a wide range of marginal environments [3]. It possesses high salinity tolerance and is considered as a facultative halophyte crop [4,5]. Quinoa can tolerate drought [6] and has been shown to grow well in controlled high temperature environments [7–9]; however, the combination of drought and heat can considerably reduce seed yield [9,10]. Quinoa seeds are highly nutritious, containing high contents of fiber, vitamins such as ascorbic acid, alpha-tocopherol, thiamin, and riboflavin, and minerals. Quinoa protein quality is high as it contains the nine essential amino acids in significant amounts, though not always in sufficient quantities to be a complete protein source to consumers across all age groups [11]. Moreover, quinoa possesses phytochemicals such as phenolics and bioactive peptides; these components have demonstrated beneficial effects on metabolic, cardiovascular, and gastrointestinal health [12–14].

Due to the characteristics described above, quinoa production has been expanding rapidly outside of the Andean region of South America over the last decade [15,16]. Peru and Bolivia are the major quinoa producers in the world, responsible for 86,011 and 70,763 tonnes (t) in 2018, respectively [17]. Ecuador was considered the third largest quinoa producer globally [17]; however, the extent of recent quinoa expansion worldwide is difficult to measure and it is a challenge to accurately rank Ecuador in quinoa production compared to other countries. Ecuador, one of the 17 most megadiverse countries in the world, is a small country in South America, divided into three continental regions: Amazon, mountains and coast, and one maritime region, the Galapagos Islands. [18,19].

The aim of this article is to summarize the advances in quinoa research in Ecuador. We discuss diverse aspects of the current quinoa production situation, and focus on breeding progress, traditional seed production in the province of Bolívar, and the impact of the quinoa work of the nongovernmental organization (NGO) "European Committee for Training and Agriculture" (CEFA) with farmers in the province of Chimborazo. Potential quinoa pests and diseases, genetic diversity, strategies for adapting quinoa to tropical areas, and innovations in quinoa processing are reported. Moreover, we include a case study highlighting a participatory breeding project between Washington State University (WSU) and the Association of Andean Seed and Nutritional Food Producers Mushuk Yuyay (Asociación de Productores de Semillas y Alimentos Nutricionales Andinos Mushuk Yuyay (APROSANAMY)) in the province of Cañar.

#### **2. Quinoa Production in Ecuador**

Quinoa has been cultivated from southern Colombia to the coast of southcentral Chile, including parts of northwest Argentina and some subtropical lowlands in Bolivia [20]. It was one of the main crops and food sources in Incan society and later was partially displaced by cereals such as barley and wheat that were introduced during the Spanish conquest [2]. According to historians, Ecuadorian quinoa was cultivated before and after the Spanish conquest and has been one of the most important food sources in the indigenous communities in the Ecuadorian highlands. Traditionally, quinoa is cultivated in Ecuador between 2400 to 3400 m a.s.l. in different production systems [21].

Traditionally, Ecuadorian quinoa production was relatively constant and did not exceed 1000 t prior to 2010. In 2001, international partners started to work with Ecuadorian quinoa on different projects such as establishing producers associations and initiating participatory plant breeding projects [21–23]. These efforts resulted in slight production increases beginning in 2010. In 2015, quinoa production achieved a record high, reaching 12,000 t (Figure 1A). This increase was primarily due to government policies that encouraged quinoa production. The Ecuadorian Ministry of Agriculture created the project "Promotion of Quinoa Production in the Ecuadorian Sierra", which delivered production

kits that included seeds, fertilizers, and pesticides to hundreds of farmers. In 2015, the total production area reached 7800 hectares, partially fulfilling the goals of the project. However, the increased quinoa grain production did not have a readymade market; thus, a large amount of quinoa seed was stored for the following two years, and the fall in quinoa prices explains the drop in production in 2017 [16] (Figure 1A). International demand was high in 2015 and 2016, and quinoa production from Peru and Bolivia combined reached 180,000 t in 2015 [24]. that included seeds, fertilizers, and pesticides to hundreds of farmers. In 2015, the total production area reached 7800 hectares, partially fulfilling the goals of the project. However, the increased quinoa grain production did not have a readymade market; thus, a large amount of quinoa seed was stored for the following two years, and the fall in quinoa prices explains the drop in production in 2017 [16] (Figure 1A). International demand was high in 2015 and 2016, and quinoa production from Peru and Bolivia combined reached 180,000 t in 2015 [24].

**Figure 1.** Quinoa production in Ecuador. (**A**) In the period from 2007 to 2019; (**B**) map including gross sown and harvested areas by province in 2019. Source: FAO and INEC-ESPAC [17,25]. **Figure 1.** Quinoa production in Ecuador. (**A**) In the period from 2007 to 2019; (**B**) map including gross sown and harvested areas by province in 2019. Source: FAO and INEC-ESPAC [17,25].

In 2019, quinoa production increased again (4504 t) since many farmers' associations, mainly in the provinces of Chimborazo and Cotopaxi, have stable international markets. NGOs such as Fundación Mujer y Familia Andina (Andean Woman and Family Foundation), Central European Fair Alliance (CEFA), and the McKnight Foundation encouraged organic quinoa production in these two provinces [21,26]. The total area sown in 2019 was 2057 ha, led by Chimborazo province (1549 ha and 1968 t of production) and followed by Cotopaxi, Imbabura, Carchi, and Pichincha (Figure 1B). In 2019, quinoa production increased again (4504 t) since many farmers' associations, mainly in the provinces of Chimborazo and Cotopaxi, have stable international markets. NGOs such as Fundación Mujer y Familia Andina (Andean Woman and Family Foundation), European Committee for Training and Agriculture (CEFA), and the McKnight Foundation encouraged organic quinoa production in these two provinces [21,26]. The total area sown in 2019 was 2057 ha, led by Chimborazo province (1549 ha and 1968 t of production) and followed by Cotopaxi, Imbabura, Carchi, and Pichincha (Figure 1B).

#### *2.1. Quinoa Breeding in Ecuador*

*2.1. Quinoa Breeding in Ecuador*  In the 1980s, the national germplasm bank of quinoa was established at Ecuador's National Institute of Agricultural Research (Instituto Nacional de Investigaciones Agropecuarias (INIAP)) with support from international cooperators and in consultation with Bolivian scientists. The first quinoa varieties, "INIAP Cochasqui" and "INIAP Imbaya", were selected from the germplasm accessions, and in 1992 two low saponin content quinoa varieties, "INIAP Ingapirca" and "INIAP Tunkahuan", were released [21]. Currently, "INIAP Tunkahuan" is the most widely planted variety among farmers, comprising 66% of total quinoa area with an average productivity of 1300 kg ha−1. In 2007, "INIAP Pata de Venado" was released due to its excellent adaptation to and production in high In the 1980s, the national germplasm bank of quinoa was established at Ecuador's National Institute of Agricultural Research (Instituto Nacional de Investigaciones Agropecuarias (INIAP)) with support from international cooperators and in consultation with Bolivian scientists. The first quinoa varieties, "INIAP Cochasqui" and "INIAP Imbaya", were selected from the germplasm accessions, and in 1992 two low saponin content quinoa varieties, "INIAP Ingapirca" and "INIAP Tunkahuan", were released [21]. Currently, "INIAP Tunkahuan" is the most widely planted variety among farmers, comprising 66% of total quinoa area with an average productivity of 1300 kg ha−1 . In 2007, "INIAP Pata de Venado" was released due to its excellent adaptation to and production in high elevation environments (3000 to 3600 m a.s.l.) [21,24].

elevation environments (3000 to 3600 m a.s.l.) [21,24]. Quinoa hybridization started in Ecuador in 2008 at the INIAP Santa Catalina Experimental Station (Estación Experimental Santa Catalina (EESC)) at 3050 m a.s.l. using "IN-IAP Tunkahuan" and "INIAP Pata de Venado" in the crossing block. The primary breeding objective for this hybridization was to obtain early maturing lines, with large seed Quinoa hybridization started in Ecuador in 2008 at the INIAP Santa Catalina Experimental Station (Estación Experimental Santa Catalina (EESC)) at 3050 m a.s.l. using "INIAP Tunkahuan" and "INIAP Pata de Venado" in the crossing block. The primary breeding objective for this hybridization was to obtain early maturing lines, with large seed diameters and low saponin contents. In 2009, crosses were made using the Bolivian variety "Jacha" in

order to obtain larger grain size (>2.2 mm). Evaluation of this population from the F2 to F4 was carried out at EESC using the pedigree method. Later, homogenous lines (F5–F8) were evaluated using participatory selection methodology in farmers' fields in the provinces of Imbabura, Cotopaxi, Chimborazo, Cañar, and Bolívar. In 2019, the F10 lines LQEP4 and LQEP8, derived from the cross "INIAP Tunkahuan" × "INIAP Pata de Venado", and lines EQ26 and EQ28, derived from the cross "INIAP Tunkahuan" × "Jacha" were selected. All four selected lines reached maturity in less than 170 days, had plant heights < 150 cm, were resistant to downy mildew, and yielded >1500 kg ha−1 (Table 1). Researchers at INIAP plan to release one or two new quinoa varieties from the four advanced lines in 2021 [27].

**Table 1.** Average yield, plant height, days to harvest, and downy mildew severity of four promising quinoa lines evaluated during 2015–2019 in different locations in Ecuador.

1 Severity scale of mildew caused by *Peronospora variabilis*: 1 to 3, first lower third of the plant (35%); 4 to 6, second middle third (35%); 7 to 9, last upper third of the plant (30%).

Additional hybridizations using accessions from the national germplasm bank of quinoa were made in 2016. The crosses ECU255 × EQ28 and ECU-248 × ECU-6205 produced F5 lines Q1 and Q2, characterized by high yield performances, early maturity, and short statured plants. In 2018, new crosses were made to obtain lines with low saponin contents, high yields, and different colors of seed such as black, red, and yellow.

#### *2.2. Participatory Research*

Participatory research has been conducted in Ecuador for many years; for example, participatory quinoa breeding involved farmers from Cotopaxi, Chimborazo, and Cañar in order to select promising breeding lines and future varieties [22,23]. Participatory research has been carried out in the province of Bolívar. Here, we synthesize the main results from this participatory research.

Bolívar province is located in the central region of Ecuador. It has a population of 200,000, of whom 60% live in rural areas, and agriculture is the main economic activity. The primary crops are corn (*Zea mays* L.), barley (*Hordeum vulgare* L.), bean (*Phaseolus vulgaris* L.), potato (*Solanum tuberosum* L.), faba bean (*Vicia faba* L.), Andean grains including Andean lupin "chocho" or "tarwi" (*Lupinus mutabilis* Sweet), and quinoa.

Through a cooperative sustainability strategy with the Validation, Technology Transfer and Training Unit of Bolívar (Unidad de Validación, Transferencia de Tecnología y Capacitación-Bolívar (UVTT/C-B)) in Bolívar province, INIAP established local strategic alliances with the Ecuadorian Populorum Progressio Fund (Fondo Ecuatoriano Populorum Progressio (FEPP)), Ministry of Agriculture and Livestock (MAG), Instituto Técnico Superior Agropecuario "Tres de Marzo", State University of Bolívar (Universidad Estatal de Bolívar (UEB)), and Alto Guanujo producers' organizations. To initiate participatory research, the UVTT/C-B, selected two agronomic zones: (i) El Alto Guanujo (2900 to 3600 m a.s.l), its production system consisting of 98% potato and pasture, and (ii) Middle Zone (2500 to 2850 m a.s.l.), where the base crop is a monocropping of white soft corn associated with beans and to a lesser extent, wheat, barley, quinoa, and other legumes such as peas, lentil, and chocho.

Different people were involved in each zone because of diverse weather, social, culture and economic conditions. For instance, farmers from eight communities actively participated with researchers in a farmer field school in Alto Guanujo, the Comités de

Investigación Agrícola Local (CIAL) "Progressio a la Vida". In the Middle Zone, the main participants were students, professors, and researchers from UEB as well as INIAP technicians, and farmers. The result of this participatory research was the validation and technology transfer of new climate-resilient varieties of quinoa, potato, barley, faba bean, chocho, and pasture in the Alto Guanujo and quinoa, potato, bean, corn, wheat, barley, peas, lentils, and chocho in the Middle Zone. Moreover, it trained farmers to diversify their production systems by including these new varieties. Here, we summarize the main results for quinoa (Table 2).

**Table 2.** Results of the participatory research on quinoa at two agronomic zones in the province of Bolivar, Ecuador.

#### *2.3. Traditional Seed Production*

When a variety has been generated, validated, and selected by the beneficiaries, it is essential for its dissemination to be designated "Quality Seed", which must comply with specified physical, genetic, physiological, and health attributes [28]. Through a strategic alliance with MAG-Bolívar, UEB designed and implemented a small-scale seed research and production program to accelerate seed distribution to farmers. It was accredited as an official seed producer in November 2017 by the Ministry of Agriculture and Livestock under the rules and regulations of the Ecuadorian seed law. In collaboration with INIAP, MAG-Bolívar, and organizations of seed producers, UEB produces seed both in the conventional ("Certified" seed class) and artisanal models (Selected seed), whereas INIAP produces pure seed in "Basic" and "Registered" classes.

To obtain high quality quinoa seeds, negative selection was applied in field seed production; thus, atypical plants, wild quinoas, and inferior plants were eliminated. In addition, UEB designed and implemented a participatory strategy to produce seed in the local area. Specifically, seeds were delivered to trained organizations of producers and agronomy students who planted the seeds and harvested the crop. After only one growing season, they returned to UEB twice the number of seed they originally received, thereby simultaneously increasing the volume of available seed and advancing its diffusion. The success of this seed program is linked to clearly determining quinoa seed variables of production, quality, quantity, continuity, and price.

#### *2.4. The Nongovernmental Organization (NGO) "European Committee for Training and Agriculture" (CEFA) in Chimborazo*

CEFA is a nonprofit, nonpolitical, and nondenominational NGO founded in Italy in 1972 and legally recognized in Ecuador through a technical cooperation framework agreement in 2009. In 2017, CEFA created the Inclusive and Sustainable Value Chains Program (Programa Cadenas de Valor Inclusivas y Sostenibles (PCV-IS)), funded by the European Union and the Italian Agency for Development Cooperation—AICS—whose objective is to create the quinoa chain in the province of Chimborazo, and within the framework of the MAG strategy, the popular and solidarity economy, and fair trade. The PCV-IS has three main components: (a) socio-organizational strengthening; (b) productive development; (c) associative marketing. In addition, implementing adaptation measures to climate change and including young people in the value chain are two transversa axes of the PCV-IS.

#### 2.4.1. Quinoa Chain in Chimborazo

Chimborazo province is located in the central area of the Ecuadorian highlands (Figure 1B). It owes its name to the Chimborazo volcano that rises to 6268 m a.s.l. and is located 150 km south of the city of Quito in the Western Cordillera of the Andes. Chimborazo has a population of 524,004 inhabitants and is the ninth most populous province of Ecuador [29].

Cultivation of quinoa in the province of Chimborazo was promoted by the Fundación Escuelas Radiofónicas Popular del Ecuador (Popular Radio Schools of Ecuador Foundation (ERPE)) with the support of international cooperators. They led to the first export of quinoa with organic certification in 1998. Prior to 1998, quinoa was cultivated primarily for selfconsumption. In 2003, the Corporación de Productores y Comercializadores Orgánicos Bio Taita Chimborazo (Bio Taita Chimborazo Organic Producers and Marketers Corporation (COPROBICH)) was formed, an organization that brought together producer families to comply with ERPE's commercial agreements. Sumak Life Cía. Ltd. a joint venture between ERPE and COPROBICH, was formed in 2006. COPROBICH commercially dissociated itself from Sumak Life Cía. Ltd. in 2012 to enter the markets of France, Belgium, and Canada with certified organic quinoa and the Small Producers seal. In the same year, the Maquita Foundation began exporting certified organic quinoa with the fairtrade certification of the World Fair Trade Organization (WFTO).

Quinoa in Chimborazo is produced under the organic certification regulations of the EU and the USA. In economic terms, according to estimates by the PCV-IS based on information contained in the certification companies' internal control systems, it represents a gross income of approximately USD 2,000,000 each year for 1500 producer families located in the cantons of Colta, Riobamba, Guamote, and Guano. According to the General Coordination of National Agricultural Information of the MAG, in 2019 the sale of quinoa to exporters was the main source of income for 60% of these families [30].

Sumak Life Cía. Ltd., COPROBICH, and Maquita Foundation together exported around seven hundred metric tons of quinoa grain every year, mainly to the European Union and North American markets, which corresponds to around 95% of the total production in Chimborazo province. The difference is divided between self-consumption and sale to local markets. Producers generally rotate quinoa with barley, wheat, faba bean, or a mix of vetches (*Vicia* spp.) and oats (*Avena sativa*) that are used as forage for their livestock.

2.4.2. Main Actions of the PCV-IS in the Province of Chimborazo Creation of a Technical Committee of Quinoa in Chimborazo

The objective of this technical committee is to analyze problems in production, transformation, commercialization, and consumption links of the quinoa chain in order to prioritize and articulate the actions of the direct and indirect actors. The direct actors in the quinoa chain are represented by Sumak Life Cía. Ltd., COPROBICH, Maquita Foundation, Sumak Tarpuy Corporation, and the Asociación de Producción y Comercialización de Productos Alimenticios Emprendedores Nutriandina (Association for the Production and Marketing of Food Products Entrepreneurs Nutriandina). Additionally, the indirect actors are represented by those who have support, regulation, investigation, or control functions in the chain, such as INIAP, MAG, Ecuadorian Agency for Quality Assurance in Agriculture (AGROCALIDAD), and the Polytechnic Higher School of Chimborazo (ESPOCH).

Promotion of Participatory Research and Links with the academy to Improve the Dynamics of Innovation in the Value Chain

Participatory research, led by INIAP, occurred in 18 field schools that were established in representative areas of the quinoa sector. Several studies were carried out on fertilization, intercropping systems, participatory selection of Chimborazo quinoa ecotypes, validation of the adaptation of quinoa advanced lines, and planting systems. In addition, ESPOCH implemented the project "*Diseño e Implementación del Proyecto de Producción, Transformación, Comercialización y Promoción de Consumo de la Quinua y sus Derivados"* (Design and Implementation of the Project for Production, Transformation, Commercialization and Promotion

of Consumption of Quinoa and its Derivatives). This project involved seven departments and 60 researchers from different areas. Several undergraduate research and community linking projects were conducted as part of the project, engaging more than 260 students. cialization and Promotion of Consumption of Quinoa and its Derivatives). This project involved seven departments and 60 researchers from different areas. Several undergraduate research and community linking projects were conducted as part of the project, engaging more than 260 students.

Promotion of Participatory Research and Links with the academy to Improve the Dy-

Participatory research, led by INIAP, occurred in 18 field schools that were established in representative areas of the quinoa sector. Several studies were carried out on fertilization, intercropping systems, participatory selection of Chimborazo quinoa ecotypes, validation of the adaptation of quinoa advanced lines, and planting systems. In addition, ESPOCH implemented the project "*Diseño e Implementación del Proyecto de Producción, Transformación, Comercialización y Promoción de Consumo de la Quinua y sus Derivados"* (Design and Implementation of the Project for Production, Transformation, Commer-

#### Identification and Validation of Three Species for Intercropping with Quinoa Identification and Validation of Three Species for Intercropping with Quinoa

*Plants* **2021**, *10*, x 7 of 23

namics of Innovation in the Value Chain

Common vetch (*Vicia sativa*) was sown in an intercropped system at the time of quinoa hilling. After the quinoa was harvested, the vetch remained a remnant for the grazing of breeding animals. A faba bean variety, "INIAP-441 Serrana", was sown in the system comprised of one row of faba bean x three rows of quinoa, for areas located above 3200 m a.s.l. in loamy to clay loam soils. Faba bean contributes nitrogen to the quinoa crop and is used for family consumption and occasionally for the marketing of surpluses. Finally, a chocho variety, "INIAP-450 Andino", was planted in a system comprised of one row of chocho × three rows of quinoa for farms below 3200 m a.s.l. in sandy and sandy loam soils (Figure 2). Common vetch (*Vicia sativa*) was sown in an intercropped system at the time of quinoa hilling. After the quinoa was harvested, the vetch remained a remnant for the grazing of breeding animals. A faba bean variety, "INIAP-441 Serrana", was sown in the system comprised of one row of faba bean x three rows of quinoa, for areas located above 3200 m a.s.l. in loamy to clay loam soils. Faba bean contributes nitrogen to the quinoa crop and is used for family consumption and occasionally for the marketing of surpluses. Finally, a chocho variety, "INIAP-450 Andino", was planted in a system comprised of one row of chocho × three rows of quinoa for farms below 3200 m a.s.l. in sandy and sandy loam soils (Figure 2).

**Figure 2.** Intercrop system of chocho (*Lupinus mutabilis*) with quinoa. Comunidad Achullay, Guamote. Juan Yuquilema (farmer), Fausto Yumisaca (INIAP Chimborazo), Rodrigo Aucancela, and Galo Morocho (CEFA). Photo: A. Leguizamo. **Figure 2.** Intercrop system of chocho (*Lupinus mutabilis*) with quinoa. Comunidad Achullay, Guamote. Juan Yuquilema (farmer), Fausto Yumisaca (INIAP Chimborazo), Rodrigo Aucancela, and Galo Morocho (CEFA). Photo: A. Leguizamo.

#### Use of Local Organic Matter Source for Soil Improvement Use of Local Organic Matter Source for Soil Improvement

ESPOCH, through the Faculty of Sciences and the local government of Riobamba, will begin to recover the vegetable residues from local markets, compost them, and then supply the compost to quinoa farmers. Approximately 1000 t of compost per year are expected to be recovered from this initiative. ESPOCH, through the Faculty of Sciences and the local government of Riobamba, will begin to recover the vegetable residues from local markets, compost them, and then supply the compost to quinoa farmers. Approximately 1000 t of compost per year are expected to be recovered from this initiative.

#### Development and Implementation of Adaptation Measures to Climate Change

Quinoa soils are vulnerable to water erosion during intense rains, especially fields with newly planted crops. Therefore, a transition from the traditional tillage system to one of minimum tillage, including cover crops such as vetch and direct seeding of quinoa, is required.

#### **3. Pests and Diseases**

#### *3.1. Quinoa Pests in Ecuador*

A formal study of insect pests affecting quinoa in Ecuador was conducted 30 years ago [31]. Species identification was performed by international experts at that time.

Seven lepidopterans from the Noctuidae family were identified affecting quinoa. Cutworms damaging plantlets in the field such as *Agrotis deprivata* Walker and *A. ipsilon* (Hufnagel) were reported. The following leaf or grain chewers were also reported: *Peridroma saucia* (Hübner), *Dargida grammivora* Walker, *Spodoptera* sp., and two nonidentified species of *Copitarsia*. Two other lepidopterans were identified—*Scrobipalpula* sp. in the Gelechiidae family affected the leaves and an *Ephestia* sp. of the Pyralidae family damaged the stored quinoa seed. Two tachinid flies were found: *Incamyia* sp. was identified parasitizing both *Agrotis* species and *Elfia* sp. was a parasitoid of *Scrobipalpula* sp. Two species within the Coleoptera order were identified: the curculionid *Naupactus* sp., which eats quinoa leaves and grains, and *Oryzaephilus surinamensis* (L.) (Cucujoidea), which affects stored quinoa flour. *Liriomyza* sp. (Diptera: Agromyzidae) is a leaf miner reported in quinoa. The sap feeders *Paratanus yust* Young (Hemiptera: Cicadellidae) and *Proba sallei* (Stal) (Hemiptera: Miridae) were identified as affecting quinoa plants in the field. The same study reported the presence of a few natural enemies of quinoa pests—two tachinid flies were found: *Incamyia* sp. was identified as parasitizing both *Agrotis* species and *Elfia* sp. as a parasitoid of *Scrobipalpula* sp. [31].

Primary pests of quinoa are only briefly mentioned in crop management documents. One publication reports cutworms (*Agrotis* sp.) in young plants and birds are the most common pests [32]. Recently, between June and August 2020, an insect inventory was carried out to survey entomofauna associated with organic quinoa fields at six locations in the province of Chimborazo. The most abundant insects collected in this inventory were from the following orders: Hemiptera (one family), Coleoptera (three families), Lepidoptera (two families); parasitoid insects from the order Hymenoptera (one family), and predatory insects from the Neuroptera (one family) (Table 3). The main phytophagous insects observed on the plants belonged to the family Aphididae (Hemiptera), which attack the leaves, and Gelechiidae (Lepidoptera), which feeds on the inflorescence (Table 3). It is also important to point out the presence of parasitoids the of Braconidae family (Hymenoptera).

**Table 3.** Families of insects collected in destructive sampling of quinoa plants. The families are ordered in relation to abundance (from highest to lowest).

1 Different morphospecies.

Insects of the family Cucurlionidae were the most abundant phytophagous insects found in the soil around the roots (Table 4). Cucurlionidae fed on the external parts of roots and did not bore into the roots. Additionally, a greater quantity of Annelidae (earth worms) was found in the plots from the lower elevation locations.

Insects from the following orders were collected at the lower elevation locations using yellow bowl pan traps placed 40 cm above the soil: Coleoptera (two families), Diptera (16 families), Hemiptera (four families), Hymenoptera (15 families), Lepidoptera (one family), and Thysanoptera (one family). In the traps of the plots in the location at the highest altitude, insects from the following orders were collected: Coleoptera (one family), Diptera (21 families), Hemiptera (three families), Hymenoptera with 10 families (Table 5), Neuroptera (one family), and Thysanoptera (one family).

**Table 4.** Insect families collected in soil blocks of quinoa plants removed from plots. The families are ordered in relation to abundance (from highest to lowest).

1 Different morphospecies.

**Table 5.** Families of Hymenoptera collected in yellow bowl pan traps in the province of Chimborazo. The families are ordered in relation to abundance (from highest to lowest).

The richness of individuals collected of Hymenoptera may indicate the good health of the plots sampled, since we know that individuals of this order are sensitive to the application of insecticides. In the plots from the lower altitude locations, most of the Hymenoptera belonged to Halictidae, Braconidae, Chalcididae, and Ichneuminidae, whereas most of the Hymenoptera in the plots from the highest altitude locations belonged to Crabronidae, Pteromalidae, Bethylidae, and Megaspilidae (Table 5).

#### *3.2. Quinoa Diseases in Ecuador*

In most cultivated plants, diseases are major biotic constraints usually involving a wide range of pathogens. Unlike most crops, quinoa is in general a marginal host for plant pathogens. In Ecuador, neither viruses nor bacteria have been reported to infect quinoa [33], though this could be due to a lack of pathology research. *Cercospora* sp. and *Phoma* sp., the most common foliar fungi [34], are low damaging pathogens. In Ecuador, the most economically important quinoa disease is downy mildew (DM), caused by the oomycete *Peronospora variabilis*.

This low range of pathogens infecting quinoa in Ecuador agrees to some extent with the worldwide quinoa disease review reported by Danielsen et al. [35]. However, *Sclerotium rolfsii* and *Pythium zingiberum,* causing seedling diseases, and *Ascochyta hyalospora* and *Phoma exigua* var. *foveata*, causing ascochyta leaf spot and brown stalk rot, respectively, have not been reported in Ecuador. *Rhizoctonia solani* and *Fusarium* sp. causing damping off have been observed sporadically but are not considered threats for quinoa in Ecuador.

#### 3.2.1. Downy Mildew

Downy mildew (DM) is the most damaging disease that threatens quinoa production worldwide [35]. In Ecuador, this disease is especially important in commercial monoculture of uniform varieties. The Department of Plant Pathology and the Quinoa Breeding Program of INIAP have been conducting research to identify and introgress durable resistance (DR) into quinoa DM, which is highlighted in this report.

*Peronospora variabilis*, previously classified as *P*. *farinosa* f. sp. *chenopodii*, was considered specific to quinoa; however, the pathogen has also been found infecting *Chenopodium album* and *C. murale* [36,37]. In Ecuador, isolates infecting *C. album* belong to the same population that infects quinoa and since *C. album* is a common weed of quinoa, the secondary host status of *C. album* is an important aspect to consider for DM management as well as in breeding for DR.

*P. variabilis* reproduces by heterothallic sexual reproduction [38], and two mating types appear to occur frequently, since oospores are massively produced on the leaves and carried by the seed [39,40]. In addition, oospores transmitted by seed are the primary pathogen inocula, and seed transmission is the main dispersion strategy for the pathogen to reach regions outside the Andes.

Studies of pathogenic diversity have shown that *P. varibilis* is a variable and adaptable pathogen [41]. Four virulence groups (Vs) of *P. varibilis* have been identified with respect to R-3 factors (R). V1 infects only susceptible lines, V2 infects susceptible lines and lines carrying R1, V3 infects susceptible lines and lines carrying R1 and R2, and V4 infects susceptible lines and lines carrying R1, R2, and R3. Pathogen evolution that has taken place in Ecuador is associated with single successive step mutations, which is in turn associated with the breeding initiatives carried out during the 1980s and early 1990s, which exploited the three R-factors, and R2 and R3 were released in varieties INIAP Ingapirca and INIAP Imbaya, respectively. Levels of resistance to DM at time of variety release were lower than 4 on a 0–9 scale, values located in the resistance fraction of the scale [21].

*P. varibilis* evolution is associated with mutation events. Although genetic recombination has not yet been evident, heterothallism is a potential mechanism by which the pathogen evolves beyond R-factor adaptation to include improved adaptation to environmental conditions and increased aggressiveness. Efficient seed transmission of the pathogen facilitates this potential mode of pathogen evolution. These epidemiological aspects are primarily associated with modern quinoa cultivation, in which cultivating genetically uniform varieties drives higher selection pressures on the pathogen.

Development of strategies to search for DR to DM quinoa should be an important objective of modern breeding programs. Since R-factors are often ephemeral, screening for new sources of R-factors is not advisable. Therefore, non-race-specific resistance appears to be the most effective strategy to attain DR in the quinoa/downy mildew pathosystem. In Ecuador, "INIAP Tunkahuan" carries this type of resistance and has remained effective since variety release in 1992. Resistance of "INIAP Tunkahuan" can be considered durable since it aligns with the requirements for durable resistance as proposed by Johnson [42]. Thus, "INIAP Tunkahuan" has been hegemonically cultivated in commercial monoculture in a relatively large area, in conditions conducive to the disease, and for a long period of time (more than 25 years).

Levels of non-race-specific resistance were studied in four locations in Ecuador conducive to DM [22]. Resistance varied from low to high in lines without R-factors and in lines with defeated R-factors (residual resistance). Furthermore, lines carrying non-race specific resistance scored area under disease progress curve (AUDPC) values as low as lines carrying efficient R-factors, indicating that non-race-specific resistance can provide the same high level of protection as provided by R-factors. This resistance of non-race-specific type is quantitative in nature, and in other downy mildews, as in lettuce, this resistance is polygenic, with major and minor effects [43,44]. The high variation of AUDPC in lines with non-race-specific resistance suggests that this type of resistance is polygenic. In this study, lines were found with similar or higher levels of resistance than "INIAP Tunkahuan", suggesting that sources of non-race-specific resistance are available in quinoa germplasm of the Quinoa Breeding Program at INIAP.

The early Ecuadorian experience in selection for DM resistance showed that breeders are interested in high levels of resistance, and by selecting lines with low disease severities, they are unconsciously favoring R-factors. However, they eventually also select high levels of non-race-specific resistance when lines carry similar disease severities to R-factors, which is the case of "INIAP-Tunkahuan". Since R-factors resistance appears ephemeral, non-race-specific resistance is a more durable resistance strategy. Discarding the most susceptible and most resistant lines, as proposed by Parlevliet (1993) appears a practical strategy to favor selection of non-specific resistance and to select for DR to DM [45]. However, by discarding resistant lines, other promising traits and residual resistance can be inadvertently discarded. Therefore, to select non-race-specific resistance more efficiently, discard the most susceptible lines with the assistance of R-factors postulation to assess residual resistance in the field. This strategy is presently being implemented by the Quinoa Breeding Program of INIAP.

#### 3.2.2. Minor Diseases

Quinoa leaf spot caused by *Cercospora* sp is considered a minor disease in Ecuador [34]. Symptoms regularly develop late in the season on leaves of the upper part of the plant. Symptoms are characterized by round to oval and brown to grey lesions with diameters of less than 1 cm with darker brown reddish margins. Similar symptoms are also found in Ecuador on *Chenopodium album* [34]. This pathogen was also detected in the USA on quinoa, and the fungus was phylogenetically similar to *Passalora dubia*, which infects *C. album*; therefore, *P. dubia* is presently the taxonomical status of the causal agent of the quinoa leaf spot, known in the USA as *Passalora* leaf spot [46]. The incubation period of *P. dubia* of around 30 days [46] is significantly longer than that of *Cercospora* sp., which might explain its limited pathogenicity and why epidemics appear late in the quinoa growing season.

Eye-like stalk spot caused by *Phoma* sp. is also a disease that affect quinoa late in the cropping season. Symptoms are gray spots with diameters of 1–3 cm surrounded by brown to dark margins. Dark pycnidia of the pathogen become regularly visible in the gray background of the lesion [34,35]. In conducive conditions, lesions can join and cover a considerable area of stems. Lesions can also be produced by mechanical injuries such as those caused by hailstorms.

#### **4. Genetic Diversity**

Ecuadorian quinoa belongs to the valley ecotype. This quinoa ecotype is characterized by tall, late-maturing plants with high seed yield potentials and biomass accumulation [21]. Seed yield is negatively affected when the photoperiods exceeds 14 h in high temperature environment [2,47]. Currently, the quinoa germplasm at INIAP contains 608 accessions— 283 from Ecuador (46%) and the rest from other countries, primarily Peru and Bolivia [48].

In recent decades, significant genetic erosion of quinoa landraces has been observed in the field. For instance, 30 years ago, variation in quinoa grain color was easy to find, including colors such as white, cream, brown, yellow, red, pink, and black. Currently, just white- or cream-colored quinoa grains are grown by most farmers, due to market demand and eating patterns in production areas [49]. A core quinoa collection using 469 quinoa accessions from the Ecuadorian quinoa germplasm bank was evaluated, and discriminating traits such as downy mildew reaction, seed color, days to flowering, and seed yield differentiated four genetic groups [50].

Traditionally, Ecuadorian quinoa is positioned to have limited genetic diversity, with Altiplano (Peru–Bolivia) quinoa indicated as the most probable point of introduction for Ecuadorian accessions [20]. This evidence came from the molecular and genetic diversity studies where just two Ecuadorian quinoa accessions were included [51,52]. Recently, a molecular characterization of diversity of Ecuadorian quinoa was conducted using 15 species-specific SSR markers on 84 quinoa accessions [53]. The results showed that Ecuadorian quinoa was highly diverse—196 alleles were detected, and the genetic heterozygosity (*HE*) was 0.71. Three subgroups were determined by a phenetic analysis one group corresponded to the "INIAP Tunkahuan" variety that has been exposed to intense inbreeding and selection. The three groups included quinoa from the seven provinces

where seed was collected, and indicated that ancestral landrace populations had been disseminated throughout Ecuador mainly due to farmers' seed interchange.

#### *Wild Quinoa Relatives*

Wild quinoa is known as "ashpa quinoa" in the Quichua language or "ajara" in the Aymara language. The wild quinoa in Ecuador has been poorly studied; however, there is a report that a free-living (wild, weedy, spontaneous) quinoa population named as *C. quinoa* ssp. *milleanum* Aellen or var. *melanospermum* is distributed mainly in the mountains of Ecuador [54]. There is a hypothesis that *C. quinoa* ssp. *milleanum* could be closely related to the North American tetraploid species *C. berlandieri* subs. *zschachkei* and the South American tetraploid species *C. hircinum* ssp. *catamarcense* [54,55].

A phylogenetic study focused on the amplification and sequences of gene rpoB in 32 wild quinoa accessions collected from four provinces including Carchi, Imbabura, Cotopaxi, and Chimborazo showed high inter- and intrapopulation genetic diversity across the accessions [56]. Accessions from Carchi and Imbabura had 77.4% of the variability and genotypes from Cotopaxi and Chimborazo had 70.2%. In this study, all the wild quinoas were designated as *C. quinoa*; this description resulted in a misunderstanding between the cultivated quinoa and the wild quinoa in Ecuador. Thus, it is necessary to conduct more molecular studies including *C. berlandieri*, *C. hircinum,* and quinoa varieties as controls to understand the evolutionary origin of Ecuadorian quinoa.