Characterization of Spanish Lentil Germplasm: Seed Composition and Agronomic Performance Evaluation
by
, , and
Pilar Brun
1
,
Marcelino de los Mozos
2,
Maria Cristina Alcántara
2,
Francisco Perea
1,
María Camacho
1,* and
Dulce Nombre Rodriguez Navarro
1,* 1
Andalusian Institute of Agricultural and Fisheries Research and Training (IFAPA), Center Las Torres, Ctra. Sevilla-Cazalla, Km 12.2, 41200 Seville, Spain
2
Department of Management and Conservation of Plant Genetic Resources, Center of Agroforestry Research Albaladejito, Ctra. Toledo-Cuenca, Km 174, 16194 Cuenca, Spain
*
Authors to whom correspondence should be addressed.
Sustainability 2024, 16(6), 2548; https://doi.org/10.3390/su16062548
Submission received: 22 January 2024
/
Revised: 11 March 2024
/
Accepted: 12 March 2024
/
Published: 20 March 2024
(This article belongs to the Section Sustainable Agriculture)
Abstract
:Lentil (Lens culinaris Medik.) is a resilient annual herb belonging to the Fabaceae family. Known for their ability to fix atmospheric nitrogen in symbiosis with rhizobia, lentils demonstrate moderate drought tolerance. Legumes are key crops in sustainable agriculture due to their low water and N requirements. This study evaluates the symbiotic responsiveness of various lentil accessions from the Spanish germplasm bank to different rhizobia strains. Additionally, the nutritional profile of seeds was determined, encompassing energy, fat, available carbohydrates, sugars, proteins, fibre, mineral content, and macro and micronutrients. Phenolic compound content was assessed using advanced UHPLC-HRMS techniques. The agronomic performance of six selected accessions was evaluated across two distinct locations under rainfed conditions and varying management systems. Notably, the protein content of the evaluated accessions exceeded 25%, particularly in two standout accessions, namely BGE025596 and BGE026702, with protein levels surpassing 30% and fat content below 2%. Furthermore, accessions BGE016362 and BGE026702 exhibited exceptional iron (Fe) content, exceeding 1 g/100 g of seed flour. It was observed that coloured microsperma lentil accessions harboured higher concentrations of phenolic compounds than non-coloured macrosperma seeds’ antioxidants and anti-inflammatories. Agronomic performance varied based on cropping region and accession origin.
1. Introduction
Lentil (Lens culinaris Medik.) is an annual herb that belongs to the Fabaceae family and is moderately tolerant of drought. Canada is the largest lentil producer in the world, boasting an annual production of 2 million tonnes. Following closely behind, India ranks second with an annual production of 1.6 million tonnes, while Australia is the third largest producer globally with 853,641 tonnes [1]. Lentil yield across the ten top world producers ranges from 730 to 1400 kg/ha. In 2022, Spain had a lentil acreage of 42,200 ha [2]. Two autonomous regions of Central and Northern Spain, Castilla-León and Castilla-La Mancha, are the most important production areas, with 12% of the national acreage dedicated to lentil cropping. Since most legume cultivation in Spain primarily occurs under rainfed conditions, lentils are predominantly grown in similar environments.
Rhizospheric bacteria named rhizobia form mutualistic associations with legume plants. During such interactions, bacteria fix atmospheric nitrogen and provide it to plants in a readily available form in exchange for carbon compounds from photosynthesis [3]. In most cases, N-fertilisers are not needed or recommended to complete the crop cycle. Due to climate change, legumes’ low water characteristics and N requirements make them key crops for sustainable and hard forecast scenarios in agricultural practices. Selecting specific and highly efficient rhizobia strains compatible with the target legume is a critical step when new species or plant accessions relying on nitrogen fixation are intended to be cropped. Additionally, legumes’ advantages positively contribute to intercropping systems. Altieri et al. [4], among others, advocated for the imperative shift towards more sustainable and responsible agriculture as well as the development of resilient arable cropping systems. These principles include diversifying the agroecosystem by enhancing biodiversity at landscape, farm, and field levels, both spatially and temporally. In addition, optimizing the beneficial biological interactions inherent in agroecosystems can maximise ecological services. Intercropping, defined as the simultaneous cultivation of two or more crop species within the same field, strongly aligns with these principles. Despite being a widespread global practice historically, intercropping has been largely disregarded in Europe due to intensification efforts. However, it is now experiencing a resurgence of interest in the transition from intensive to low-input agricultural systems [5].
Many nations grapple with various forms of malnutrition, spanning from inadequate nutrition and deficiencies in essential nutrients to the prevalence of obesity and diseases linked to diet. Within this framework, legumes emerge as significant food crops and should be integrated into a balanced diet as accessible sources of protein, complex carbohydrates, fibres, vitamins, minerals, and bioactive compounds [6], alongside their low fat content [6,7,8]. Nutrition experts regard grain legumes as a healthy dietary choice that is rich in nutrients such as protein, which can notably reduce the risk of stroke and heart disease. Despite being staples in numerous traditional diets, global legume consumption has declined.
Spanish landraces are categorised according to seed weight rather than phenology [9]. This finding supports Erskine’s conclusion [10] that phenology is a significant factor in adaptation on a macrogeographic scale; however, its importance diminishes at the microgeographic level. Seed weight, being a trait of low adaptive value, is easily observed [9,11]. Human preferences may have been a relevant factor in selecting and preserving Spanish lentil landraces. Some studies [9,10,12] found that the microsperma type was more polymorphic than the macrosperma type. In our case, the three microsperma accessions were coloured, unlike the macrosperma ones. In this study, we worked with lentil germplasm from the Plant Genetic Resources Centre (CRF) to contribute to the conservation of food–legume genetic diversity and its exploitation in food production [13]. In turn, agricultural sustainability and the availability of healthier food products will also increase. To the best of our knowledge, this is the first study concerning agronomic performance evaluation, nutritional, mineral, and phenolic compounds (antioxidants and anti-inflammatories) that characterize local lentil landraces or accessions. At the same time, responses to rhizobia strains were also addressed. These findings provide crucial insights into lentil cultivation practices and germplasm conservation strategies.
2. Materials and Methods
2.1. Plant Material
Seeds of lentil accessions were provided by the Plant Genetic Resources Centre (CRF), Madrid, Spain. Seed augmentation was conducted at the Agriculture Experimental Station Albaladejito (IRIAF, Cuenca, Spain) in 2021. The accession number code and origin of the lentils used in this work are listed in Table 1.
2.2. Rhizobium Strains and Culture Media
Rhizobium leguminosarum strains, namely HA-2 and GU-2, isolated from Vicia narborensis L. and Pisum sativum L., respectively [14], and ISL37 and ISL55 [15], isolated from Vicia faba L., were used in this work. Bacterial strains were routinely grown in yeast–mannitol (YM) [16] medium or tryptone–yeast extract (TY) medium [17].
2.3. Plant Test under Controlled Conditions
Two plant trials, under greenhouse or plant growth chamber conditions, were conducted on lentil plants growing in 0.5 l Leonard jars [16], filled with a perlite-vermiculite mixture (1:2, v/v) and watered with N-free nutrient solution [18]. Plant tests aimed at determining the symbiotic capacity of R. leguminosarum strains were conducted on disinfected and pre-germinated lentil accession surfaces. The number of native rhizobia in soil samples was estimated by the most probable number (MPN) counting technique using the commercial small-seeded Pardina as the plant host [19].
2.4. Soil Characteristics and Climate of Experimental Areas
The main soil characteristics were analysed at the IFAPA Laboratory of Soil and Water Management employing standardized methodologies (Table 2). The accumulated rainfall and monthly averages of maximum and minimum temperatures throughout the crop setting were measured at each experimental station (Figures S1 and S2).
2.5. Field Experiments
Field experiments (FE) were conducted at two locations with different edaphoclimatic conditions: the Agriculture Research Stations IRIAF-Albaladejito (Cuenca, Spain) (40°04′ N, 2°08′ W, 997 masl) and IFAPA-Tomejil (Carmona, Seville, Spain) (37°28′ N, −5°38′ W, 253 masl) from 2021 to 2022. Experimental fields were laid out in a randomised complete block design with three replicates. In each block, there were plots of 1.2 × 7 m (divided into rows, spaced 0.5 m apart). A space of 0.5 m was allowed between plots and 2.5 m between blocks. Three management systems were assessed: legume monocrop, inoculated-legume monocrop, and intercropping with barley (commercial cultivar Baliner). A planting density of 300 plants/m2 for the monocrop system and 200 plants/m2 for the intercropping system (160 lentil plants and 40 barley plants) was used. In all inoculated treatments, seeds were inoculated with perlite-based inoculants following [20] and consisted of two strains (GU-2 and HA-2) [14].
2.6. Proximate and Mineral Composition of Seeds
Dry and raw seeds of lentil accessions were finely ground and filtered through a 1 mm sieve to obtain the corresponding flour. These flour samples were then sent to the accredited specialised analysis unit Laboratorio Agroalimentario de Córdoba, (AGAPA) to determine proximate and mineral composition. The analysis included the mandatory nutrition components outlined in EU Regulation No. 1169/2011 (art. 30) [21] in addition to fibre content, ash, and humidity. Mineral elements such as nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), sodium (Na), iron (Fe), manganese (Mn), copper (Cu), and zinc (Zn) were quantified using ICP-OES (Inductively Coupled Plasma–Optical Emission Spectrometer).
2.7. Phenolic Compounds Determination
Phenolic compounds were analysed using a liquid chromatography system consisting of a binary UHPLC Dionex Ultimate 3000 RS connected to a quadrupole–orbitrap QExactive hybrid mass spectrometer (ThermoFisher Scientific, Waltham, MA, USA) with HESI ionization probe, at CITIUS (Centre of Research, Technology and Innovation, University of Seville, Seville, Spain). Xcalibur software 4.3 was used for instrument control and data acquisition. Separation was conducted using an Acquity BEH C18 column (1.7 µm particle size, 100 × 2.1 mm) (Waters) at 40 °C at a flow rate of 0.5 mL/min. A binary gradient consisting of (A) water and (B) methanol, both containing 0.1% formic acid, was used with the following elution profile: 5% B (1 min), linear gradient to 100% B (9 min), 100% B (2 min), and finally 5% B (3 min). The injection volume was 5 µL. A data-dependent acquisition method (Top5) was used in negative mode at resolutions of 70,000 and 17,500 at m/z 200 FWHM for Full Scan and Product Ion Scan, respectively. HESI source parameters were: spray voltage, 3.0 kV; S lens level, 50; capillary temperature, 320 °C; sheath and auxiliary gas flow, 60 and 25, respectively (arbitrary units); and probe heater temperature, 400 °C. Trace Finder 5.1 software was used for data treatment. The identification was made by comparing retention time, the exact masses of pseudomolecular ions and their fragment ions (maximum deviation of 5 ppm) with data contained in a phenolic compounds database with 87 compounds. Isotopic pattern scores higher than 80% were also required.
2.8. Statistical Analysis
Statistical analysis was conducted employing a linear model analysis of variance (ANOVA). Multiple comparisons of treatment means were conducted using Fisher’s protected Least Significant Difference (LSD) method at a significance level of 5%. Linear correlations between two variables were analysed using Pearson’s linear model analysis.
All the analyses were performed in Statistix 9.1 software (http://www.statistix.com/, accessed on 15 January 2023).
3. Results and Discussion
In this study, we employed eight accessions of Lens culinaris Medik. from Central Spain (Castilla-La Mancha region): Guadalajara, Ciudad Real, and Albacete provinces; Southern Spain (Andalusia region): Jaén and Granada provinces; Canary Islands: Lanzarote and Fuerteventura Islands, and one from León Province (Northern Spain) (Table 1). These accessions belong to a wide geographical area with different ecological conditions. To our knowledge, this is the first investigation assessing the symbiotic reaction of Spanish lentil accessions to rhizobia strains. Previous studies, which focused on evaluating agro/morphological traits in one hundred Spanish landraces [9], did not assess symbiotic performance.
3.1. Plant Test under Controlled Conditions
In these assays, all studied accessions positively responded to the inoculation of several rhizobia strains under controlled conditions, albeit with significant differences in symbiotic efficiency, indicating a high plant genotype x rhizobia strain interaction.
The Rhizobium leguminosarum strains used in this study were obtained from root nodules of various plant hosts (Vicia ervilia, Pisum sativum, and Vicia faba), which are distinct from lentils. Strains HA-2 and GU-2 demonstrated effectiveness in nodulation and nitrogen fixation in lentils and several Vicia spp. [16]. By contrast, ISL strains were not previously tested in lentils and induced effective nodules in Pisum sativum [15]. It is well-known that strains within the R. leguminosarum complex exhibit a broad host range. Our first trial involved the inoculation of four different lentil accessions, two macrosperma (from Albacete and Jaén) and two microsperma (from Granada and León) types, with four specific rhizobia strains (Table 3). Compared to non-inoculated plants, it yielded a positive response in terms of shoot biomass accumulation, except for some combinations, such as ISL37 x Albacete and GU-2 and ISL55 x Jaén. In most accessions, the nodulation capacity (number of nodules and nodule mass) was superior using the HA-2 strain, as reflected in its symbiotic efficiency, with some exceptions such as combinations ISL37 x Jaén and GU-2 x Granada. In the latter accession, the symbiotic efficiency was higher when combined with the strain ISL37. In general, lentil accessions of microsperma types showed higher symbiotic efficiency than all inoculant strains compared to macrosperma types. In addition, these two microsperma accessions (local denomination verdina) had dark-green pigmented testa, which is consistent with other works [22,23] showing that coloured seed varieties of legume plants, namely Phaseolus vulgaris and Vigna subterranea, had superior nodulation and N2 fixation capacity. Correlations between variables, plant biomass, and nodule biomass were high and positive for all rhizobia strains, where r = 0.78–0.90. Lower but positive correlations were found between variables, plant biomass, and number of nodules, where r = 0.33–0.66. Based on the results of this trial, strains HA-2 and GU-2 were chosen to expand the assessment to other accessions and conduct further evaluations under field conditions.
In a second plant trial, six lentil accessions chosen based on their geographical origin and seed type (three macrosperma and three microsperma) were evaluated for their symbiotic response to the selected strains HA-2 and GU-2 (Table 4). Compared to non-inoculated plants, all accessions responded positively to inoculation in terms of shoot biomass accumulation. In general, plants accumulated more biomass when inoculated with strain GU-2, as reflected in their high symbiotic efficiency. This fact is especially evident in accessions from León and Granada, confirming the data obtained in the previous trial and from other authors, where coloured varieties showed superior symbiotic efficiency [22,23]. Surprisingly, the nodulation capacity (number of nodules and nodule mass) of both strains was similar in each accession. Correlations between plant biomass and nodule biomass were high and positive for both strains (r = 0.75–0.79), whereas correlations between plant biomass and the number of nodules, where r = 0.41–0.50, were lower but still positive.
3.2. Field Experiments
Three management systems were assessed for both field experiments: legume monocrop, inoculated-legume monocrop, and intercropping with barley (commercial cultivar Baliner).
In the field experiments, the same six accessions of Lens culinaris Medik. were employed. Fe results at IFAPA-Tomejil (Table 5) showed that grain yields of accessions from the Canary Islands and those of the verdina type (from León and Granada) significantly outstood grain yields of two other accessions under conventional monocropping systems. Seed inoculation with a specifically composed inoculant of two R. leguminosarum strains (GU-2 and HA-2) did not increase seed yield over non-inoculated monocrop treatment. Under intercropping management with barley, accessions from Fuerteventura Island and Granada both outstood the yields of the others. Under intercropping management, lentil yields were not reduced in most accessions, except Lanzarote Island and León accessions. Under climate conditions in the experimental area, which was located in Southern Spain at low altitude with less rainfall and high minimum temperatures (Figure S1), accessions originating from the provinces of Guadalajara and Ciudad Real in Northern Spain yielded significantly less than the other accessions, contrasting grain yields obtained in FE-Albaladejito (see below).
Barley yield results (FE-IFAPA-Tomejil) under intercropping management (lentil: barley) are shown in Table S1. No differences were observed between the grain yields of unfertilized monocrop barley and barley intercropped with lentils. Furthermore, barley intercropped with lentils from Lanzarote Island did not exhibit significant differences in yield compared to barley grown under fertilized monocrop conditions.
FE results at IRIAF-Albaladejito (Table 6) showed that under conventional monocrop conditions, grain yields of accessions from Guadalajara and Granada significantly outstood the others (average 1.5–4.7-fold). Moreover, accessions from the Canary Islands did not perform well in the Northern Spain cultivation area. Under these climate conditions, temperatures were lower (minimum temperatures below zero) during crop development in the winter season (Figure S2) compared to the prevalent climate conditions in Southern Spain. Two accessions (from Ciudad Real and León) positively responded to inoculation with specific rhizobia strains (inoculated monocrop vs. monocrop), with grain gains of 61 and 21%, respectively. which resulted unpredictable as the size of rhizobia population in this soil (Table 2) exceeded the recommended rhizobia density, that may potentially preclude the use of inoculants, or a positive response to inoculation [24]. In [24], the authors showed that the probability of enhancing yield decreases dramatically with increasing amounts of indigenous rhizobia. The response to inoculation and competitive success of inoculant rhizobia were inversely related to the amount of indigenous rhizobia. In general, intercropping management systems did not negatively affect the grain yield of most accessions, except for Fuerteventura and Granada. Contrasting the results obtained in Southern Spain, accessions from warmer climate conditions (Fuerteventura and Lanzarote) adapted less (less mean yield) to the prevalent conditions in Cuenca.
In summary, the Seville field area accumulated less rainfall during the cropping season than Cuenca (321 mm vs. 421 mm), of which 107 mm and 70 mm, respectively, fell during the full pod development period in March. It is possible that the clay-type soil of the Seville experimental area contributed to a better water reservoir till harvest, which could partially explain the generally higher yields to some extent. The average yield of four Spanish accessions grown in Southern Spain aligns with reported yield values [8] for 13 lentil breeding lines, ranging from 1182 to 1529 kg/ha. However, only accessions from Guadalajara yielded over 1000 kg/ha in the experimental field in Northern Spain.
3.3. Proximate and Mineral Composition of Seeds
The increasing world population has led to a fast increase in food demand, posing a significant challenge to meet nutritionally balanced diet needs. Pulses have the potential to address these challenges and offer substantial nutritional and physiological benefits, making them crucial components of the human diet. Chickpeas, green gram, peas, horse gram, beans, lentils, black gram, and similar legumes are abundant in protein (190–260 g kg−1), carbohydrates (600–630 g kg−1), dietary fibres, vitamins, and bioactive compounds [6]. In addition, seed legumes are also a rich source of essential micronutrients, such as iron, potassium, magnesium, zinc, and boron. Daily mineral intake requirements can be fulfilled by consuming 100–200 g of pulses such as lentils, cowpeas, and chickpeas.Thus, they have gained more interest in the field of developing healthy and functional foods. The proximate composition of lentil seeds is presented in Table 7. We determined the constituents referred to as mandatory nutrition declaration in [21], plus fibre, ash, and humidity content.
The nutritional constituents of lentil accessions contrast the values reported in FAO [7] for 21 lentil entries. All comparisons were made based on mature, whole, dried, and raw seed data. The energy values of our samples averaged 343 kcal, which is below but consistent with reported values of 355–375 kcal. Available carbohydrates and fat content were higher (averages of 55.7 and 2.05 g/100 g, respectively) in our samples than reported values. Some differences were found when comparing our results to those reported by [25] for whole lentils; thus, Spanish lentils have twice the fat content, higher protein content, and lower available carbohydrates. The two verdina accessions had the lowest fat (<2%) and carbohydrate contents of the analysed Spanish accessions. Reported values in the FAO food composition guide, such as total dietary fibre (11.7–19 g), and those reported by [25] (30.5 g) are superior to our samples (average 4.4 g). The average protein content was similar to those reported in both surveys (28–25.8%), while verdina accessions showed the highest protein content (>30%). The mineral fraction of seeds (ash content) was superior in Spanish microsperma (4.7%) than in macrosperma-type lentils (3.3%), with an overall average of 4.0 g/100 g, while the reported FAO values are between 2 and 3.3%. Additionally, Regulation (EC) No. 1924/2006 [26], in the provision of food information to consumers, established that when claiming a significant amount of listed nutrients, the food should meet 15% of the nutrient reference values (NRV) supplied per 100 g (Annex XIII). The studied Spanish lentils can claim to be low-fat, low-sugar, very low-salt, high-protein food and a source of minerals. Macro and micronutrients in seed concentrations are summarized in Table 8. Nitrogen content ranged from 323 to 515 mg/100 g, in accordance with the protein content. P and K levels were quite uniform across all accessions, following those reported by [8]. Ca, Mg, and Na were superior in microsperma-type accessions (from Fuerteventura, León, and Granada provinces). In relation to micronutrients, the Fe content (>1 g/100 g) of accessions from Fuerteventura Island and Granada province exceeded the reported values of 7–10 mg/100 g in other surveys [6,7,8,25,27]. Mn content averaged 2.93 mg/100 g, two times higher than the reported FAO values and two microsperma accessions (from Fuerteventura Island and Granada) almost duplicated the average concentration. Cu content was uniform across the studied accessions and corresponded with other surveys [7,8]. Zn content in macrosperma seeds (from Guadalajara, Ciudad Real, and Lanzarote) was almost double the concentration in microsperma seeds. These differences may be due to genetic variation among accessions rather than chemical characteristics of the soil, as the lentils in this study were cultivated in the same area. Based on EU Regulation No. 1169/2011 [21], all lentil accessions contain significant amounts of all analysed minerals (Table 8). When comparing these data with those obtained in a recent study on the seed composition of Spanish chickpeas [28], several differences emerged. Lentils were found to contain lower levels of fat, total sugars, and energy than chickpeas. Both types of seeds exhibited similar available carbohydrates and crude fibre contents. However, a primary distinction was observed in protein content, which was higher in lentils (averaging 28.3%) than in chickpeas (averaging 24.6%). In terms of mineral composition, lentil seeds demonstrated higher levels of P and Ca but lower levels of Na than chickpeas. Regarding micronutrients, lentils exhibited notably high Fe content, with two accessions exceeding 1 g/100 g. Other micronutrients, such as Mn, Cu, and Zn, were present at similar levels in both legumes.
Table 8.
Seed mineral content of lentil accessions.
| Macronutrients (mg/100 g) | Micronutrients (mg/100 g) | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Origin | N | P | K | Ca | Mg | Na | Fe | Mn | Cu | Zn |
| Guadalajara | 402 | 510 | 1343 | 163 | 137 | 4.8 | 183 | 1.67 | 0.61 | 5.57 |
| Ciudad Real | 405 | 458 | 1273 | 104 | 126 | 6.7 | 95 | 1.90 | 0.56 | 4.96 |
| Lanzarote | 455 | 444 | 1243 | 132 | 141 | 5.6 | 179 | 2.04 | 0.53 | 4.21 |
| Fuerteventura | 323 | 338 | 1198 | 813 | 172 | 8.0 | 1581 | 4.84 | 0.51 | 2.47 |
| León | 515 | 461 | 1222 | 386 | 151 | 5.6 | 674 | 2.75 | 0.54 | 2.98 |
| Granada | 471 | 443 | 1298 | 733 | 176 | 7.6 | 1308 | 4.43 | 0.52 | 2.62 |
| Mean | 428 | 442 | 1263 | 388 | 150 | 6.4 | 670 | 2.93 | 0.55 | 3.80 |
| 15% of Reference intakes 1 | -- | 105 | 300 | 120 | 56.30 | -- | 2.10 | 0.30 | 0.15 | 1.50 |
1 Reference intakes (EU Regulation No. 1169/2011).
Table 9.
Phenolic compounds in lentil seeds.
| Phenolic Acids | Origin of Lentil Accessions | |||||
|---|---|---|---|---|---|---|
| Guadalajara | Ciudad Real | Lanzarote | Fuerteventura | León | Granada | |
| Gallocatechin | + | + | + | - | + | + |
| Protocatechuic | - | - | - | + | + | + |
| Procyanidin B1 | + | - | - | - | * | - |
| Gentisic | * | - | - | - | - | - |
| 4-hydroxybenzoic | + | + | + | + | + | + |
| Catechin | + | + | + | + | + | + |
| 2,4-Dihydroxybenzoic | - | + | + | - | - | - |
| Vanillic | - | - | - | * | - | - |
| Epicatechin | + | + | - | - | + | + |
| Flavanomarein | + | + | + | + | + | + |
| p-Cumaric | + | + | + | + | + | + |
| Phloretic | - | - | - | - | + | + |
| Luteolin-7-O-Glc | - | - | - | - | + | + |
| Aromadendrene | - | * | - | - | - | - |
| Salicylic | + | + | + | + | + | + |
| Quercetin-4′-O-Glc | - | - | - | - | + | + |
| Kaempferol-3-O-Glc | - | - | - | + | + | + |
| Luteolin-4′-O-Glc | - | - | - | + | + | + |
| Quercetin-3-O-rhamnoside | - | - | - | + | + | + |
| Total | 9 | 9 | 7 | 10 | 15 | 14 |
(+) presence, (-) absence. (*) Unique compound among the studied genotypes. Compounds are ordered upward RT (retention time).
However, grain legumes are typically consumed following various processing steps such as soaking, boiling and cooking, which are known to cause nutrient loss [27,29,30]. Despite this, the FAO user guide [7] has established nutrient retention factors (RFs), which are coefficients indicating the preservation of nutrients in a food or dish following storage, preparation, warming, or reheating. Specifically, for water-soaked and boiled pulses, RFs are applied to minerals, vitamins, and inositol.
After applying these range factors (0.7–0.9) to minerals found in the studied lentil samples, they retained significant amounts of minerals even after processing, in accordance with EU Regulation No 1169/2011.
3.4. Phenolic Compound in Lentil Accessions
Legumes have garnered attention for their rich bioactive compounds, making them valuable sources of ingredients for functional foods and various applications. A focused analysis encompassing over 90 polyphenolic compounds was conducted on the methanolic extracts of six lentil accessions. The total number of phenolic compounds varied among accessions (Table 9). Macrosperma type seeds with non-coloured testa contained 7–9 compounds, whereas 10–15 compounds were detected in microsperma types. Accessions from Fuerteventura Island had reddish-yellow testa and accessions from León and Granada had dark-green testa. These three accessions exclusively contained protocatechuic acid, kaempferol-3-O-glucoside, luteolin-4′-O-glucoside, quercitin-3-O-rhamnoside, and flavonoid and flavone derivative compounds with recognized antioxidant and anti-inflammatory activity. All accessions shared 4-hydroxybenzoic acid, catechin, flavanomarein, p-cumaric acid, and salicylic acid. Gallocatechin was common in these lentil seeds but not detected in Fuerteventura Island accessions. Both verdina type accessions had the highest phenolic compound content and exclusively shared phloretic acid, luteolin-7-O-glucoside and quercetin-4′-O-glucoside. Aromadendrene (di-hydrokaempferol), a flavanol compound, was exclusively detected in Ciudad Real lentils. Vanillic acid was only detected in Fuerteventura seeds. The notable presence of bioactive compounds in coloured lentil seeds appears to be a common trait among other legumes such as Phaseolus vulgaris and Vigna subterranea, called black soybeans and azuki beans [22,23,31]. These findings further emphasize the globally recognized significance of consuming grain legumes as sources of bioactive compounds with strong antioxidant properties, complementing their nutritional and mineral provisions.
4. Conclusions
To our knowledge, this is the first study on lentil accessions from Spanish gene banks that details the nutrients, minerals, and phenolic compound composition of seeds. We found higher Fe content in two lentil accessions as well as higher protein content than in previous reports for two lentil accessions. Moreover, exclusive bioactive compounds were found, especially in microsperma types, making them valuable sources of ingredients for functional foods and various applications. Consequently, our findings could help broaden lentil integration into dairy diets. Additionally, symbiotic compatibility with specific N2-fixing bacteria is seldom incorporated into legume breeding programs. Our findings highlight the importance of this plant–microbe interaction, which contributes to several Environmental Sustainability Goals, such as enhancing food security, improving nutrition, promoting sustainable agriculture, and mitigating biodiversity loss by aiding in the protection and sustainable use of terrestrial ecosystems.
Supplementary Materials
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/su16062548/s1, Figure S1. Climate data Tomejil, Figure S2. Albaladejito climate data, Table S1. Barley yields in intercropping with lentil accessions.
Author Contributions
Methodology and draft preparation, P.B.; methodology and formal analysis, M.d.l.M., M.C.A. and F.P.; funding acquisition and data curation, M.C.; Funding acquisition, conceptualization, and writing—original draft preparation, D.N.R.N. All authors have read and agreed to the published version of the manuscript.
Funding
This work was supported by PRIMA PCI2020-112151 project.
Institutional Review Board Statement
Not applicable.
Informed Consent Statement
Not applicable.
Data Availability Statement
Data are contained within the article and Supplementary Materials.
Acknowledgments
The authors acknowledge Laboratorio Agroalimentario de Córdoba (AGAPA-Andalucía) for proximate composition assessment.
Conflicts of Interest
Authors declare that they do not have any financial and personal relationships with other people or organizations that could inappropriately influence (bias) their work. We have not used AI-assisted technologies in the writing process.
References
- Available online: https://www.atlasbig.com/en-gb/countries-by-lentil-production (accessed on 8 February 2024).
- Catalogo de Publicaciones de la Administración General del Estado. Ministerio de Agricultura, Pesca y Alimentación. Available online: http://cepage.mpr.gob.es/ (accessed on 8 January 2024).
- Poole, P.; Ramachandran, V.; Terpolilli, J. Rhizobia: From saprophytes to endosymbionts. Nat. Rev. Microbiol. 2018, 16, 291–303. [Google Scholar] [CrossRef]
- Altieri, M.A.; Nicholls, C.I.; Montalba, R. Technological approaches to sustainable agriculture at a crossroads: An agroecological perspective. Sustainability 2017, 9, 349. [Google Scholar] [CrossRef]
- Moutier, N.; Baranger, A.; Fall, S.; Hanocq, E.; Marget, P.; Floriot, M.; Gauffreteau, A. Mixing Ability of Intercropped Wheat Varieties: Stability Across Environments and Tester Legume Species. Front. Plant Sci. 2022, 13, 877791. [Google Scholar] [CrossRef]
- Langyan, S.; Yadava, P.; Khan, F.N.; Bhardwaj, R.; Tripathi, K.; Bhardwaj, V.; Bhardwaj, R.; Gautam, R.K.; Kumar, A. Nutritional and food composition survey of major pulses toward healthy, sustainable, and biofortified diets. Front. Sustain. Food Syst. 2022, 6, 878269. [Google Scholar] [CrossRef]
- FAO/INFOODS. Global Food Composition Data Base for Pulses. User Guide. 2017. Available online: https://www.fao.org/infoods/infoods/tables-and-databases/faoinfoods-databases (accessed on 15 November 2023).
- Vandemark, G.J.; Grusak, M.A.; McGee, R.J. Mineral concentrations of chickpea and lentil cultivars and breeding lines grown in the U.S. Pacific Northwest. Crop J. 2018, 6, 253–262. [Google Scholar] [CrossRef]
- Lázaro, A.; Ruiz, M.; de la Rosa, L.; Martín, I. Relationships between agro/morphological characters and climatic parameters in Spanish landraces of lentil (Lens culinaris Medik.). Genet. Resour. Crop Evol. 2001, 48, 239–249. [Google Scholar] [CrossRef]
- Erskine, W. Lessons for breeders from landraces of lentil. Euphytica 1997, 93, 107–112. [Google Scholar] [CrossRef]
- Bejiga, G.; Tsegaye, S.; Tullu, A.; Erskine, W. Quantitative evaluation of Ethiopian landraces of lentil (Lens culinaris). Genet. Resour. Crop Evol. 1996, 43, 293–301. [Google Scholar] [CrossRef]
- Piergiovanni, A.R.; Laghetti, G.; Olita, G.; Monti, M.; Preiti, G.; Di Prima, G. Screening for agronomic and biochemical traits in a lentil germplasm collection. In Proceedings of the 3rd European Conference on Grain Legumes Proceedings, Valladolid, Spain, 14–19 November 1998; p. 206. [Google Scholar]
- Bellucci, E.; Aguilar, O.M.; Alseekh, S.; Bett, K.; Brezeanu, C.; Cook, D.; De la Rosa, L.; Delledonne, M.; Dostatny, D.F.; Ferreira, J.J.; et al. The INCREASE project: Intelligent collections of food-legume genetic resources for European grifood systems. Plant J. 2021, 108, 646–660. [Google Scholar] [CrossRef]
- Ruiz-Díez, B.; Fajardo, S.; Fernández-Pascual, M. Selection of Rhizobia from Agronomic Legumes Grown in Semiarid Soils to be Employed as Bioinoculants. Agron. J. 2012, 104, 550–559. [Google Scholar] [CrossRef]
- Van Berkum, P.; Beyene, D.; Temprano Vera, F.; Keyser, H.H. Variability among Rhizobium Strains Originating from Nodules of Vicia faba. Appl. Environ. Microbiol. 1995, 61, 2649–2653. [Google Scholar] [CrossRef]
- Vincent, J.M. A Manual for the Practical Study of Root Nodule Bacteria; Blackwell Scientific Publications: Oxford, UK, 1970. [Google Scholar]
- Beringer, J.E. R factor transfer in Rhizobium leguminosarum and Rhizobium phaseoli. J. Gen. Microbiol. 1974, 84, 188–198. [Google Scholar]
- Rigaud, J.; Puppo, A. Indole-3-acetic acid catabolism by soybean bacteroids. J. Gen. Microbiol. 1975, 88, 223–228. [Google Scholar] [CrossRef]
- Somasegaran, P.; Hoben, H.J. Methods in Legume-Rhizobium Technology; University of Hawaii NifTAL Project and MIRCEN, Department of Agronomy and Soil Science, Hawaii Institute of Tropical Agriculture and Human Resources: Paia, HI, USA, 1985. [Google Scholar]
- Albareda, M.; Rodríguez-Navarro, D.N.; Camacho, M.; Temprano, F.J. Alternatives to peat as a carrier for rhizobia inoculants: Solid and liquid formulations. Soil Biol. Biochem. 2008, 40, 2771–2779. [Google Scholar] [CrossRef]
- Regulation (EU) No 1169/2011 of the European Parliament and of the Council of 25 October 2011 on the Provision of Food Information to Consumers. 2011. Available online: https://eur-lex.europa.eu/legal-content/en/ALL/?uri=CELEX%3A32011R1169 (accessed on 8 January 2023).
- Hungría, M.A.; Phillips, D.A. Effects of a seed colour mutation on Rhizobial nod-gene inducing flavonoids and nodulation in common bean. Mol. Plant-Microbe Interact. 1993, 6, 418–422. [Google Scholar] [CrossRef]
- Puozza, D.K.; Jaiswal, S.K.; Dakora, F.D. Black seedcoat pigmentation is a marker for enhanced nodulation and N2 fixation in Bambara groundnut (Vigna subterranea L. Verdc.) landraces. Front. Agron. 2021, 3, 692238. [Google Scholar] [CrossRef]
- Thies, J.E.; Singleton, P.W.; Bohlool, B.B. Influence of the size of indigenous Rhizobial populations on establishment and symbiotic performance of introduced rhizobia on field-grown legumes. Appl. Environ. Microbiol. 1991, 57, 19–28. [Google Scholar] [CrossRef]
- Faris, M.A.-I.E.; Takruri, H.R.; Issa, A.Y. Role of lentils (Lens culinaris L.) in human health and nutrition: A review. Mediterr. J. Nutr. Metab. 2013, 6, 3–16. [Google Scholar] [CrossRef]
- Regulation (EC) No 1924/2006 of the European Parliament and of the Council of 20 December 2006 on Nutrition and Health Claims Made on Foods. 2006. Available online: https://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32006R1924 (accessed on 8 January 2023).
- Avola, G.; Patanè, C.; Barbagallo, R.N. Effect of water cooking on proximate composition of grain in three Sicilian chickpeas (Cicer arietinum L.). Food Sci. Technol. 2012, 49, 217–220. [Google Scholar] [CrossRef]
- Brun, P.; Camacho, M.; Perea, F.; Rubio, M.J. Rodríguez Navarro, D.N. Characterization of Spanish chickpea genotypes (Cicer arietinum L.): Proximate, mineral, and phenolic compounds composition. Eur. Food Res. Technol. 2024, 250, 1007–1016. [Google Scholar] [CrossRef]
- Güzel, D.; Sayar, S. Effect of cooking methods on selected physicochemical and nutritional properties of barlotto bean, chickpea, faba bean, and white kidney bean. J. Food Sci. Technol. 2012, 49, 89–95. [Google Scholar] [CrossRef] [PubMed]
- Margier, M.; Georgé, S.; Hafnaoui, N.; Remond, D.; Nowicki, M.; Du Chaffaut, L.; Amiot, M.J.; Reboul, E. Nutritional composition and bioactive content of legumes: Characterization of pulses frequently consumed in France and effect of the cooking method. Nutrients 2018, 10, 1668. [Google Scholar] [CrossRef] [PubMed]
- Lin, P.-Y.; Lai, H.-M. Bioactive compounds in legumes and their germinated products. J. Agric. Food Chem. 2006, 54, 3807–3814. [Google Scholar] [CrossRef] [PubMed]
Table 1.
Geographical origin and seed characteristics of lentil accessions used in this work.
| Accession Code 1 | Origin Province/Locality | 100 SW 2 (g) | Seed Type Local Name |
|---|---|---|---|
| BGE001908 | Guadalajara (Villanueva de Argecilla) | 4.8 ± 0.30 | Macrosperma |
| BGE011096 | Ciudad Real (Alcolea de Calatrava) | 4.7 ± 0.30 | Macrosperma |
| BGE001867 | Albacete (Tarazona de la Mancha) | 5.8 ± 0.30 | Macrosperma |
| BGE001055 | Jaén (Torredonjimeno) | 6.1 ± 0.20 | Macrosperma |
| BGE023655 | Lanzarote Island (Yaiza) | 4.4 ± 0.30 | Macrosperma |
| BGE016362 | Fuerteventura Island (La Oliva) | 1.9 ± 0.06 | Microsperma majorera |
| BGE025596 | León (Gusendos de los Oteros) | 1.8 ± 0.03 | Microsperma verdina |
| BGE026702 | Granada (Huéneja) | 1.9 ± 0.03 | Microsperma verdina |
1 Number code of the Spanish Germplasm Bank. 2 100 seed weight ± standard deviation of three replicates.
Table 2.
Soil characteristics of field experimental areas.
| Province | Texture % | Type | pH | Conductivity (µS/cm) | o.m.% | P (ppm) | N% | MPN 3 | ||
|---|---|---|---|---|---|---|---|---|---|---|
| Sand | Clay | Silt | ||||||||
| Cuenca 1 | 48 | 30 | 22 | Sandy Clay Loam | 8.75 | 150.90 | 2.09 | 19.40 | 0.06 | 5.85 |
| Seville 2 | 30 | 56 | 14 | Clay | 8.69 | 202.60 | 1.32 | 16.20 | 0.09 | 1.1 3 |
1 IRIAF-Albaladejito. 2 IFAPA-Las Torres. 3 MPN, Most Probable Number.
Table 3.
Symbiotic response of lentil accessions to inoculation with Rhizobium leguminosarum strains.
Table 3.
Symbiotic response of lentil accessions to inoculation with Rhizobium leguminosarum strains.
| NNod | NodDW (mg) | SDW (mg) | Symbiotic Efficiency (%) | |
|---|---|---|---|---|
| Strains | Lentil Accessions Origin | |||
| Albacete | ||||
| GU-2 | 66.7 ± 12.7 bc | 40.1 ± 6.7 ab | 480.0 ± 95.0 ab | 279 |
| HA-2 | 94.3 ± 4.9 a | 53.2 ± 3.7 a | 676.7 ± 92.6 a | 434 |
| ISL37 | 45.0 ± 7.6 c | 20.8 ± 2.8 c | 320.0 ± 70.0 bc | 152 |
| ISL55 | 91.7 ± 5.4 ab | 35.8 ± 2.4 b | 493.3 ± 29.6 ab | 289 |
| NI | -- | -- | 126.7 ± 17.6 c | -- |
| Jaén | ||||
| GU-2 | 12.5 ± 0.5 b | 18.1 ± 10.0 a | 306.7 ± 68.9 ab | 115 |
| HA-2 | 57.0 ± 17.7 a | 41.5 ± 17.2 a | 570.0 ± 216.6 a | 300 |
| ISL37 | 76.0 ± 6.0 a | 34.6 ± 7.8 a | 553.3 ± 129.9 a | 289 |
| ISL55 | 46.0 ± 11.7 ab | 23.2 ± 5.0 a | 376.7 ± 58.9 ab | 165 |
| NI | -- | -- | 142.3 ± 10.8 b | -- |
| Granada | ||||
| GU-2 | 80.0 ± 19.1 a | 21.3 ± 0.2 a | 160.0 ± 25.2 a | 356 |
| HA-2 | 26.7 ± 5.8 c | 9.5 ± 2.7 b | 170.0 ± 43.6 a | 384 |
| ISL37 | 50.7 ± 7.9 ab | 14.0 ± 2.2 b | 186.7 ± 20.3 a | 432 |
| ISL55 | -- | -- | -- | -- |
| NI | -- | -- | 35. 1 ± 6.2 b | -- |
| León | ||||
| GU-2 | 36.7 ± 9.9 ab | 11.4 ± 3.2 a | 240.0 ± 5.8 a | 523 |
| HA-2 | 60.0 ± 32.0 a | 12.3 ± 6.1 a | 135.0 ± 15.0 b | 250 |
| ISL37 | 43.3 ± 11.2 ab | 13.3 ± 3.1 a | 233.3 ± 58.9 a | 506 |
| ISL55 | 20.3 ± 3.3 b | 5.7 ± 0.1 b | 133.3 ± 18.5 b | 246 |
| NI | -- | -- | 38.5 ± 0.4 c | -- |
Data are means of three replicates (2 plants/replicate). Values followed by the same letter in each column and lentil accession are not significantly different at p < 0.05. NI, non-inoculated treatment; NNod, number of nodules; NodDW, nodules dry weight; SDW, shoot dry weight; Symbiotic efficiency, (SDW inoculated plants-SDW NI plants)/SDW NI plants × 100.
Table 4.
Symbiotic response of lentil accessions to inoculation with Rhizobium leguminosarum strains.
Table 4.
Symbiotic response of lentil accessions to inoculation with Rhizobium leguminosarum strains.
| NNod | NodDW (mg) | SDW (mg) | Symbiotic Efficiency (%) | |
|---|---|---|---|---|
| Strains | Lentil Accessions Origin | |||
| Guadalajara | ||||
| HA-2 | 53.0 ± 19.1 a | 10.7 ± 4.8 a | 160.0 ± 23.1 b | 68 |
| GU-2 | 51.7 ± 6.5 a | 13.4 ± 1.9 a | 217.5 ± 14.4 a | 129 |
| NI | -- | -- | 95.0 ± 7.3 c | -- |
| Ciudad Real | ||||
| HA-2 | 37.0 ± 3.0 a | 12.3 ± 1.9 a | 243.3 ± 1.4 a | 143 |
| GU-2 | 26.3 ± 3.9 a | 11.2 ± 1.5 a | 252.5 ± 7.5 a | 152 |
| NI | -- | -- | 100.0 ± 13.5 b | -- |
| Lanzarote Island | ||||
| HA-2 | 35.7 ± 5.9 a | 9.3 ± 0.8 a | 246.7 ± 59.2 a | 190 |
| GU-2 | 38.0 ± 3.5 a | 11.1 ± 1.4 a | 205.0 ± 13.2 a | 141 |
| NI | -- | -- | 85.0 ± 8.6 b | -- |
| Fuerteventura Island | ||||
| HA-2 | 30.7 ± 12.7 a | 7.0 ± 1.6 a | 130.0 ± 5.8 a | 206 |
| GU-2 | 42.0 ± 5.7 a | 12.6 ± 3.8 a | 173.3 ± 44.8 a | 308 |
| NI | -- | -- | 42.5 ± 2.5 b | -- |
| León | ||||
| HA-2 | 16.0 ± 2.0 a | 2.7 ± 0.4 a | 30.0 ± 5.7 b | 9 |
| GU-2 | 31.0 ± 5.7 a | 6.8 ± 1.5 a | 110.0 ± 7.1 a | 300 |
| NI | -- | -- | 27.5 ± 4.7 b | -- |
| Granada | ||||
| HA-2 | 22.3 ± 1.2 a | 5.0 ± 0.6 a | 50.0 ± 5.8 b | 186 |
| GU-2 | 22.3 ± 1.8 a | 6.7 ± 1.6 a | 110.0 ± 35.1 a | 528 |
| NI | -- | -- | 17.5 ± 4.8 b | -- |
Data are means of four replicates (2 plants/replicate). Values followed by the same letter in each column and lentil accession are not significantly different at p < 0.05. NI, non-inoculated treatment; NNod, number of nodules; NodDW, nodules dry weight; SDW, shoot dry weight; Symbiotic efficiency, (SDW inoculated plants-SDW NI plants)/SDW NI plants × 100.
Table 5.
Lentil grain yields under different management. Field Experiment, Seville (2021–2022).
| Origin of Accessions | Conventional Monocrop | Conventional Inoculated Monocrop | Intercropping | Mean Yield per Accession |
|---|---|---|---|---|
| Grain (kg/ha) | ||||
| Guadalajara | 697 ± 289 cA | 718 ± 262 bA | 457 ± 178 dA | 624 |
| Ciudad Real | 664 ± 305 cA | 680 ± 239 bA | 867 ± 362 cA | 737 |
| Lanzarote Island | 1522 ± 73 bA | 1219 ± 442 abAB | 728 ± 214 cdB | 1156 |
| Fuerteventura Island | 2028 ± 230 aA | 1800 ± 398 aA | 1766 ± 133 aA | 1865 |
| León | 1509 ± 71 bAB | 1648 ± 427 aA | 844 ± 159 cdB | 1334 |
| Granada | 1331 ± 309 bA | 1552 ± 414 aA | 1297 ± 179 bA | 1393 |
Data are means of three replicates (blocks). In each column, values were followed by the same lower case letter are not significantly different (p < 0.05). In each file, values followed by the same capital letter are not significantly different (p < 0.05).
Table 6.
Lentil grain yields under different management. Field trial, Cuenca (2021–2022).
| Origin of Accessions | Conventional Monocrop | Conventional Monocrop Inoculated | Intercropping | Mean Yield per Accession |
|---|---|---|---|---|
| kg/ha | ||||
| Guadalajara | 1301 aA | 1319 aA | 931 aA | 1184 |
| Ciudad Real | 484 cdB | 1250 aA | 429 bcB | 722 |
| Lanzarote Island | 474 cdA | 449 bcA | 764 aA | 563 |
| Fuerteventura Island | 310 dA | 307 cA | 130 cB 1 | 249 |
| León | 847 bcAB | 1070 aA | 646 abB | 854 |
| Granada | 1214 abA | 883 abAB | 668 abB | 922 |
Data are means of three replicates (blocks). In each column, values followed by the same lower case letter are not significantly different (p < 0.05). In each file, values followed by the same capital letter are not significantly different (p < 0.05). 1 This value was not considered in mean yield per accession calculation.
Table 7.
Nutritional (proximate) composition of seeds from lentil accessions.
| Origin | Energy (Kcal) | Fat 1 | CH-AVD * | Total Sugars | Protein | Salt 2 | Crude Fibre | Ash | Humidity (%) |
|---|---|---|---|---|---|---|---|---|---|
| Guadalajara | 348 | 2.5 | 55.7 | 5.4 | 28.7 | 12 | 4.6 | 3.5 | 10.0 |
| Ciudad Real | 348 | 2.1 | 56.6 | 5.5 | 28.3 | 17 | 4.2 | 3.3 | 9.9 |
| Lanzarote | 345 | 1.8 | 58.2 | 3.8 | 26.6 | 14 | 4.6 | 3.1 | 10.4 |
| Fuerteventura | 340 | 2.3 | 57.7 | 4.2 | 24.8 | 20 | 4.5 | 4.6 | 10.7 |
| León | 341 | 1.9 | 53.0 | 3.2 | 30.5 | 14 | 4.3 | 4.3 | 10.5 |
| Granada | 341 | 1.7 | 52.8 | 3.7 | 31.0 | 19 | 4.2 | 5.4 | 9.3 |
Data: g/100 g of seed flour. 1 Fat: crude fat plus saturated fat. 2 Equivalent content of sodium × 2.5 (mg/100 g). * CH-AVD, carbohydrates available by difference (FAO/INFOOD equation 4 = 100-water-total fat-total protein-fibre-ash).
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2024 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/).
Share and Cite
MDPI and ACS Style
Brun, P.; de los Mozos, M.; Alcántara, M.C.; Perea, F.; Camacho, M.; Rodriguez Navarro, D.N. Characterization of Spanish Lentil Germplasm: Seed Composition and Agronomic Performance Evaluation. Sustainability 2024, 16, 2548. https://doi.org/10.3390/su16062548
AMA Style
Brun P, de los Mozos M, Alcántara MC, Perea F, Camacho M, Rodriguez Navarro DN. Characterization of Spanish Lentil Germplasm: Seed Composition and Agronomic Performance Evaluation. Sustainability. 2024; 16(6):2548. https://doi.org/10.3390/su16062548
Chicago/Turabian StyleBrun, Pilar, Marcelino de los Mozos, Maria Cristina Alcántara, Francisco Perea, María Camacho, and Dulce Nombre Rodriguez Navarro. 2024. "Characterization of Spanish Lentil Germplasm: Seed Composition and Agronomic Performance Evaluation" Sustainability 16, no. 6: 2548. https://doi.org/10.3390/su16062548
Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.
