https://doi.org/10.22319/rmcp.v16i3.6883

Technical note

Underground biomass of perennial ryegrass with different nitrogenous sources and associated with white clover and red clover

 

Francisco Gutiérrez León ^a

Mónica Sacido ^b

Susana Feldman ^b

 

^a Universidad Central del Ecuador. Facultad de Ciencias Agrícolas. Ciudad de Quito, Ecuador.

^b Universidad Nacional de Rosario. Facultad de Ciencias Agrarias, Ciudad de Rosario, Argentina. 

 

*Corresponding author: fgutierrez@uce.edu.ec

 

Abstract:

This research aimed to evaluate the root biomass of perennial ryegrass (R) (Lolium perenne) with different nitrogenous sources and associations with white clover (Wc) (Trifolium repens) and red clover (Rc) (Trifolium pratense), as well as the nodulation capacity of white or red clover. Two experiments were conducted: (Exp1) monoculture of perennial ryegrass, fertilized with nitrogen (urea, slow-release urea, ammonium nitrate, foliar nitrogen, ammonium nitrate + foliar nitrogen, and a control or without application), and (Exp 2) mixtures of ryegrass with white or red clover, in low and high density, and a control. They were replicated in 2 localities: Tumbaco (Tm) and Machachi (Mc), in Pichincha, Ecuador. The statistical design was a randomized block design (n=3), and a soil analysis was performed at the beginning. Root biomass (Rb) was evaluated on a dry basis in the 2 experiments; for its part, in Exp 2, the number (Nn) and weight of nodules (Nw) were also evaluated. The results indicate that species modify their root growth as an adaptation to environmental conditions. In Exp 1 in Tm, where there was a higher temperature, there was a higher Rb. In Exp 2, the mixtures of R and Rc accumulated greater root biomass. For Nn and Nw, Wc had the highest values. Based on these results, it can be concluded that the environment plays a preponderant role in root biomass and that species adapt by modifying this trait and the number and dry weight of nodules.

Keywords: Nodulation, Roots, Adaptation, Environment.

 

Received: 18/03/2025

Accepted: 14/07/2025

 

The shoots and roots have complementary functions, as the aerial stem harvests radiant energy from the sun, while the roots absorb mineral nutrients and water⁽¹⁾. According to Ramos-Hernández and Martínez-Sánchez⁽²⁾, underground biomass is usually equal to or greater than aboveground biomass. Poorter and Nagel⁽³⁾ state that, in the face of low nutrient availability, the root has priority in the allocation of carbohydrates over the aerial part. Likewise, the physical, chemical, and biological properties of soils play an essential role in root growth⁽⁴⁾. Root systems allow plants to grow and nourish themselves, so they are decisive in the yield of agricultural crops⁽⁵⁾. Nevertheless, it is argued⁽⁶⁾ that, in forage plants, the growth and production of roots is much less studied and understood than that of leaves.

Roots have intense biological activity; among the most important is the symbiotic association between members of the family Leguminosae and soil diazotrophic bacteria (rhizobia), which are well known for their ability to fix atmospheric nitrogen⁽⁷⁾. Legumes, therefore, play a key role in the sustainable intensification of agricultural systems, improving biodiversity and ecosystem services, and reducing the dependence of food production on nitrogen fertilizers⁽⁸⁾. For this reason, forage legumes have been selected for their nodulation capacity, with white clover developing the greatest capacity⁽⁹⁾. Nonetheless, N fixation varies greatly depending on rhizobia; there are several factors that determine the success of the legume-rhizobia symbiotic relationship⁽¹⁰⁾, one of the most important being to match suitable symbionts with the host plants⁽¹¹⁾.

Therefore, the objective of this research was to assess the root biomass of perennial ryegrass (Lolium perenne L.) as a monoculture, with different nitrogenous sources and in mixtures with white clover (Trifolium repens) and red clover (Trifolium pratense), as well as to evaluate the nodulation capacity of white or red clovers in two contrasting localities.

This research was conducted in Ecuador in the province of Pichincha in two experimental sites, in the parishes of Machachi, Canton Mejía, and Tumbaco, Canton Quito.

According to INEC’s⁽¹²⁾ agricultural census, pastures occupy 58.3 % of the area subjected to agricultural work; therefore, livestock production is carried out in a pastoral system. It is also mentioned that 79.5 % of milk production is from the Sierra region, and in it, the province of Pichincha covers 18 % of the total, being the province with the highest national production.

The climate in the province of Pichincha varies according to altitude; the climate of the locality of Tumbaco is classified as dry mesothermal, while that of Machachi as humid mesothermal⁽¹³⁾. Machachi is located at 0°30'36.4'' S; 78°34'1.6'' W, and has a precipitation of 1,043 mm and an average temperature of 12 °C; for its part, Quito is situated at 0°13'52.5'' S; 78°11'0.3'' W, and has a precipitation of 954 mm and an average temperature of 16 °C.

The origin of soils in the inter-Andean region is volcanic, and climate is the most critical factor in their development, so the specific environmental conditions of each site have given rise to different processes that have influenced soil formation⁽¹⁴⁾. Based on this, soil samples were taken from each locality, in which the following were determined: organic carbon (by wet combustion, Walkley-Black Method), pH (with a potentiometer, in aqueous solution), electrical conductivity (using a conductivity meter with a saturated paste extract), total N (by calculation based on the percentage of organic matter), available P (with a photocolorimeter, modified Olsen Method), and exchangeable cations (K, Ca, Mg; atomic absorption spectrophotometry) (Table 1).

 

 

Table 1: Physicochemical characteristics of the soils of the experimental sites

EC= electrical conductivity; SOC= soil organic carbon; SOM= soil organic matter; dS= deciSiemens; ppm= parts per million; cmol= centimoles.

 

 

Experiment 1: Effect of different nitrogen sources on perennial ryegrass (R) monoculture. The following was used: a randomized block design (n=3, plots of 7 x 4 m), 30 kg ha^-1 as planting density, and fertilization with nitrogen sources with the sources specified in Table 2.

 

 

Table 2: Type and concentration of fertilizers used in perennial ryegrass monoculture in the two experimental sites

(1)= sulfur-rich polymer that allows a controlled release of urea, limiting leaching.

 

 

Experiment 2: Effect of forage mixtures, ryegrass with white clover and red clover at different planting densities. The following was used: a randomized block design (n=3, plots of 7 x 4 m) and mixtures of grasses and legumes, as detailed in Table 3. These two clover species were selected for their different growth habits: red clover has more erect growth, whereas white clover is more creeping. The clovers were not inoculated with bacteria of the genus Rhizobium, as this is not a common practice in Ecuador, since there are native strains, but they have been little studied⁽¹⁵⁾. Clover planting densities were adjusted according to León et al⁽¹⁶⁾, who consider that they should range between 2.5 and 5 kg per hectare.

 

 

Table 3: Forage species used in experiment 2 and their planting densities

 

 

Based on the information provided by a tensiometer, the experimental plots of both experiments were maintained at field capacity through the use of a sprinkler irrigation system. At the beginning of the experiments, fertilization was carried out with 80 kg P ha^-1 (triple superphosphate plus microelements) and 60 kg K ha^-1 (potassium muriate). Both experiments were conducted at each experimental site, and they were planted in August 2021 using the broadcast seeding method. During 2022, the aerial growth of the experiments in the two localities was evaluated monthly, and the underground biomass was assessed in February 2023.

Root biomass (g DM L^-1 soil): Six random samples were taken from each treatment and washed in plastic bags with 3 mm diamond-type perforations, roots were manually separated from the soil and dried with filter paper, the total weight of the roots was determined without separation by crop (fresh weight), and finally they were dehydrated in a forced-air oven at 70 °C for 24 h; they were weighed, and the percentage of dry matter was calculated⁽¹⁷⁾.

Number of nodules (No. of nodules L^-1 soil): Six samples were taken from each treatment, with red or white clover, and the number of nodules was determined by manually separating the roots and nodules⁽¹⁸⁾.

Nodule weight (mg DM L^-1 soil): Six samples were taken from each treatment, with red or white clover; the washed nodules were dehydrated in a forced-air oven at 70 °C for 24 h to determine the dry weight⁽¹⁹⁾.

All measurements were made in February 2023 on samples of 20 x 20 x 20 cm⁽²⁰⁾; this sampling depth was used because, in perennial ryegrass and clovers, more than 80 % of the roots grow to a depth of less than 20 cm⁽²¹⁾.

Statistical analysis. For each variable analyzed, the normal distribution was determined with the Shapiro-Wilks test and homogeneity of variance with the Levene test. Data analysis was performed using analysis of variance, and variables with statistical differences (P<0.05) were compared with a Tukey test. The statistical program INFOSTAT⁽²²⁾ was employed.

 

The mathematical model used for experiment 1 (perennial ryegrass monoculture) was as follows:

Y_ijk=μ + L_i + B_j (L_i)+ F_k + L_i x F_k + E_ijk

Where:

μ= total mean effect,

Li= effect of locality i,

Bj (Li)= effect of block j nested in Locality i,

Fk= effect of the fertilization source caused by level k,

Eijk= experimental error at level i of the cut, level j of the block, and level k of the source.

 

The mathematical model used for experiment 2 (forage mixture) was as follows:

Yijk=μ + Li + Bj (Li) + Mk + Li x Mk + Eijk

Where:

μ= total mean effect,

Li= effect of locality i,

Bj (Li)= effect of block j nested in Locality i,

Mk= effect of ryegrass and clover mixture caused by level k,

Eijk= experimental error at level i of the cut, level j of the block, and level k of the mixture of ryegrass and clover.

 

Figure 1 presents the root biomass (Rb) data from experiment 1, expressed as grams of DM of roots per unit volume (L) of soil. Section 1A shows the differences between localities on the amount of Rb, with Tumbaco having a higher value, 4.04 g DM of roots L^-1 soil, whereas Machachi registered 3.22 g. Section 1B illustrates the data by treatment; the foliar N treatment had the highest Rb, 5.13 g, followed by urea, ammonium nitrate plus foliar N, and the control (T0) (4.09; 3.58; 3.07 g), and in lesser amounts, ammonium nitrate and protected urea (2.99 and 2.92 g). Section 1C presents the interaction (locality x source); the leaf N had the highest Rb in the two localities, 4.64 g DM of roots L^-1 soil in Machachi and 5.62 g in Tumbaco, whereas the control and ammonium nitrate obtained the lowest Rb values in Machachi (2.34 and 2.15 g).

 

Figure 1: Effect of nitrogen fertilizer sources on root biomass (g DM of roots L^-1): (1A) effect of the localities of Machachi and Tumbaco; (1B) effect of the treatments of the sources evaluated; (1C) locality x treatment interaction

The values are means + 1 standard error. Different letters indicate significant differences between treatments (P<0.05). In each figure, the general means are represented by solid lines.

 

 

Figure 2 shows the root biomass (Rb) of the different forage mixtures of perennial ryegrass (R) with/without white clover (Wc) and with/without red clover (Rc). There were no significant differences between localities. In contrast, differences (P<0.05) between treatments are observed in 2A; R + 2.5 Rc had the highest root biomass (4.76 g DM of roots L^-1 soil), and the lowest value was recorded in R + 2.5 Wc (2.35 g). 2B exhibits the locality x mixture interaction, showing that R + Rc 2.5 kg had the highest Rb in both localities, 4.98 g in Machachi and 4.53 g in Tumbaco. The control was one of the highest in Rb in Tumbaco (4.79 g), and the lowest value of Rb was observed in Machachi, in the plots with R+ Wc 2.5 kg (1.37 g). Likewise, the root biomass in mixtures of ryegrass with clover varied depending on the species, observing that mixtures with red clover had a higher root biomass, 4.76 g, compared to that of ryegrass with white clover, 2.35 g DM of roots L^-1.

 

 

Figure 2: Effect of mixtures of ryegrass and white or red clover on root biomass (g DM of roots L^-1) (2A) differences between mixtures; (2B) locality x mixture interaction

The values are means + 1 standard error. Different letters indicate significant differences between treatments (P<0.05). A solid line on the Y-axis represents the general mean. R= ryegrass, Tb= white clover (Wc), and Tr= red clover (Rc).

 

 

Figure 3A shows the statistical differences (P<0.05) for the number of nodules (Nn) in the locality x mixture interaction. In Machachi, the mixture of R + Wc 5 kg had 90 Nn L^-1 soil, and the mixture of R + Wc 2.5 kg + Rc 2.5 kg had 70 Nn, the highest Nd, whereas in Tumbaco, it was R + Rc 5 kg, which had 64 Nn. Nodule weight (Nw) showed differences in the locality x mixture interaction (P<0.05). In 3B, it is observed that, in Machachi, the mixture of R + Wc 5 kg had 62 mg DM of nodules L^-1 soil, being the highest Nw; in contrast, in Tumbaco, it was the mixture of R + Rc 5 kg, which had 41 mg nodules.

 

 

Figure 3: (3A) Effect of the mixture of ryegrass and white or red clover on the number of nodules (No. of nodules/L soil) in the locality x mixture interaction. (3B) Effect of the mixture of ryegrass and white or red clover on nodule weight (mg nodules on a DM basis/L soil) and of the locality x mixture interaction

The values are means + 1 standard error. Different letters indicate significant differences between treatments (P<0.05). A solid line on the Y-axis represents the general mean. R= ryegrass, Tb= white clover (Wc), and Tr= red clover (Rc).

 

 

The differences in temperatures, 16 °C in Tumbaco and 12 °C in Machachi, could be responsible for the higher root biomass in the former compared to the latter locality (4.04 vs. 3.02 g DM of roots L^-1 soil, respectively). These results are related to Walne and Reddy⁽²³⁾, who determined that temperature is a critical environmental factor that regulates crop growth and yield. For example, in corn (Zea mays) crops, when temperatures increased, they detected longer, thicker, and denser roots. Similarly, in crops of perennial forage plants, plant growth was activated by temperature⁽²⁴⁾. In megathermal forage plants, it has been established that species adapt to the environment by modifying the depth and length of their root development⁽²⁵⁾. This is corroborated by Cougnon et al⁽²⁶⁾, who, when studying temperate climate pastures, such as perennial ryegrass and fescue (Festuca arundinacea Schreb), determined that they have a higher root biomass when soil temperatures increase, such as in spring.

The root biomass in mixtures of ryegrass and clover varied according to the species, observing that mixtures with red clover had a higher root biomass, 4.76 g DM of roots L^-1, compared to that of ryegrass with white clover, 2.35 g. An important factor in the development and growth of white clover roots is, according to Nichols et al⁽²⁷⁾, the death of the taproot, which occurs between 12 and 18 mo after the formation of the plant, after which the plant depends only on the nodal roots. The nodal roots are smaller and shallower, which limits the search for water and nutrients, making white clover more vulnerable⁽²⁸⁾.

The nodulation of legumes is a critical factor in the ability to fix N, as stated by Echeverría and Sainz Rosas⁽²⁹⁾, who mentioned that the bacteria in the nodules could fix between 25 and 80 % of the N required by the plant, and that this variability was due to environmental and soil factors and rhizobia strains. It should also be borne in mind that nitrogen transfer between legumes and grasses in mixed grasslands played a vital role in the nitrogen cycle of sustainable agricultural systems⁽³⁰⁾. Reilly et al⁽³¹⁾ stated that the biological fixation of N by legumes increased over time, and that the combination of grasses and legumes increased the total biomass yield compared to monocultures.

The average number of nodules was higher in Machachi, 53.82 nodules L^-1 soil, whereas in Tumbaco, it was 41.86. While the number of nodules could indicate the legume’s ability to fix N, the fixation of N₂ by rhizobia has a high energy cost for the plant. According to Dicenzo et al⁽³²⁾, fixing an atmospheric N₂ molecule requires the investment of 16 ATP molecules; however, other researchers⁽³³⁾ stated that if all the processes associated with biological fixation are considered, the energy cost amounts to 30 ATP molecules. In any case, the high energy cost would force the legume to control the number of nodules, avoiding excessive energy expenditure that would compromise its development and survival⁽³⁴⁾. On the other hand, if there are areas rich in assimilable nitrogen in the soil, legumes obtain this nutrient through a non-symbiotic pathway, avoiding extra energy expenditure⁽³⁵⁾.

Likewise, the plant provides the nodule with constant amounts of phosphate for the development of the nodule, so phosphate deficiencies activate a process of self-regulation of nodules in legumes^(35,36). The availability of P in its soils has a regulatory effect on nodulation⁽³⁷⁾, so the greater availability of P in the Machachi soil could explain the greater number of nodules in this locality, since P deficiencies reduce the rhizobial infection process.

The average weight of the nodules was higher in Machachi than in Tumbaco, 30.24 vs. 25.97 mg of nodules on a dry basis L^-1 soil. According to Dubach and Russelle⁽³⁸⁾, when comparing the weight of nodules in alfalfa (Medicago sativa) and bird’s foot clover (Lotus corniculatus), the weight of the nodules was related to the higher concentration of N. In another report⁽³⁹⁾, by evaluating peas (Pisum sativum ssp. sativum), they stated that the weight of the nodules was positively related to the green matter yield of the crop. On the other hand, in peanut (Arachis hypogaea L.) crops, the presence of mycorrhizae influenced the weight of the nodules⁽⁴⁰⁾.

It has been suggested that legume root nodules establish an ecological niche for the survival and growth of other bacteria^(41,42). Wigley et al⁽⁴³⁾ concluded that nodules contain various bacterial species in alfalfa (Medicago sativa) and that the population density of these bacteria is in the range of 10⁴ viable bacteria per gram of fresh legume nodule tissue⁽⁴⁴⁾.

The term consortium of microorganisms (CMs) has been assigned to the mixture of two or more microbial species or strains that live together symbiotically; a CM generally works better than the inoculation of a single species⁽⁴⁵⁾. Ye et al⁽⁴⁶⁾ also found a higher abundance of microbe-derived molecules in legume plantations. By evaluating the impact of the co-inoculation of Rhizobium sp. and Azospirillum sp. on red clover growth, they obtained greater nodule development than by inoculating Rhizobium alone⁽⁴⁷⁾. The interaction of these strains forms a CM that enhances root growth and crop yields⁽⁴⁸⁾. On the other hand, it is claimed⁽⁴⁹⁾ that the organic carbon content of the soil improves the activities of microorganisms, and that it acts as a reservoir of soil nutrients and biologically increases the energy supply to microbes⁽⁵⁰⁾.

Therefore, the greater weight of the nodules in the Machachi locality could be due to the better availability of phosphorus and organic carbon in the soil, allowing the development of microorganisms in general, and they enhanced the legume-Rhizobium association.

The inoculation of forage legumes should be a strategy to improve N fixation, since soils may be deficient in the number and quality of native rhizobia to improve plant productivity⁽⁵¹⁾. Studies carried out in Ecuador inoculating native strains of Bradyrhizobium japonicum in soybean (Glycine max L.) crops showed greater crop growth⁽⁵²⁾. Granda-Mora et al⁽¹⁵⁾ selected native strains of Rhizobium sp. for beans (Phaseolus vulgaris L.) in Ecuador, assuring that the bacterial inoculant obtained stimulated the growth and yield of beans. This opens the door to the potential use of native strains for forage plants, such as in Argentina, where they obtained improvements of 73 % in Desmanthus virgatus, a native forage plant inoculated with native rhizobia⁽⁵³⁾, or in Brazil, where they identified native strains for Leucaena leucocephala and obtained greater biomass production and accumulation of N⁽⁵⁴⁾.

Enhancing the N fixation capacity in legumes is a tool that allows reducing the use of N fertilizer in pasture production⁽⁵⁵⁾. Forage associations of grasses and legumes increase the production and quality of forage for livestock⁽⁵⁶⁾. They also have advantages over nitrogen fertilization, which has adverse side effects: water contamination by NO₃^-(57) and production of N₂O, a greenhouse gas⁽⁵⁸⁾.

Temperature plays a vital role in ryegrass monoculture. Differences were detected between species regarding the accumulation of root biomass: higher in red clover. In soils of greater fertility, such as those of Machachi, a greater number and weight of nodules were observed. Nodulation capacity was different among legumes; in general terms, white clover had a greater number and weight of nodules than red clover. The species were able to adapt to the different environments; the red clover had better nodulation in the conditions of Tumbaco, whereas the white clover was better in Machachi. The most advisable amount of clover seed to use in the mixture with perennial ryegrass is 5 kg per hectare, which makes it possible to achieve a greater number and weight of nodules; likewise, it is also recommended to make mixtures with white and red clover in 2.5 kg of each.

 

Acknowledgements

The authors thank the Research Directorate of the Central University of Ecuador for funding the research.

 

Conflict of interest

The authors state that there are no conflicts of interest in this study.

 

Literatura citada:

1.       Paredes M, Becerra V, Donoso ÑG, Olmos S, Rodríguez R. Morfología y estados de crecimiento y desarrollo de la planta de arroz. 100 años del cultivo del arroz en Chile en un contexto internacional. 1920-2020 / Paredes M, et al. editores. INIA Chile, 2021;Tomo II, 408-445. INIA Chile. https://repositorio.inta.gob.ar/handle/20.500.12123/15751.

2.       Ramos-Hernández E, Martínez-Sánchez JL. Almacenes de biomasa y carbono aéreo y radicular en pastizales de Urochloa decumbens y Paspalum notatum (Poaceae) en el sureste de México. Rev Biol Trop 2020;68(2):440-451.

3.       Poorter H, Nagel O. The role of biomass allocation in the growth response of plants to different levels of light, CO₂, nutrients and water: a quantitative review. Functional Plant Biol 2000;27(12):1191-1191. https://www.publish.csiro.au/fp/pp99173_co.

4.       Rasche AJW, Gomez EJ, Fatecha Fois DA, Leguizamón RCA, Rasche AJW, Gomez EJ, et al. Compactación del suelo y su efecto en el crecimiento vegetativo de soja, maíz y guandú. Invest Agraria 2020;22(1):13-21.

5.       Viana W, Scharwies JD, Dinneny JR. Deconstructing the root system of grasses through an exploration of development, anatomy and function. Plant, Cell Environ 2022;45(3):602-19. https://onlinelibrary.wiley.com/doi/abs/10.1111/pce.14270.

6.       Lattanzi F. Crecimiento de plantas forrajeras. En: Producción animal en pastoreo. 2.^a ed. Buenos Aires, Argentina: Instituto Nacional de Tecnología Agropecuaria; 2011.

7.       Rodríguez-Navarro D, Lorite MJ, Temprano Vera FJ, Camacho M. Selection and characterization of Spanish Trifolium-nodulating rhizobia for pasture inoculation. Syst Applied Microbiol 2022;45(2):126290. https://www.sciencedirect.com/science/article/pii/S0723202021001132.

8.       Palmero F, Fernandez JA, Garcia FO, Haro RJ, Prasad PVV, Salvagiotti F, et al. A quantitative review into the contributions of biological nitrogen fixation to agricultural systems by grain legumes. European J Agr 2022;136:126514. https://www.sciencedirect.com/science/article/pii/S1161030122000624.

9.       Matse DT, Huang CH, Huang YM, Yen MY. Nitrogen uptake and growth of white clover inoculated with indigenous and exotic Rhizobium strains. J Plant Nut 2020; 43(13):2013-27. https://doi.org/10.1080/01904167.2020.1758134.

10.   Goyal RK, Mattoo AK, Schmidt MA. Rhizobial–host interactions and symbiotic nitrogen fixation in legume crops toward agriculture sustainability. Front Microbiol 2021;2.  https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2021.669404/full.

11.   Ramana JV, Griffiths AG, Hofmann RW, Ridgway HJ. Nodule occupancy of effective rhizobia vary between historical and modern cultivars of white clover. NZ J Agr Res 2024;0(0):1-15.  https://doi.org/10.1080/00288233.2024.2331687.

12.   INEC. Instituto nacional de estadísticas y censos. 2022. Encuesta de superficie y producción agropecuaria continua. https://www.ecuadorencifras.gob.ec/estadisticas-agropecuarias-2/.

13.   INAMHI. Atlas climático de Pichincha. Gobierno autónomo descentralizado de Pichincha, editor. Instituto Nacional de Meteorología E Hidrología; 2018 https://www.pichincha.gob.ec/pichincha/datos-de-la-provincia/95-informacion-general.

14.   García L, Schlatter J. Caracterización de suelos a lo largo de un gradiente altitudinal en Ecuador. Rev Brasil Ciênc Agr 2012;7(3):456-64. http://www.agraria.pro.br/ojs32/index.php/RBCA/article/view/v7i3a1736.

15.   Granda-Mora K, Correa-Ullauri C, Collahuazo-Reinoso Y, Robles-Carrión Á. Inoculantes microbianos comerciales con PGPR sobre variables productivas y económicas de fríjol común (Phaseolus vulgaris L.). Agr Mesoamericana 2024;55654-55654. https://revistas.ucr.ac.cr/index.php/agromeso/article/view/55654.

16.   León R, Bonifaz N, Gutiérrez F. Pastos forrajes del Ecuador: siembra y producción de pasturas. 2018. http://dspace.ups.edu.ec/handle/123456789/19019.

17.   Rodríguez I, Hernández L, Crespo G, Sandríno B. Comportamiento de la fitomasa subterránea en diferentes pastizales de la provincia Mayabeque, Cuba. 2013; https://www.redalyc.org/articulo.oa?id=193028751016.

18.   Bécquer C. Potencial de formación de nódulos en leguminosas forrajeras y de granos de rizobios, nativos de Sancti Spíritus, Cuba. Rev Cubana Cienc Agr 2014;48(3):301-307. https://www.redalyc.org/pdf/1930/193032133017.pdf.

19.   Eastman I, Alonso V, Armand Ugón V, Rondoni M, Ferreira V, Rodriguez C, et al. Capacidad de promoción del crecimiento vegetal de beta-rizobios simbiontes de leguminosas nativas. Segundo Simposio de Microorganismos para la Agricultura INIA Las Brujas, 2022. https://redi.anii.org.uy/jspui/handle/20.500.12381/3620.

20.   Ansín OE, Oyhamburu EM, Hoffmann EA, Vecchio MC, Ferragine MC. Distribución de raíces en pastizales naturales y pasturas cultivadas de La Pampa Deprimida Bonaerense y su relación con la biomasa forrajera. Rev Facultad Agr La Plata.  2015;103(2):141-148. http://revista-vieja.agro.unlp.edu.ar/index.php/revagro/article/view/221

21.   Wedderburn M, Crush J, Pengelly W, Walcroft J. Root growth patterns of perennial ryegrasses under well-watered and drought conditions. NZ J Agr Res 2010;53(4):377-388. https://doi.org/10.1080/00288233.2010.514927.

22.   Di Rienzo J, Balzarini M, Gonzalez L, Casanoves F, Tablada M, Walter Robledo C. Infostat: software para análisis estadístico. Universidad Nacional de Cordova. 2010. https://www.infostat.com.ar/.

23.   Walne C, Reddy K. Temperature effects on the shoot and root growth, development, and biomass accumulation of corn (Zea mays L.). Agriculture 2022;12(4):443. https://www.mdpi.com/2077-0472/12/4/443.

24.   Houde S, Thivierge MN, Fort F, Bélanger G, Chantigny MH, Angers DA, et al. Root growth and turnover in perennial forages as affected by management systems and soil depth. Plant Soil 2020;451(1):371-387. https://doi.org/10.1007/s11104-020-04532-1.

25.   Huot C, Zhou Y, Philp JNM, Denton MD. Root depth development in tropical perennial forage grasses is related to root angle, root diameter and leaf area. Plant Soil 2020;456(1):145-58. https://doi.org/10.1007/s11104-020-04701-2.

26.   Cougnon M, Deru J, Eekeren N van, Baert J, Reheul D. Root depth and biomass of tall fescue vs. perennial ryegrass. In: Helgadóttir A, Hpkins A eds. The role of grasslands in a green future: threats and perspectives in less favoured areas. Proceed 17th Symp European Grassland Federation. Akureyri, Iceland; 2013. https://www.cabidigitallibrary.org/doi/full/10.5555/20133335529.

27.   Nichols S, Hofmann R, Williams W. Effect of hybridization with Trifolium uniflorum on tap root survival in white clover. NZ J Agr Res 2015;58(4):371-383. https://doi.org/10.1080/00288233.2015.1071711.

28.   Janssen PWL, Hoekstra NJ, van der Schoot JR, van Eekeren N. White clover (Trifolium repens) population dynamics are partly dependent on timing of seminal taproot death. Grass Forage Sci 2023;78(1):173-183. https://onlinelibrary.wiley.com/doi/abs/10.1111/gfs.12598.

29.   Echeverría H, Sainz Rosas H. Nitrógeno. En: Echeverría H, García F, editores. Fertilidad de suelos y fertilización de cultivos. 2.^a ed. Buenos Aires, Argentina: Instituto Nacional de Tecnología Agropecuaria; 2014.

30.   Xie K, Wang Y, Wan J, Zhang S, Sui X. Mechanisms and factors affecting nitrogen transfer in mixed legume/grass swards: A review. 2020;29(3):157-70. https://www.cabidigitallibrary.org/doi/full/10.5555/20219975556.

31.   Reilly EC, Gutknecht JL, Tautges NE, Sheaffer CC, Jungers JM. Nitrogen transfer and yield effects of legumes intercropped with the perennial grain crop intermediate wheatgrass. Field Crops Res 2022;286:108627. https://www.sciencedirect.com/science/article/pii/S0378429022001988.

32.   Dicenzo G, Tesi M, Pfau T, Mengoni A, Fondi M. Genome-scale metabolic reconstruction of the symbiosis between a leguminous plant and a nitrogen-fixing bacterium. Nat Commun 2020;11(1):2574. https://www.nature.com/articles/s41467-020-16484-2.

33.   Ferguson B, Mens C, Hastwell A, Zhang M, Su H, Jones C, et al. Legume nodulation: The host controls the party. Plant Cell Environ 2019;42(1):41-51. https://onlinelibrary.wiley.com/doi/abs/10.1111/pce.13348.

34.   Isidra-Arellano MC, Reyero-Saavedra MDR, Sánchez-Correa MDS, Pingault L, Sen S, Joshi T, et al. Phosphate deficiency negatively affects early steps of the symbiosis between common bean and rhizobia. Genes 2018;9(10):498. https://www.mdpi.com/2073-4425/9/10/498.

35.   Reid D, Ferguson B, Gresshoff PM. Inoculation- and Nitrate-induced CLE peptides of soybean control NARK-dependent nodule formation. Mol Plant-Microbe Int 2011;24(5):606-618. https://apsjournals.apsnet.org/doi/abs/10.1094/MPMI-09-10-0207.

36.   Dixon R, Kahn D. Genetic regulation of biological nitrogen fixation. Nat Rev Microbiol  2004;2(8):621-631. https://www.nature.com/articles/nrmicro954.

37.   Hernández G, Valdés-López O, Ramírez M, Goffard N, Weiller G, Aparicio-Fabre R, et al. Global changes in the transcript and metabolic profiles during symbiotic nitrogen fixation in phosphorus-stressed common bean plants. Plant Physiol 2009;151(3):1221-1238. https://doi.org/10.1104/pp.109.143842.

38.   Dubach M, Russelle MP. Forage legume roots and nodules and their role in nitrogen transfer. Agronomy J 1994;86(2):259-66. https://onlinelibrary.wiley.com/doi/abs/10.2134/agronj1994.00021962008600020010x.

39.   Vasileva V, Kosev V. Evaluation of nodule related components and forage productivity in Pea (Pisum sativum L.) genotypes. Int J Pharmacy Life Sci 2015;6(2):4230-4237. http://www.ijplsjournal.com/issues%20PDF%20files/2015/February-2015/3.pdf.

40.   Adinurani PG, Rahayu S, Purbajanti ED, Siskawardani DD, Stankevica K, Setyobudi RH. Enhanced of root nodules, uptake NPK, and yield of peanut plant (Arachis hypogaea L.) using rhizobium and mycorrhizae applications. SJA 2021;37(s1). http://researcherslinks.com/current-issues/Enhanced-Root-Nodules-Uptake-NPK/14/29/3832/html.

41.   Muresu R, Polone E, Sulas L, Baldan B, Tondello A, Delogu G, et al. Coexistence of predominantly nonculturable rhizobia with diverse, endophytic bacterial taxa within nodules of wild legumes. FEMS Microbiol Ecol 2008;63(3):383-400. https://doi.org/10.1111/j.1574-6941.2007.00424.x.

42.   Deng Z, Zhao L, Kong Z, Yang W, Lindström K, Wang E, et al. Diversity of endophytic bacteria within nodules of the Sphaerophysa salsula in different regions of Loess Plateau in China. FEMS Microbiol Ecol 2011;76(3):463-475. https://doi.org/10.1111/j.1574-6941.2011.01063.x.

43.   Wigley K, Moot D, Wakelin SA, Laugraud A, Blond C, Seth K, et al. Diverse bacterial taxa inhabit root nodules of lucerne (Medicago sativa L.) in New Zealand pastoral soils. Plant Soil 2017;420(1):253-262. https://doi.org/10.1007/s11104-017-3395-6.

44.   Sturz AV, Christie BR, Matheson BG, Nowak J. Biodiversity of endophytic bacteria which colonize red clover nodules, roots, stems and foliage and their influence on host growth. Biol Fertil Soils 1997;25(1):13-19. https://doi.org/10.1007/s003740050273.

45.   Behera B, Das T, Raj R, Ghosh S, Raza M, Sen S. Microbial consortia for sustaining productivity of non-legume crops: prospects and challenges. Agric Res 2021;10(1):1-14. https://doi.org/10.1007/s40003-020-00482-3.

46.   Ye Q, Wang Y, Huang W, Li L, Li J, Zheng Y, et al. Dissolved organic matter characteristics in soils of tropical legume and non-legume tree plantations. Soil Biol Biochem 2020;148:107880. https://www.sciencedirect.com/science/article/abs/pii/S0038071720301772.

47.   Furtak K, Gawryjole K, Galazka A, Gradziel J. The response of red clover (Trifolium pratense L.) To separate and mixed inoculations with Rhizobium leguminosarum and Azospirillum brasilense in presence of polycyclic aromatic hydrocarbons. Int J Environm Res Pub Health 2020;17(16):5751. https://www.mdpi.com/1660-4601/17/16/5751

48.   Mir M, Mukkamula N, Kiran B, Raghu K, Parray J, Khan I, et al. Impact of microbial consortium of Rhizobium tropici and Rhizobium mayense on the growth of Phaseolus vulgaris L. South African J Botany 2024;168:562-572. https://www.sciencedirect.com/science/article/pii/S0254629924002217.

49.   Al Virk A, Lin B, Kan Z, Qi J, Dang Y, Lal R, et al. Simultaneous effects of legume cultivation on carbon and nitrogen accumulation in soil. In: Advances in Agronomy. Academic Press; 2022:75-110. https://www.sciencedirect.com/science/article/abs/pii/S006521132100081X.

50.   Bailey V, Smith J, Bolton H. Fungal-to-bacterial ratios in soils investigated for enhanced C sequestration. Soil Biol Biochem 2002;34(7):997-1007. https://www.sciencedirect.com/science/article/abs/pii/S0038071702000330.

51.   Abdulahi A, Howieson J, O´Hara G, Terpolilli J, Tiwari R, Yusuf A. History of rhizobia inoculants use for improving performance of grain legumes based on experience from Nigeria. En: Sutton M, Mason K, editors. Just enough nitrogen perspectives on how to get there for regions with too much and too little nitrogen. Just Enough Nitrogen. Cham, Switzerland: Springer International Publishing; 2020. https://doi.org/10.1007/978-3-030-58065-0.

52.   Soto-Valenzuela J, Catuto-Suárez A, Álvarez-Vera M. Evaluación del crecimiento y nodulación de plantas de soya (Glycine max) inoculadas con Rhizobium y Bradyrhizobium japonicum en Manglaralto, Santa Elena (Ecuador). Rev Cient Tecnol UPSE 2021;8(2):27-32. https://incyt.upse.edu.ec/ciencia/revistas/index.php/rctu/article/view/577.

53.   Fornasero LV, Zuber NE, Toniutti MA, del Papa MF, Pensiero JF, Lagares A. Caracterización feno-genotipica y simbiótica de rizobios aislados de Desmanthus Virgatus (l.) Willd. en suelos de la provincia de Jujuy (Argentina). Fave Sección Ciencias Agrarias. 2021;20(1):247-64. http://www.scielo.org.ar/scielo.php?script=sci_abstract&pid=S1666-77192021000100247&lng=es&nrm=iso&tlng=es.

54.   Hinojosa JGC, Araujo FDS, Sá ELSD. Rizóbios nativos eficientes en la fijación de nitrógeno en Leucaena leucocephala en Rio Grande do Sul, Brasil. Biotecnol Sector Agropecu y Agroind 2021;19(1):128-138. https://revistas.unicauca.edu.co/index.php/biotecnologia/article/view/1482.

55.   Hasanuzzaman M, Araújo S, Sarvajeet S. The plant family fabaceae biology and physiological responses to environmental stresses. Singapore: Springer Nature Singapore; 2020.  https://doi.org/10.1007/978-981-15-4752-2.

56.   Bondaruk V, Lezama F, del Pino A, Piñeiro G. Overseeding legumes in natural grasslands: Impacts on root biomass and soil organic matter of commercial farms. Sci Total Environ 2020;15:140771. https://www.sciencedirect.com/science/article/pii/S0048969720342959.

57.   Lv H, Lin S, Wang Y, Lian X, Zhao Y, Li Y, et al. Drip fertigation significantly reduces nitrogen leaching in solar greenhouse vegetable production system. Environm Pollution 2019;245:694-701. https://www.sciencedirect.com/science/article/pii/S0269749118340405.

58.   Gutiérrez-León F, Lastra-Bravo X, Cali V. Estimación de la huella de carbono de las ganaderías de leche de la zona andina del ecuador: huella de carbono de ganaderías de leche del Ecuador. Chilean J Agr Anim Sci 2023;39(3):305-318. https://revistas.udec.cl/index.php/chjaas/article/view/10600.