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Cuban Journal of Agricultural Science

versión impresa ISSN 0864-0408versión On-line ISSN 2079-3480

Cuban J. Agric. Sci. vol.54 no.3 Mayabeque sept.-dic. 2020  Epub 01-Sep-2020


Pasture Science

Selection of promising rhizobia to inoculate herbaceous legumes in saline soils

I. Hernández1  * 

Susana L. Estévez2

Maida D. Peña2

María C. Nápoles1

1Instituto Nacional de Ciencias Agrícolas, Gaveta postal 1, San José de las Lajas, Mayabeque, C.P. 32 700

2Universidad de Holguín, Sede “Oscar Lucero Moya”. Gaveta postal 57, Holguín, C.P. 80100


Salinity of soils constitutes a limiting factor for growth and development of legumes of agricultural and livestock interest. The objective of this study was to select rhizobia isolates with potentialities to improve the establishment of herbaceous legumes in saline soils. Nodulation pattern of the herbaceous legumes Desmodium triflorum (L.) DC., Alysicarpus vaginalis (L.) DC. and Crotalaria retusa L., established in a strongly saline soil and under water deficit conditions, was studied. Possible rhizobia were isolated from the rhizosphere and from nodules of these plants, and were culturally, morphologically and physiologically characterized. In addition, nodulation tests of rhizobia isolates were carried out in siratro plants under controlled conditions. The three legumes presented small nodules and scarce nodulation. Twelve possible isolates of rhizobia were obtained. Of these, 66.7% showed cultural and physiological characteristics similar to strains of Rhizobiaceae family and 33.3% similar to Bradyrhizobium strains. Out of these isolates, 100% produced effective nodules for biological nitrogen fixation in siratro plants. Plants inoculated with Riz 5-1, Riz 8-11 and Riz 8-12 isolates presented higher number and dry mass of nodules, aerial dry mass and radical dry mass. These isolates survive saprophytically in soils affected by water deficit and salinity, which makes them attractive for the preparation of bio-preparations that allow the rehabilitation of pastures and forages under the previously mentioned conditions.

Key words: Rhizobium; salinity; forage; characterization; growth

Traditionally, herbaceous legumes of Desmodium, Alysicarpus and Crotalaria genera have been used in the control of soil erosion, restoration of its chemical properties, as forage and green manure (Midega et al. 2017 and DemLew et al. 2019). Furthermore, they increase the availability of macro and micronutrients for companion crops (Chidowe et al. 2014 and Trevisan et al. 2017). Desmodium and Alysicarpus have broad prospects for the development of cattle rearing, since they are non-toxic, have high palatability, high nutritional value and good digestibility of dry matter (Arango et al. 2016). Biopreparations made from Crotalaria cause mortality to pest insects populations and herbicides (Atuesta et al. 2017 and da Costa et al. 2019).

Much of the benefits of these legumes in soils dedicated to cattle rearing are due to the symbiotic association they establish with nitrogen-fixing bacteria, called rhizobia (García et al. 2018). However, many of these soils are affected by factors such as water deficit and salinity, which explain the low quality of grasslands and their high degradation level, leading to insufficient livestock productivity (Machado 2009). Previous studies found that water deficits and salinity affect legumes, rhizobia and, more drastically, the symbiosis between both organisms (Yamal et al. 2016 and Fuskhah et al. 2019).

The multiple experiences in the use of legumes to reforest degraded areas, soils lacking organic matter and affected by salinity, serve as an indication of the potential of this group of plants in the sustainability of agricultural and livestock production systems (Castro et al. 2017). One way of making this possible is to inoculate forage legumes with previously selected rhizobia strains, which are adapted to soil stress conditions and have the ability to colonize and form effective nodules for biological nitrogen fixation (BNF) (Franzini et al. 2019 and Tewari and Sharma 2020).

Recent research has shown that inoculation of legumes with rhizobia under salinity and water deficit conditions produces a decrease of ethylene, increases tolerance of plants to stress and increases nodulation and photosynthesis (Khaitov et al. 2020). Therefore, the objective of this research was to select rhizobia isolates with potentialities to improve the establishment of herbaceous legumes in saline soils.

Materials and Methods

The study was carried out with nodules and rhizospheric soil of three herbaceous legumes genera, taxonomically identified as Desmodium triflorum (L.) DC., Alysicarpus vaginalis (L.) DC. and Crotalaria retusa L, belonging to Fabaceae family (Romero et al. 2015 and Almaguer et al. 2017). These plants were established in a strongly saline Solonchak soil (Hernández et al. 2015), from the Unidad Empresarial de Base (UEB) Ganadera “Rafael Quintana”, in Sabanazo popular council, Calixto García municipality, Holguín province. The taxonomic study was developed during drought period, between January and February, 2014. Although permanence time of legumes in this ecosystem is not known, it is suggested that they were subjected to moderate-severe drought, with accumulated mean precipitations of only 200 mm (Romero et al. 2015).

Characterization of herbaceous leguminous nodules established in saline soil. Three healthy plants of each species were selected, with no signs of pest attack or presence of diseases, from six sampling points of the UEB Ganadera "Rafael Quintana". Variables related to nodulation were determined: density of total nodulation and of nodulation in the main root, size and shape of nodules.

Regarding density of total nodulation and in the main root, plants presenting from 0 to 10 nodules were classified as scarce, medium for those from 11 to 50, and abundant for plants with more than 50 nodules. Nodules with a diameter between 1.0 and 1.5 mm were considered small; from 1.5 to 3.0 mm medium, and large those over 3.0 mm of diameter. They were also classified as spherical or polymorphic, according to their external morphology (Bécquer et al. 2016). At the time of sampling, they were placed in closed containers that contained calcium chloride (CaCl2) and were kept at 4 oC until they were transferred to the Microbiology Laboratory of the Department of Physiology and Biochemistry of the National Institute of Agricultural Sciences (INCA, initials in Spanish).

Isolation and characterization of possible rhizobia associated with herbaceous legumes established in saline soil. The possible rhizobia were isolated from the rhizospheric soil and from nodules of the three species of legume. For isolation from rhizospheric soil, serial decimal dilutions were made from 1 g of soil. Dilutions were cultivated by dissemination in Petri dishes, containing mannitol yeast (MY) solid medium (Vincent 1970) and incubated at 30 oC for 10 d.

Isolation from the nodules began with their hydration in distilled water, for 5 min. Then, they were superficially disinfected with 95% ethanol, for 45 sec. and with 0.1% (w/v) sodium hypochlorite for 4 min. Then, nodules were washed six times with sterile distilled water under aseptic conditions. They were transferred to hemolysis tubes with sterile forceps, containing 500 µL of saline solution (NaCl 8.5 g L-1). Nodules were macerated and 0.1 mL of the suspension was cultivated by dissemination in plates containing MY solid medium, which were incubated at 30 oC for ten days (Sosa et al. 2004).

From the bacterial colonies resulting from both processes, those with similar cultivation characteristics to those described in the literature for rhizobia group were selected (Wang and Martínez-Romero 2001). Successive cultures of isolates were carried out in MY solid medium to guarantee their purity, and they were stored at 4 oC in test tubes containing the same culture medium.

Later, a cultural, morphological and physiological characterization of the previously selected bacterial isolates was carried out, in order to identify them as possible rhizobia. Isolates that did not present similar characteristics to those described above for this group of bacteria (Wang and Martínez-Romero 2001) were discarded from the following determinations.

For cultural characterization, isolates were grown by triplicate, in MY solid medium, and incubated at 30 oC for 10 d. Color, mucus and size of colonies were considered as cultural aspects. Regarding diameter, those with a diameter greater than 2.0 mm were considered as large colonies, between 1 and 2 mm as medium, and less than 1 mm as small. Gram staining was used for the morphological characterization of isolates, which took into account cell morphology, staining response and endospore presence.

From the physiological point of view, time of colony appearance, acid or base production and capacity to produce nodules in a model legume were determined. For the first case, isolates were cultivated in the MY solid medium and the moment when they became visible was recorded. Bacterial colonies could be observed every 24 h, during 10 d of incubation, at 30 oC. Isolates, which colonies could be seen in the medium after two or three days, were considered as fast growing. Isolates with colonies that appeared after four and five days, were classified as moderate growth, and those that were observed after seven days, were classified as slow growing microorganisms (Wang and Martínez-Romero 2001).

To determine the capacity of bacterial isolates to produce acid or base, they were cultivated by triplicate and by exhaustion in the MY solid medium, which was supplemented with bromothymol blue indicator (0.5% in 0.016 N NaOH). Dishes were incubated at 30 oC, for 10 d. The change of coloration of the medium, from green to yellow, was interpreted as the production of organic acids, and the change from green to blue as the production of bases (Sosa et al. 2004).

As part of the physiological characterization, the ability of bacterial isolates to form nodules in plants of Macroptillium atropurpureum (Moc. and Sessé ex DC.) Urb. (siratro) was also evaluated. Firstly, inocula of isolates were prepared from the inoculation of a loop of these in 100 mL Erlenmeyers, containing 20 mL of MY medium. Flasks were kept shaking, in an orbital shaker at 150 rpm. and 30 oC, between 18 and 20 h for fast growing isolates, and from 56 to 58 h for slow growing isolates. Purity of cultures was determined by Gram staining. Finally, absorbance of the bacterial cultures was adjusted to an OD=0.1 (λ=600 nm), in a UV-Visible Light Spectrophotometer (GENESYS 6).

At the same time, surface disinfection and pre-germination of siratro seeds were carried out. The seeds were disinfected with 70% ethanol, for 5 min. Then, they were washed with distilled water and immersed in concentrated sulfuric acid for 10 min. Then, seeds were kept in a 25% (v/v) sodium hypochlorite solution for 15 min. and washed ten times with sterile distilled water. Finally, they were placed on dishes with water-agar (0.75% v/v) and incubated at 30 oC in a dark place for 24 h, to favor their germination (Pérez et al. 2008).

Pregerminated seeds with approximately 1 cm of emerging root were placed in 70 mL volume bottles, containing 50 mL of Norris and Date semi-solid medium (Norris and Date 1967), at a rate of one seed per bottle, and inoculated with 1 mL of bacterial inocula. Seedlings inoculated with 1 mL of sterile MY medium were considered as a negative control of the experiment. Plants were kept under controlled growth conditions, with a photoperiod of 16 h light/8 h dark, at a day/night temperature of 26 oC / 22 oC and relative humidity of 70%. Four weeks after inoculation, the number of nodules in the main root (NNRP), number of total nodules (NNT), effectiveness of nodules and dry mass of total nodules (MSNT) were determined. In addition, aerial dry mass (MSA) (g) and radical dry mass (MSR) (g) of siratro plants were determined.

Statistical design and analysis. Inoculation test of bacterial isolates in siratro plants was carried out using a completely randomized design. Seven replicates per treatment were used. Resulting data was subjected to normality test (Babtlett test, Babtlett 1937) and homogeneity of variance (Kormogorov-Smirnov test. Kolmogorov 1933). One way classification analysis of variance was applied by Tukey test, for P <0.05, in order to determine differences between means (Sigarroa 1985). Statgraphic plus, version 5.0 was used for statistical data processing. These were plotted in Sigmaplot for Windows, version 11.0.


The three herbaceous legumes, established in a strongly saline soils and under water deficit conditions, have scarce nodulation. Results showed that all plants had scarce total nodulation. This performance was also seen in the main root. Even at collection point number 5 (p5), A. vaginalis (L.) DC. plants lacked nodules. D. triflorum (L.) DC. and A. vaginalis (L.) DC. presented small and spherical nodules. However, medium and polymorphic nodules appeared in the roots of C. retusa L. (table 1).

Table 1 Nodulation pattern of herbaceous legumes Desmodium triflorum (L) DC., Alysicarpus vaginalis (L) DC. and Crotalaria retusa L. established under water deficit conditions in a strongly saline soil 

Sampling points Herbaceous legumes Nodulation
Total Main root Nodule size Nodule shape
P1 Desmodium triflorum (L.) DC. Scarce Scarce Small Spherical
P2 Desmodium triflorum (L.) DC.
P3 Alysicarpus vaginalis (L.) DC. Scarce Small Spherical
P4 Alysicarpus vaginalis (L.) DC. Scarce Small Spherical
P5 Alysicarpus vaginalis (L.) DC. Without nodules Small Spherical
P6 Crotalaria retusa L. Scarce Medium Polymorphic

Populations of possible rhizobia inhabit the rhizosphere and interior of nodules of the three herbaceous legumes.

A total of 15 possible rhizobia isolates were obtained from the rhizosphere and from the nodules of the three studied herbaceous legumes. Out of these, 20% came from D. Triflorum (L.) DC., 53.3% from A. vaginalis (l.) DC. and 26.7% from C. retusa L. Most of isolates from Alysicarpus were obtained from their nodules, while it was 100% in C. retusa L. (table 2).

Table 2 Characterization and possible identification of bacterial isolates from rhizosphere and nodules of the herbaceous legumes Desmodium triflorum (L.) DC., Alysicarpus vaginalis (L.) DC. and Crotalaria retusa L., established in saline soils 

Isolation place Isolates Characterization Possible identification
Cultural Morphological Physiological
Type of growth Acid or base
Desmodium triflorum (L.) DC.
Nodules Riz 1-1 Semi-translucent, mucus, large Bacilli, G -, not sporulated Fast Acid Rhizobaceae
Rhizospheric soil Riz 1-2 Semi-translucent, mucus, large Bacilli, G -, not sporulated Fast Acid Rhizobaceae
Riz 2-1 Semi-translucent, mucus, large Cocobacilli, G -, sporulated - - -
Alysicarpus vaginalis (L.) DC.
Nodules Riz 5 Semi-translucent, mucus, large Bacilli, G -, not sporulated Fast Acid Rhizobaceae
Riz 5-1 Pale pink, dry, small Bacilli, G -, not sporulated Slow Basic Bradyrhizobium
Riz 5-2 Semi-translucent, mucus, large Bacilli, G -, not sporulated Fast Acid Rhizobaceae
Riz 6 Semi-translucent, mucus, large Bacilli, G -, not sporulated Fast Acid Rhizobaceae
Riz 6-1 Semi-translucent, mucus, large Bacilli, G -, not sporulated Fast Acid Rhizobaceae
Riz 7-1 Semi-translucent, mucus, large Bacilli, G -, not sporulated Fast Acid Rhizobaceae
Riz 7-2 Semi-translucent, mucus, large Bacilli, G -, not sporulated Fast Acid Rhizobaceae
Rhizospheric soil Riz 5-0 Beige, dry, large - - - -
Crotalaria retusa L.
Nodules Riz 8-11 Pale pink, dry, small Bacilli, G -, not sporulated Slow Basic Bradyrhizobium
Riz 8-12 Pale pink, dry, small Bacilli, G -, not sporulated Slow Basic Bradyrhizobium
Riz 8-13 Beige, dry, large - - - -
Riz 8-14 Beige, dry, large - - - -

G:Gram positive

Most of the isolates were characterized by forming large, mucus, semi-translucent colonies in the MY solid culture medium. However, isolates Riz 5-1, Riz 8-11, and Riz 8-12 formed small, dry, pale pink colonies. Isolates Riz 8-13 and Riz 8-14 presented a third pattern from a cultural point of view, as they formed large, dry, and beige colonies.

Gram staining showed that the majority of the isolates were Gram negative bacilli, not sporulated. Only Riz 2-1 isolate was observed as sporulated Gram negative cocobacilli. Results also showed two patterns regarding the time of appearance of colonies in the MY solid medium and acid and base production. Eight isolates showed fast growth and produced acid, while another group grew slowly and produced base.

Taking into account the results of the cultural, morphological and physiological characterization of the bacterial isolates, only those with similar characteristics to those described for rhizobia group were selected, with which inoculation tests with syratro plants would be carried out. Results showed that, except Riz 7-1, all isolates produced nodules in the main root of the plants. In addition, effective nodules were visualized in the BNF in all the plants, except for those inoculated with Riz 7-1 and Riz 6 isolates (figure 1).

Mean with equal letters have no statistical difference

sex (number of nodules in the main root) = 0.29*;

sex (number of effective nodules in the main root) = 0.21*

(Tukey P = 0.012, N=7)

Figure 1 Number and effectiveness of nodules in the main root of siratro plants, inoculated with bacterial isolates from a rhizospheric soil and from nodules of the herbaceous legumes Desmodium triflorum (L.) DC., Alysicarpus vaginalis (L.) D.C. and Crotalaria retusa L, at 30 d of cultivation under controlled conditions 

Plants inoculated with Riz 5-1, Riz 8-11 and Riz 8-12 isolates presented the highest values of number of nodules in the main root and number of effective nodules in the BNF. In these variables, no significant differences were observed between plants treated with Riz 8-11 isolate and those that used Riz 8-12.

When analyzing total nodulation of siratro plants, results showed a similar performance to that found for the nodulation in the main root. Plants inoculated with Riz 5-1, Riz 8-11, and Riz 8-12 isolates produced the highest number of total and total effective nodules. No significant differences in these variables were observed among the plants treated with Riz 8-11 and Riz 8-12 isolates. There were differences between the latter and those where Riz 5-1 isolate was inoculated (figure 2).

Mean with equal letters have no statistical difference

sex (number of total nodules) = 0.43*;

sex (number of effective total nodules) = 0.38*

(Tukey P=0.015, n=7)

Figure 2 Number and effectiveness of total nodules in siratro plants, inoculated with bacterial isolates from a rhizospheric soil and from nodules of the herbaceous legumes Desmodium triflorum (L.) DC., Alysicarpus vaginalis (L.) DC. and Crotalaria retusa L, at 30 d of cultivation under controlled conditions 

Regarding dry mass of nodules, results demonstrated that the use of Riz 5-1, Riz 8-11 and Riz 8-12 isolates produced the highest dry mass values of nodules in the main root and total nodulation (table 3).

Tabla 3 Dry mass of nodules in the main root and total nodules, and dry mass of the aerial part and of roots of siratro plants, inoculated with possible isolates of rhizobia from rhizosphere and nodules from the herbaceous legumes Desmodium triflorum (L.) DC., Alysicarpus vaginalis (L.) DC. and Crotalaria retusa L. at 30 d of cultivation under controlled conditions 

Treatments DMNMR (g) DMTN (g) ADM (g) RDM (g)
Control 0.0000 ͤ 0.0005 ᶠ 0.022 de 0.020 cd
Riz 1-1 0.0013 ᵈ 0.0024 de 0.028 d 0.024 c
Riz 1-2 0.0006 de 0.0028 ͩ 0.021 de 0.020 cd
Riz 5 0.0009 de 0.0020 def 0.020 de 0.016 d
Riz 5-1 0.0196 ᶜ 0.0196 c 0.156 c 0.039 b
Riz 5-2 0.0007 de 0.0014 def 0.017 e 0.017 d
Riz 6 0.0002 de 0.0010 ef 0.021 de 0.019 cd
Riz 6-1 0.0003 de 0.0010 ef 0.014 e 0.016 d
Riz 7-1 0.0000 ͤ 0.0006 f 0.020 de 0.020 cd
Riz 7-2 0.0009 de 0.0016 def 0.015 e 0.017 d
Riz 8-11 0.0238 ͣ 0.0248 a 0.212 a 0.054 a
Riz 8-12 0.0214 ᵇ 0.0229 b 0.184 b 0.038 b
sex 0.0004* 0.0006* 0.003* 0.002*

Mean with equal letters have no statistical difference (Tukey P = 0.034, n = 7)

DMNMR: dry mass of nodules in the main root

DMTN: dry mass of total nodules

ADM: aerial dry mass

RDM: root dry mass

Something similar was found with the aerial and radical dry mass of plants, since those that were inoculated with these three isolates significantly increased both variables. Those inoculated with Riz 8-11 isolate were highlighted, with the highest values of ADM and RDM.


In this investigation, a study of nodulation pattern and rhizobia populations associated with three herbaceous legumes established in a very strongly saline Solonchak soil was carried out. The use of rhizobia-based inoculants could form part of the integrated management of these ecosystems to increase the establishment of legumes, even under salinity limiting conditions, and thereby increase availability of pasture for cattle rearing.

At the time of sampling, the studied plants were established under conditions of water deficit and soil salinity, factors that can limit growth and development of legumes and the microorganisms associated with it. It is known that water deficit limits the mobility, viability and concentration of rhizobia in the rhizosphere of legumes (Hussain et al. 2014 and Gopalakrishnan et al. 2015). Previous studies show a decrease of nodulation in bean plants (Phaseolus vulgaris l.), grown under water deficit conditions (Estrada et al. 2017). This condition causes hormonal (abscisic acid) and enzymatic changes (sucrose synthase) in the legume, which brings about photosynthetic effects and ensures the survival of bacteroids inside the nodule. As a result, rhizobia multiplication rate decreases and, therefore, inhibition of nodule growth occurs (Liu et al. 2005, Ladrera et al. 2007 and Azcón-Bieto and Tolón, 2010).

Salinity also causes negative effects on legume growth and on rhizobia multiplication. Previous research showed a decrease of number and size of nodules in cowpea plants (Vigna unguiculata), with the increase of salinity in the substrate (Al-Saedi et al. 2016). This factor decreases the colonization of rhizosphere by rhizobia, since it inhibits the synthesis of glucans, lipopolysaccharides (LPS) and exopolysaccharides, which are superficial molecules of the bacterium that are essential for its interaction with the plant (Tewari and Sharma 2020). These events cause a decrease of number and mass of nodules, of leghemoglobin synthesis and of nitrogenase activity (Ballhorn et al. 2018 and Sunita et al. 2019).

This research also found differences in the shape and size of nodules found in C. retusa (L) with respect to D. triflorum (L.) DC. and A. vaginalis (L.) DC. Crotalaria produces indeterminate and polymorphic nodules that present a permanent meristem, tissue that allows constant growth of the organ (Renier et al. 2011). This capacity, maintained even under limiting conditions of drought and salinity, suggests greater osmotolerance of indeterminate nodules with respect to the determined ones (Fernández Pascual et al. 2007, Selami et al. 2014 and Huang et al. 2018). These morphological and physiological characteristics could explain the large size of Crotalaria nodules, even under water deficit and soil salinity conditions.

Despite the negative effects of water deficit and salinity for legumes and rhizobia, situations such as these favor the natural selection of bacterial strains that establish symbiosis with legumes with which they were not compatible (Andrews and Andrews 2017). Isolating rhizobia strains adapted to stressful conditions and increasing their concentration in these soils from their inoculation, could have a positive ecological effect on ecosystems.

Considering the above, isolation of possible rhizobia was carried out from nodules and from the rhizospheric soil of three herbaceous legume genera. For this purpose, conventional taxonomy methods were applied, including the study of phenotypic characters that allow an approach to bacterial strain identification.

Nodules are structures that are formed in the symbiosis between rhizobia and legumes. Together with the rhizosphere of these plants, they constitute the most frequent habitats for rhizobia. In fact, the way to authenticate whether a bacterium belongs to rhizobia group is to confirm its ability to form nodules in legumes under aseptic conditions (Howieson and Dilworth 2016). This is explained because, in recent years, it has been demonstrated that populations of bacteria that do not have the ability to form these structures coexist, together with rhizobia, in the nodules of many legumes (Peix et al. 2015 and Velázquez et al. 2017).

As a primary criterion, cultural characteristics described for rhizobia were used, when they grow in the MY solid culture medium. According to Wang and Martínez-Romero (2001), rhizobia form two fundamental phenotypes of colonies in this medium: semi-translucent, large and mucous colonies, and semi-translucent, small and dry colonies. According to the above, Riz 8-13 and Riz 8-14 isolates were ruled out as possible rhizobia, as they formed large, dry, beige colonies (table 2) and were eliminated from subsequent determinations.

Gram staining was used as a second criterion. According to Madigan et al. (2019), rhizobia are Gram negative bacilli or cocobacilli, and not sporulated. Therefore, the observation of endospores in the cells of Riz 2-1 isolate (table 2) allowed to remove them as possible rhizobia, and it was not considered in the rest of the characterization either.

Determination of physiological aspects of rhizobia, such as the time of appearance of the colonies on the MY solid medium and acid or base production, also contribute to the grouping of these bacteria in different genera (Wang and Martínez-Romero 2001). In this research, two cultivation and physiological patterns are distinguished: the first and most numerous are the fast-growing, acid-producing isolates that form large, mucous and semi-translucent colonies (table 1). This pattern agrees with rhizobia strains that have previously been located in Rhizobiaceae family, which groups Ensifer, Rhizobium and Shinella genera (Sahgal and Jaggi 2018).

Riz 5-1, Riz 8-11 and Riz 8-12 isolates presented characteristics that agree with those of the second cultural and physiological pattern. This pattern consists of slow-growing, base-producing isolates that form small, dry and semi-translucent colonies in the MY solid medium. Strains with these characteristics have been grouped into Bradyrhizobium genus (Sahgal and Jaggi 2018).

Regarding the results of cultural, morphological and physiological characterization, 80% of total isolates were selected as possible rhizobia. All isolates from D. triflorum (l.) DC. were identified as part of Rhizobiaceae family. Previous research has shown the presence of Rhizobium genus, which belongs to this family, in Desmodium oldhami and Desmodium sequax species (Xu et al. 2016). In the case of the legume C. retusa L., all bacterial isolates were classified in the Bradyrhizobium genus. During the symbiosis, prevalence of some genera with respect to others is explained, among other aspects, by the specificity of molecular signals between legume and rhizobia (Liu and Murray 2016, Andrews and Andrews 2017 and Salas et al. 2017). Some chemical groups in the structure of the so-called nodulation factors, better known determinants that govern the rhizobium-legume interaction, have a very important role in this specificity (Wang and Zhu 2018).

Unlike D. triflorum (L.) DC. and C. retusa L., rhizobia of Rhizobiaceae family and Bradyrhizobium genus coexist in nodules and rhizosphere of the legume A. vaginalis (L.) DC. Establishing symbiosis with a wide range of rhizobia genera does not necessarily imply that an efficient BNF is performed with all microsymbionts (Terpolilli et al. 2008 and Chibeba et al. 2017). Taking into account the above, it is necessary to select and inoculate efficient strains in colonization, establishment and FBN, in order to obtain an increase of growth and yield of legumes. Native strains and the necessary concentration of them for provoking the desired effects on plants are not always found in the soil (Lodeiro 2015). In this study, inoculation tests were carried out on the twelve possible isolates of rhizobia in siratro plants, in order to determine their ability to produce effective nodules in the BNF and promote growth of this plant.

Siratro is used as a model legume to evaluate nodulation, due to its ability to establish symbiosis with a wide range of genera from rhizobia group (Lima et al. 2009). The ability to form nodules in legumes is one of the most widely considered criteria for classifying bacteria as rhizobia (Howieson and Dilworth 2016). Therefore, the presence of nodules in siratro roots, inoculated with the twelve isolates (figures 1 and 2), constitutes an important evidence to consider these microorganisms as rhizobia.

Riz 5-1, Riz 8-11 and Riz 8-12 isolates stood out in all the evaluated variables, both in nodulation and in growth promotion of siratro plants. The ability of strains to nodule the main root is one of the most important criteria in strain selection studies, since nodules located in this position show greater activity in the BNF (Samrudhi et al. 2013). The main amount of nutrients resulting from photosynthesis flows through the main root, which are used by bacteroids to obtain energy to fix nitrogen (Azcón-Bieto and Tolón 2010).

Nodule size is directly related to their mass. The presence of high concentrations of bacteroides induces the multiplication of cells from root cortex, which implies an increase of nodular size (Patriarca et al. 2004). This would explain the significant differences found in the dry mass of nodules of plants inoculated with Riz 8-11 and Riz 8-12 isolates (table 3), independently from similarities in the number of nodules in the main root and total (figures 1 and 2) found between these plants (table 3).

Nitrogen from BNF is used for structural protein synthesis and with a catalytic function, favoring growth and development of legumes (Azcón-Bieto and Tolón 2010). The increase of ADM and RDM of siratro plants inoculated with Riz 5-1, Riz 8-11 and Riz 8-12, isolates that stood out for causing abundant effective nodulation in these plants, suggests the positive effect of the BNF in promoting plant growth. Recent studies with common bean (Phaseolus vulgaris l.) plants, inoculated with different rhizobia strains, showed that the most nodular plants with the highest nodular mass show the highest values of root and aerial dry mass (Ferreira et al. 2018 and Menge et al. 2018).

Rhizobia are considered as plant growth promoting bacteria that, in addition to FNN, have other mechanisms to promote plant growth, such as the synthesis of indole-3-acetic acid (IAA) and gibberellins (Flórez et al. 2017 and Singha et al. 2017). Both hormones favor the formation of lateral roots and increase the number and size of root hairs (Azcón-Bieto and Tolón 2010). The ability of some microorganisms to produce IAA from ammonia (Chandra et al. 2018), which, in the case of rhizobia, would come from BNF, could also contribute to the phyto-stimulating action of these microorganisms. The combined action of these mechanisms would explain the growth promotion produced by the inoculation of rhizobia strains such as Riz 5-1, Riz 8-11 and Riz 8-12 in siratro plants.

Research aimed at finding ecologically viable solutions for productive use of land dedicated to cattle rearing is insufficient in Cuba. Crop productivity in saline areas is limited, fundamentally due to the lack of nitrogen in the soil, which underlies the importance of symbiotic nitrogen fixation in affected areas. This study suggests the use of bacteria from rhizobia group for the inoculation of herbaceous legumes, which naturally establish in saline soils, and would allow the rescue of areas to increase cattle rearing and agricultural productivity in Cuba.


All the results of this study allow to select Riz 5-1 (from Alysicarpus nodules), and Riz 8-11 and Riz 8-12 (from Crotalaria nodules) isolates as promising for the inoculation of herbaceous legumes. These isolates saprophytically survive in soils affected by water deficit and salinity, a quality that makes them attractive for producing bio-preparations that allow the rehabilitation of pastures and forages in these soils.


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Received: February 28, 2020; Accepted: June 22, 2020


Declaración de conflicto de intereses: Los autores declaran no presentar conflicto de intereses

Contribución de los autores: Los autores declaran presentar contribución igualitaria en la concepción de la investigación, obtención y procesamiento de los datos y redacción del documento

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