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Revista mexicana de ciencias agrícolas

versão impressa ISSN 2007-0934

Rev. Mex. Cienc. Agríc vol.7 no.4 Texcoco Mai./Jun. 2016



Mitigating the effect of chloride salts and sulfáticas germination of Salicornia bigelovii (Torr.) by beneficial bacteria in vitro

Ma. Magdalena Hernández Perales1 

Rodolfo Cisneros Almazán1 

Jesús Ortega-García2 

Cándido Márquez Hernández3 

Juan José Reyes-Pérez4 

Bernardo Murillo-Amador5 

Luis G. Hernández Montiel4 

Alejandra Nieto-Garibay5 

Edgar O. Rueda-Puente2  § 

1 Universidad Autónoma de San Luis Potosí-Departamento de Biotecnología. Álvaro Obregón 64, Centro Histórico, 78000 San Luis Potosí. México. (;

2 Universidad de Sonora, Departamento de Agricultura y Ganadería. Boulevard Luis Encinas y Rosales, Hermosillo, Sonora, México. (jortega@

3 Universidad Juárez del Estado de Durango. Colonia Filadelfia, Gómez Palacio, Durango. México. 35056. (

4 Technical University of Cotopaxi, Ecuador. Av. Simón Rodríguez s/n Barrio El Ejido Sector San Felipe. Latacunga-Ecuador. (;

5 Centro de Investigaciones Biológicas del Noroeste S. C. CIBNOR. Av. Instituto Politécnico Nacional 195, Playa Palo de Santa Rita Sur; La Paz, B C S, México, C. P. 23096. (;


In Mexico 30% of its agricultural land is affected by salinity. This affects exposed to conventional agriculture based glycophyte not be profitable and productive. In the interest of expanding knowledge with halophytes of agroindustrial interest and to propose them to the productive sector presents problems of salts, this study under conditions of hydrochloric salinity and induced sulfate developed, evaluated two genotypes of Salicornia bigelovii in the stages of germination and development (improved genotype SOS-7 and wild genotype Bay Kino= BK=), developing in salinity conditions: two sodium chloride (NaCl) at 0.5 molar and 1 molar and two calcium sulfate (Ca2SO4+2H2O) to 0.5 Molar and 1 Molar, with inoculation of two plant growth promoting bacteria (PGPR) (Azospirillum halopraeferens= Ah and Klebsiella pneumoniae= Kp) and an uninoculated control. The results indicate that when evaluating Salicornia in saline conditions sulfate and hydrochloric inoculation with BPCV, including the results include a greater percentage of germination was observed in the presence of BPCV, excelling those inoculated with Kp. It was observed that as the salinity increases, Kp presents be more effective with wild genotype BK. Studies concerning as possible bio-fertilizers are needed in different phenological stages with genotypes studied, who for the germination stage, turned out to have expectations.

Keywords: Salicornia bigelovii; Azospirillum halopraeferens; Klebsiella pneumoniae; germination; genotypes


En México 30% de su superficie agrícola está afectada por la salinidad. Lo anterior expuesto repercute para que una agricultura convencional a base de glicófitas, no sea redituable y productiva. Con el interés de ampliar el conocimiento con halófitas de interés agroindustrial y poder proponerlas al sector productivo que presenta problemas de sales, se desarrolló el presente estudio bajo condiciones de salinidad clorhídrica y sulfática inducida, evaluando dos genotipos de Salicornia bigelovii en las etapas de germinación y desarrollo (genotipo mejorado SOS-7 y genotipo silvestre Bahía de Kino= BK=), desarrollándola en condiciones de salinidad: dos de cloruro de sodio (NaCl) a 0.5 molar y a 1 molar y dos de sulfato de calcio (Ca2SO4+2H2O) a 0.5 Molar y a 1 Molar, con la inoculación de dos bacterias promotoras del crecimiento vegetal (BPCV) (Azospirillum halopraeferens= Ah y Klebsiella pneumoniae= Kp) y un control sin inocular. Los resultados indican que al evaluar a Salicornia en condiciones salinas clorhídricas y sulfáticas con la inoculación de BPCV, entre los resultados obtenidos figuran que un porcentaje mayor de germinación fue observado en presencia de las BPCV, sobresaliendo aquellos inoculados con Kp. Se observó, que conforme la salinidad incrementa, Kp presenta tener mayor efectividad con el genotipo silvestre BK. Estudios concernientes como posibles biofertilizantes son necesarios en las diferentes etapas fenológicas con los genotipos en estudio, quienes para la etapa de germinación, resultaron tener expectativas.

Palabras clave: Salicornia bigelovii; Azospirillum halopraeferens; Klebsiella pneumoniae; germinación; genotipos


The areas considered to have high agricultural potential aridity in 43% of the total area of the world (Rueda et al., 2003). In Mexico solonchaks soils are combined with calcisols and it is estimated that around 1.7 million hectares of the 6.2 million ha of irrigation problems presented average high salinity. Also, these areas are characterized by large quantity of salts dissolved carbonate type, sulphates and gypsum at a depth of 8 to 10 mm in the soil, and water wells for agricultural use, with high salt content (SEMARNAT, 1999). These soils are located in the Chihuahuan Desert and in the states of Aguascalientes, Baja California, Baja California Sur, Chihuahua, Coahuila, Durango, Nuevo Leon, Sonora, Zacatecas and San Luis Potosi (INEGI, 2013). In the state of San Luis Potosi, Mexico, there are problems areas with medium to high salinity (4-8 g L of water), a situation that has affected the productivity of conventional farming.

With the presence of these salts the establishment of traditional crops is limited by decreasing their yields, which are required to supply the food needs of the population, with plants that tolerate high salt concentrations of both soil and water, and thus incorporate agricultural or low production harvestable crop development systems land.

An alternative may be the halophytes, which grow naturally in saline arid environments. A representative genus with exploitable potential is Salicornia bigelovii, which has been proposed as an alternative for agro-arid areas and use of brackish water. However, to develop it as a monoculture requires a nitrogenous chemical application ranging from 1 000-1 500 kg distributed in three stages of phenology; these high applications of chemical fertilizers bring high salinization due to agricultural soils in arid areas such as the area of San Luis Potosi state (Martinez, 1996). An alternative solution to the above plan is the application of promoting bacteria plant growth, which have the particularity to fix atmospheric nitrogen in addition to produce and / or act as inducers in the synthesis of plant hormones, which include auxin and gibberellins (AG3) that promote germination, f lowering and fruiting plants. In order to expand the knowledge of the behavior of Salicornia bigelovii with the interaction of beneficial bacteria under saline conditions set out to evaluate the effect of inoculation of Azospirillum halopraeferens and Klebsiella pneumoniae and an uninoculated control in two genotypes of Salicornia bigelovii (improved genotype SOS-7 and wild genotype Bahia Kino= BK=) in the stages of germination and early development of seedlings developing it in saline conditions: sodium chloride (NaCl) and calcium sulfate (Ca2SO4+2H2O) to 0.5 molar and 1 molar. The variables evaluated were: percentage and germination rate, height of seedling.


This work was conducted in two phenological stages: germination and early seedling greenhouses agroindustrial area of the faculty of engineering of the Autonomous University of San Luis Potosi (UASLP).

Germination stage

Collection and seed selection. First seeds mature plants of two genotypes of Salicornia bigelovii were collected (wild and improved); wild develops naturally in the Estero Bay Kino (BK) between the parallel north latitude 31° 18' 41.64 " west longitude, latitude north 113° 33' 31.88" west longitude and improved corresponds to the variety SOS-7, which it was provided by the salinity laboratory of the department of agriculture of the University of Sonora.

Sanitization seed. Prior to the selection of seeds, in the case of wild genotype, seed collection was selecting ripe and dried plants of the above populations. Then the seeds were sieved using a mesh 2 mm in diameter. The larger seeds were selected and uniformly colored without apparent damage based on size and appearance. In the first, considering a size 2 mm using spatulas and separation in appearance, those that showed a uniform genotype of each color.

In the present study, a statistical design was applied a completely random arrangement trifactorial (2*4*3): factor A: two genotypes; factor B: saline concentrations: two sodium chloride (NaCl) at 0.5 molar and 1 molar and two calcium (Ca2SO4+2H2O) sulfate 0.5 molar and 1 molar, factor C: two bacteria (Azospirillum halopraeferens (Ah) and Klebsiella pneumoniae (Kp) and a control uninoculated. This causes 24 treatments, five replications of 50 seeds each, were considered, which shows a total of 6 000 experimental units.

Determining seed viability. They were deposited in containers with drinking water for an hour and discard those that floated. They were then disinfected in a solution of sodium hypochlorite 3% for three minutes and then rinsed with sterile distilled water three times to remove excess hypochlorite (Carrillo et al., 1998; Rueda et al., 2003).

Development of bacteria Azospirillum halopraeferens and Klebsiella pneumoniae. For growth of the microorganisms under study: Azospirillum halopraeferens and Klebsiella pneumoniae was performed using a bacterial culture medium called "RENNIE" (Rennie, 1981), which does not present an enriched nitrogen source. This culture was performed using a one liter flask, then 1 ml of the bacteria deposited under study (each separately) and allowed to proceed time 16 h bacterial kinetic growth as this is the limit for bacterial cells mature. After 10 ml of each of the bacteria individually to a 20 ml test tube were transferred and centrifuged at 4 500 revolutions per minute (rpm) for 10 minutes, the bacterial pellet was removed and added to 10 ml of saline 0.85% to resuspend the cell pellet (bacterial pellet). The button is resuspended and the optical density at 540 nm was measured using a spectrophotometer Roy Company Spectronic mark 20 D Milton. The optical density reading was considered one, which indicates that the sample of the inoculum is about 109 colony forming units (UFC/ml). Then 1 ml of the adjusted inoculum was transferred to a test tube with 9 ml of 0.85% saline to obtain a dilution of 107 UFC/ml. (Carrillo et al., 1998).

Inoculating seed. The inoculation of the seeds by Klebsiella and Azospirillum bacteria was carried out using the technique called "vacuum" as described by Carrillo et al. (1998), which consists of using a pump Gast brand, model 5KH33GN293KX. The individual groups of 100 seeds of each genotype, after 24 hours after disinfection were placed in a 500 ml flask, then 100 ml of media enriched with bacteria at a concentration of 107 UFC/ml was added. The flasks seeded and bacterial medium subjected to the action of vacuum (600 mmHg) for 5 min. Each seed groups (100) of their respective treatment (genotype-bacteria-salinity) were deposited in Petri dishes previously sterilized presenting sponge support.

Conditions in vitro. The petri dishes were placed in an incubator (Mca. Shel Lab model 1380 FM) at a temperature of 25 °C and a relative humidity of 35% for 15 days; each of the boxes of each genotype was irrigated in initially with an amount of 25 ml daily during the course of the study (considering the type of saline: sodium chloride (NaCl) at 0.5 molar and 1 molar and calcium sulfate (Ca2SO4+2H2O) 0.5 molar and 1 molar, respectively). Germination was under continuous darkness.

Variables evaluated in vitro. The study variables were: percentage and germination rate; to obtain this information the Petri dishes were examined every other day until the end of the study. The root length and seedling height were measured with a vernier brand JONSHON; for the number of bacteria adhering to the root five seedlings per repetition were taken and were stirred for 10 s in test tubes in a sterile saline 0.85% NaCl. 0.1 ml of the suspension was taken and planted quadrant streaking in Petri dishes with bacteria culture medium "Rennie". They were incubated for 36 h at a temperature of 28 °C. Later, they were counted with a manual counter the colonies of bacteria. The fresh weight was weighed on an analytical balance Mettler-Toledo GmbH, recital 5 seedlings per repetition; these were finally dried in a drying chamber (Mca. Shel Lab model 1380 FM) at 80 °C to take your weight again.

A completely randomized statistical design with 16 treatments and five replications each was used in this study. Using this same design, analysis of variance transforming previously germination percentage percentage values arcsin (Sokal and Rohf l, 1988) were performed. The germination rate, which is the sum of germinated seeds in a time was counted daily for the first four days, and from the same (4th day), readings were taken every third day. The germination rate was also analyzed. The least significant difference between treatment means, of the variables (percent and germination rate, height, root length, fresh weight of seedling, seedling dry weight and number of bacterial cells adhering to the root), were evaluated by the multiple range test of duncan 0.05%. Data were analyzed using statistical computer program SAS (SAS, 2001).

Results and discussion

According to the methodology referred above, the results shown below.

Germination of genotypes of Salicornia bigelvii (Bahia de Kino= BK= and SOS-7) with inoculation of Azospirillum halopraeferens (Ah) and Klebsiella pneumoniae (Kp) under conditions of salinity (0.5 and 1 M sodium chloride= NaCl= and calcium sulfate= Ca2SO4+2H2O.

The results obtained by making use of a salt chloride of sodium (0.5 M) on the two genotypes of Salicornia bigelovii (BK and SOS-7) with the inoculation of bacteria, the SOS-7 genotype showed the best germination percentage (91.3%) inoculated with Kp, following him in the background, the same genotype SOS-7 with 82.9 inoculated with Ah (Figure 1); similar behavior to the wild genotype was observed (BK), that inoculated with protruding Kp compared Ah (79.2 against 77.6%, respectively). Regarding the use of calcium sulphate (0.5 of Ca2SO4+2H2O), SOS-7 showed values of 80.1 against 79.3, inoculated with Kp and Ah, respectively. While BK ecotype was lower compared SOS-7, yielding values of 69.1 and 68.3, both inoculated with Kp and ah, respectively. Meanwhile uninoculated ecotypes in both saline solutions showed the lowest values (Figure 1).

Figure 1 Percentage of germination of Salicornia bigelovii genotypes (wild material Kino Bay= BK and improved material SOS- 7), inoculated with bacteria Klebsiella pneumoniae= kp= y Ah= Azospirillum halopraeferens to 0.5 and 1 M of NaCl and 0.5 and 1 Ca2SO4+2H2O. The letters indicate significant differences (p< 0.005). 

To a salinity of 1 M of NaCl, genotype uninoculated BK, reduced the percentage of germination up to 75% compared to 0.5M. However, being inoculated with beneficial bacteria, the germination percentage is even reduced by 50% (48.2% with 45.3K pand with Ah). With regard to vegetative material SOS-7, 1 M of NaCl, who was not inoculated was significantly affected germination, achieving only 10% germination. However, to be innoculated percentage of germination was favored by 55.3 and 57.2 with Ah and Kp, respectively. The same genotype BK 1M of CaSO4 (inoculated with Kp and Ah) reduced the percentage of germination by 55%, compared with 0.5 M of Ca2SO4+2H2O. However, SOS-7 uninoculated was affected up to 74% less (18%).

Regarding SOS-7, subjected to 1 M of Ca2SO4+2H2O, there were no significant differences between SOS-7 inoculated with Kp and Ah (35.1 against 34.2), respectively. SOS-7 uninoculated, reduced germination up to 60% less (11%) compared with the inoculated Kp and Ah. The results of inoculation match Bashan and Holguin (1997); Meza et al. (2015), which indicate the use of bacterium induces germination because phytohormone synthesis released. Another factor observed in this study and which other research glisten (Besnier, 1988; Camacho, 1994), is the germination inhibition effect of some abiotic factors such as temperature, humidity, light and salinity. The latter is an important factor that did not allow the genotypes in study Salicornia bigelovii germinate properly as salinity increased (Allison et al., 1980; Ungar, 2000).

Germination rate of genotypes of Salicornia bigelovii (Bahia Kino= BK= and SOS-7) with the inoculation of bacteria (Azospirillum halopraeferens (Ah) and Klebsiella pneumoniae (Kp)) to 0.5 M of NaCl and Ca2SO4+2H2O.

The behavior of the different treatments on the germination rate at a salinity of 0.5 M of NaCl, the KB genotype inoculated with Kp, behaved significantly higher compared with the same genotype inoculated with Ah and SOS-7 with two inoculants as the tenth day reached its highest percentage (79.2%). However, it is important to note that the maximum percentage of germination was achieved by SOS-7 inoculated with Kp (91.3%) at 15 days after planting. Meanwhile genotypes (BK and SOS-7) uninoculated, where germination rate showed no favored dynamic, initiating the germination process on the sixth day.

Regarding the germination rate at a salinity of 0.5 M of CaSO4, the behavior was similar to 0.5 M of NaCl; i.e. the BK genotype inoculated with Kp showed an initial germination on the fourth day and ending at the twelfth day to 69.1%. For its part inoculated BK with Ah, she started sprouting on the fifth day, totaling 68.3% with no significant difference with BK + Kp. Notwithstanding the foregoing, even when SOS-7, in its germination rate has not been observed favored the eighth day showed a final germination of 80.1 by Kp against 79.3 inoculated with Ah, not having shown statistical differences between these two treatments. Those dynamically uninoculated treatments showed not favored in the germination rate.

Germination rate of genotypes of Salicornia bigelovii (Bahia Kino= BK= and SOS-7) with the inoculation of bacteria (Azospirillum halopraeferens (Ah) and Klebsiella pneumoniae (Kp) to 1 M of NaCl and Ca2SO4+2H2O.

According to the germination rate of both genotypes (Bahia de Kino= BK= and improved genotype SOS-7), subject to a salinity of 1 M of NaCl and CaSO4 and inoculated with Ah and Kp, wild genotype SOS-7+Kp , behaved higher germination rate with 57.2% of germinated seeds 12 days after sowing in the NaCl solution compared the remaining genotypes. The SOS-7+Ah, genotype showed maximum germination percentage in the 13 days to 1 M of NaCl. Moreover, this same salinity (1 M of NaCl), treatments with genotype BK, inoculated with Kp and Ah, initiated germination on the ninth day and finalizing on the fourteenth day with a 48.2 and 45.3%, respectively. The uninoculated treatments showed dynamically lower compared to those inoculated. With regard to the dynamics of germination 1M solution Ca2SO4+2H2O, it was similar to those treatments to 1 M of NaCl, but with significant results in the final percentage of germination behavior. In salinities applied to the genotypes of Salicornia bigelovii clearly seen where bacteria promoted seed germination. According to Lira (2003), it indicates that hormones, specifically gibberellins and auxins in germination are synthesized in seeds; coupled with the production of the same by bacteria, the effect on the rate and % of germination is intrinsically expressed as the rate of cell division increases (mitosis) (Fallik et al., 2000).

Seedling height and root length of Salicornia bigelovii genotypes (Kino Bay= BK= and SOS-7), inoculated with bacteria Klebsiella pneumoniae and Azospirillum halopraeferens 0.5 and 1 M of NaCl and Ca2SO4+2H2O.

The height of the genotypes studied at a salinity of 0.5 M of NaCl, BK+Kp showed the highest values (38.2 mm) compared BK+Ah, SOS-7+Ah, SOS-7-Kp with 32.3, 35.4 and 35.7 mm, respectively. A similar behavior was observed with Ca2SO4+2H2O to the 0.5 M solution; there were no significant differences, however, the highest numerical values were genotyped for the BK+Kp with 33.2 mm compared BK+Ah, SOS-7+Ah, SOS-7-Kp with 29.3, 30.4 and 31.4, respectively . To this salt concentration, there were no significant differences between the type of salt; But not this, was identified genotypes in solving Ca2SO4+2H2O numerical values are lower compared NaCl. The uninoculated treatments showed 25% less in the variable height 0.5M conditions both salts, compared to those inoculated.

Meanwhile, the same variable height, at a salinity of 1 M of NaCl, genotype BK+Kp showed the highest values (18.1 mm) compared BK+Ah, SOS-7+Ah, SOS-7-Kp with 12.2, 15.3 and 15.4, respectively. A similar behavior was observed with Ca2SO4+2H2O solution 1M with no significant differences, however, higher values were genotyped for the BK+Kp with 13.1 mm compared BK+Ah, SOS-7+Ah, SOS-7-Kp with 9.1, 10.4 and 11.4, respectively. Just as in the concentration of this salt concentration 0.5M, there was no significant difference between the types of salt; but not this, was identified genotypes in solving Ca2SO4+2H2O, numerical values were lower compared NaCl. The uninoculated treatments were lower in height variable conditions 1M both salts. With regard to root length, SOS- 7+Ah (62.9 mm) genotype showed significant higher values compared to SOS-7+Kp and SOS-7 (44.12 and 41.09 mm respectively) at 0.5 M of NaCl, without significant differences between solutions (SOS-7+Ah (59.7 mm) revealed significant higher values compared to SOS-7+Kp and SOS-7 (39.11 and 38.02 mm, respectively). with regard to the concentration of 1 M of NaCl and Ca2SO4+2H2O to 1M, treatment with BK+Kp was the one that presented greater root length (35.8 mm) compared BK+Ah, SOS-7-Kp and SOS-7+Ah, with 15.1, 12.7 and 11.2 mm respectively. the uninoculated treatments showed similar numerically to SOS-7+Ah treatment 11.01 and 11.00 mm (BK and SOS-7, respectively). the previously mentioned values generated significant results with p< 0.05 between salt concentrations (0.5 with 1M).

According to the results, genotypes as SOS BK-7 and the Ah and Kp bacteria can clearly see the effect on height growth and root length were taken into the salt concentrations. These findings by the effect of bacteria correspond to that in other studies, which show that auxins, cytokinins and gibberellins produced by microorganisms, as well as inducing them in vegetative organs such as seeds, exhibit strong regulating properties growth and each with its particular structure and active at very low concentrations in the plant (Bashan et al., 2000; Lira, 2003; Rueda et al., 2004).

Cool and dry weight seedling genotypes of Salicornia bigelovii (Bahia Kino= BK= and SOS-7), inoculated with bacteria (Klebsiella pneumoniae and Azospirillum halopraeferens) at 0.5 and 1 M of NaCl and Ca2SO4+2H2O.

In the variables fresh weight, considering the treatments with inoculants inoculants 0.5 M of NaCl, genotype presented the highest values was the BK+Kp, with an average of 0.0335 mg, followed in second order SOS-7+Kp with 0.0329 mg. Those inoculated with Ah, were numerically lower than those inoculated with Kb, without significant differences. Meanwhile treatments without inoculants 0.5 M of NaCl, genotype presented the highest values was the BK, with an average of 0.0242 mg, followed in second order SOS-7. The results show no significant differences between inoculated and uninoculated treatments. Relative to 0.5M of Ca2SO4+2H2O solution, the results show no significant difference between the type of salt (NaCl against Ca2SO4+2H2O) to 0.5M. Low and values presented for those not inoculated (Bk and SOS-7 with 0.0191 and 0.0188 mg respectively).

Also, the concentration of 1 M of NaCl against Ca2SO4+2H2O, between treatments inoculated bacteria with Kp, similarly Bk continued to behave superior compared to the other genotypes (fresh weight= 0.0274 mg, dry weight= 0.0032 mg). Regarding Ah inoculation, the levels obtained were fresh weight for SOS-7 genotype, while for higher dry weight was again Bk.


Salicornia bigelovii a halophyte that develops off the coast of Baja California Sur and Sonora in sight for the immediate future as an option to contribute to the agricultural economy in states with salinity problems and a potential positive impact on regional development of the sector agricultural. Although states like San Luis Potosi not develop naturally this halophyte, soils rich in salts such as chlorides and sulfates and brackish waters above 6 g of dissolved solids, allow agriculture with halophytes agroindustrial importance, they can be implemented in the agricultural sector. The results obtained in this research contribute to expand knowledge on possible alternative agricultural production and effects on the application of biofertilizers in new plant materials with productive potential of socio-economic interest to states with problems of availability of good quality water, as is the state of San Luis Potosi. The results suggest the feasibility of that in the germination stage, the use of plant growth promoting bacteria, it contributes significantly to the germination of Salicornia bigelovii genotypes. Also, the type of salt and concentration thereof, affects the germination. However, it evaluated phenological stage is mitigated when the genotypes studied (Bahia de Kino and SOS-7) Salicornia bigelovii, are inoculated with Klebsiella pneumoniae and Azospirillum halopraeferens. Notwithstanding the foregoing, as studies concerning possible bio-fertilizers, they are necessary in subsequent different phenological stages with both genotypes, who for this phenological stage, turned out to have expectations.

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Received: January 2016; Accepted: March 2016

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