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Brazilian Journal of Microbiology

Print version ISSN 1517-8382

Braz. J. Microbiol. vol.45 no.2 São Paulo Apr./June 2014 



Microorganisms associated to tomato seedlings growing in saline culture act as osmoprotectant



Daniel Cortés-JiménezII; Abril Gómez-GuzmánI, II; Gabriel IturriagaIII; Ramón SuárezIII; Gisela Montero AlpírezII; Froylán M.E. EscalanteI

ILaboratory of Microbial Processes, Department of Chemistry, Autonomous University of Guadalajara, Zapopan, Jalisco, Mexico
IIDepartment of Engineering, Autonomous University of Baja California, Mexicali, Baja California, Mexico
IIICentro de Investigación en Biotecnología, Universidad Autónoma del Estado de Morelos, Cuernavaca, Morelos, Mexico





Less than 0.5% of total water in the world is available for human consumption and agriculture. The major part of the world's water is saline and salinity in soils interferes in germination of seeds and the posterior development of the plant. In order to increase the osmotolerance of tomato, seedlings were associated with Azospirillum brasilense Cd, Azospirillum brasilense Cd transformed bacteria with a plasmid harboring a trehalose biosynthesis gene-fusion or Chlorella vulgaris. Two plant culture media: Hydroponic and Murashige and Skoog were tested. In the first set of studies seedlings were associated to single free cells meanwhile in a second set single and combined free cells were studied. A positive interaction between transformed Azospirillum and Chlorella vulagris and tomato plants was observed. Seedlings showed a salt concentration tolerance, as sodium chloride, up to 200 mM. According to our results, the association of plants with A. brasilense Cd-BIF and C. vulgaris is a viable approach to increase their salt tolerance and biomass, as consequence the possible use of sea water to irrigate horticultural plants.

Key words: hydroponic culture, microbial association, salt tolerance, Trehalose.




The negative impact of salinity on plant growth in irrigated and non-irrigated areas of the world's arid regions is a major problem for agriculture (Nasr et al., 2011; Meloni et al., 2008; Ríos-Gómez et al., 2010; Velarde et al., 2003). Salinization is a process of soil enrichment with salts more soluble than calcium sulfate, usually chlorides and sulfates of sodium and magnesium. This causes osmotic stress and plant intoxication, thus interfering with the growth of most crops (Porta et al., 1999). Plants develop a plethora of biochemical and molecular mechanisms to cope with salt stress. These processes include compartmentalization of compatible solutes, change in photosynthetic pathway, alteration in membrane structure, induction of antioxidative enzymes and plant hormones and as a consequence plants redirect their growth when exposed to stress (El-Baky et al., 2008). It is hypothesized that such morphogenic responses are part of a general acclimation strategy that constitutes the 'flight' response of plants (Potters et al., 2007). The initial effects of increasing soil salinity are very similar to those observed when plants are exposed to drought. Reductions in leaf water potential will reduce stomatal conductance and eventually inhibit photosynthetic metabolism (Zribi et al., 2009).

Numerous studies have shown that in saline environments, adaptation to salinity during germination and early stages of seedling growth are crucial in determining the success of the establishment. Even in the late stages of development salinity may affect the distribution of plants in certain species (Tobe, 2000).

Salinity tolerance and other adverse conditions in soils are currently overcome with an excess of irrigation water, thus given the increasingly shortage of fresh water at global level, it is necessary to develop strategies that include both salt-resistant crops and techniques to reduce stress injury (Bacilio et al., 2004). A major effort in research has been dedicated to organic fertilization or use of biofertilizers to increase plant development. Biofertilizers are best described as microbial communities that add, preserve and mobilize soil nutrients. Azospirillum is considered one of the most studied plant growth promoting bacteria (PGPB) given its ability to significantly increase not only plant growth development, but also the yield of numerous agricultural crops (Givaudan and Bally, 1991; Strzelczyk et al., 1994). Azospirillum brasilense promotes growth due to the accumulation and transport of indole-3-acetic acid to the plant (Umali-Garcia et al., 1980; Hartmann et al., 1983) and other plant growth regulation molecules such as absicic acid (ABA) and diamine cadaverine (CAD) (Canto Martín et al., 2004; Perrig et al., 2007).

Azospirillum spp. colonizes the roots of a wide range of crops and can efficiently colonize roots submerged in growth solutions, and thus increase plant growth. The ability of Azospirillum spp. to stimulate plant growth has been widely demonstrated in experiments both field and greenhouse. Several mechanisms have been suggested to be responsible for the stimulatory effect observed in the inoculated plants in numerous studies and inoculation with Azospirillum spp.

In the last decade it has been demonstrated that Azospirillum brasilense increases the growth parameters not only of superior plants but unicellular microalgae Chlorella spp. by changing its cytology and metabolic parameters (de Bashan et al., 2004). This phenomena is also associated with the potential of Azospirillium spp. to produce plant growth regulators, because the microalga responds to the presence of Azospirillum similarly to higher plants, increasing their growth and changing its metabolism, due to the green microalgae meet the basic requirements of a higher plant (Mazur et al., 2001; Stirk et al., 2002).

Recently, it has been shown that a recombinant Azospirillum brasilsence Cd strain expressing the omoprotectant trehalose is able to grow in salt stress conditions (Rodríguez-Salazar et al., 2009). The aim of this study is to find out if Chlorella vulgaris, Azospirillum brasilsence Cd and Azospirillum brasilense Cd-BIF, could increase the salt tolerance of tomato in hydroponic cultures.


Material and Methods

Microalgae and bacteria


Chlorella vulgaris (UTEX 2714) was grown in axenic cultures of Basal Bold Medium (Bischoff y Holtzer, 1969) at 2000 lux illumination with light/dark periods of 16/8 h for 14 days prior to its use. The cell concentration was adjusted to 1x106 cells/mL prior to its use as inoculant.


Azospirillum brasilense Cd was grown in nutrient broth with ampicillin at 30 °C for 72 h prior to its use; Azospirillum basilense Cd-BIF which enables the accumulation of trehalose in excess (Rodríguez-Salazar et al., 2009) was grown in nutrient broth supplied with ampicillin and gentamycin at 30 °C for 72 h prior to its use. The viability of the strains was tested on Congo Red agar plates. The cell concentration was adjusted to 1x109 cells/mL prior to its use as inoculant.

In vitro hydroponic culture

Hydroponic media composition

Hydroponic media was modified from (Jensen y Malter, 1995). Composition in g/L: MgSO4.7H2O, 0.5; K2HPO4, 0.27; KNO3, 0.2; K2SO4, 0.1; Ca(NO3)2, 0.5; EDTA, 0.25; 250 µL of trace minerals. Trace minerals composition in g/L: H3BO3, 16.6; MgCl2, 15.0; CuCl2, 0.82; MoO3, 0.33.

Tomato seeds germination

Seeds of Solanum lycopersicum var. cherry were germinated in sterile Petri dishes with hydroponic media for 10 days under light/dark periods of 16/8 h.

Plant growth and development

Tomato plants where placed axenically in plastic conical tubes in groups of five under different sodium chloride concentrations: 0, 50, 100, 150, 200 y 250 mM using hydroponic medium (Jensen y Malter, 1995) or Murashige and Skoog (MS) medium (Murashige y Skoog, 1962) as base. Each group of treatments was supplied with one of the following free cells cultures: AW, AB, CV, AWCV, ABCV, including a control treatment without microorganisms; note: combined microbial treatments were tested only in hydroponic culture in a second set of experiments. Tomato plants were placed in 13.5 mL of the corresponding media and 1.5 mL of the corresponding microbial culture (Table 1). Plant growth and development was monitored after 10, 20 and 30 days of culture; every period stem and root length were measured.

Experimental design and statistical analysis

In order to compare the effect of media composition, time of culture and salt concentration in stem and root elongation, a multifactorial design was used. Data was analyzed using the ANOVA and Least Significant Difference (LSD) analysis at level 0.05 of confidence.



Two sets of experiments were planned. In a first group, two mediums (Hydroponic and Murashige and Skoog), three periods of time (10, 20 and 30 days), six NaCl concentrations (0 to 250 mM) and three single cells A. brasilense Cd (AW), A. brasilense Cd-BIF (AB) and C. vulgaris (CV) were analyzed. In a second group, based on the results of group one, only hydroponic medium was used given its simplicity and microbial associations were tested with single or combined free cells.

Stem length

The ANOVA analysis of the first set of experiments showed that the associated microorganism, elapsed time and salinity were the only significant factors for plant growth, despite of the used medium (MS or hydroponic), (Table 1). It is remarkable that as long as NaCl increased in concentration form 0 to 250 mM the stem length diminished, in any case A. brasilense Cd-BIF promoted longer stems in the seedlings. C. vulgaris had the second better results in this measure (Figure 1a). Regarding salinity, two groups are clearly differentiated, Group I: NaCl concentrations 0, 50 and 100 mM with stem lengths average of 22.7 ± 0.6 mm and Group II: NaCl concentrations 150, 200 and 250 with stem lengths average of 17.8 ± 0.6 mm. When considering time as a second factor, it can be seen that as long as time passed the seedlings were less tolerant to salt, as stem lengths were shorter ~15 and 20 mm in comparison to the first ten days were stem lengths were in the range of 25 to 26 mm (Figure 1b). In general, those seedlings growing with any microorganism associated showed longer stems in comparison to the control, where no microbial association was used, which means that salt tolerance is favored by plant microbial associations.

If the average of stem elongation through the three periods of time is considered, A. brasilense Cd-BIF showed the major elongation in the stems (21.8 ± 0.07 mm) in comparison to the other microbial treatments. However, at the end of 30 days period the stems were shorter than those observed at 10 days, and this was true for almost all cases, except for A. brasilense Cd where the longitudes of the stems (22.3 ± 0.12 mm) were significantly larger (p < 0.05) than other treatments after 30 days (Figure 1b).

For the second set of experiments, where combined microorganisms were tested, according to the ANOVA analysis (Table 2) the combination of A. brasilense Cd and C. vulgaris (AWCV) had a better impact on stem growth (25.3 ± 0.7 mm) followed by the associated A. brasilense Cd-BIF and C. vulgaris (ABCV) (23.3 ± 0.8 mm) (Figure 2a). Longer stems were observed at 20 days in those seedlings associated to A. brasilense Cd and C. vulgaris (AWCV) (29.8 ± 1.3 mm), showing even higher results at NaCl concentrations below 100 mM (up to 30.6 ± 1.7 mm), but as before, in general the tendency of stems was to decrease in size as long as NaCl concentration increased; nonetheless, seedlings associated with ABCV showed longer stems than any other treatment at 250 mM of NaCl (25.7 ± 3.3 mM) (Figure 2b).

Root length

Conversely with stem length, the microbial association had no statistical significant differences (p < 0.05) in root length but growing medium (Table 3). Those seedlings grown in MS medium averaged longer roots (1.29 ± 0.03 mM) in contrast to those seedlings grown in hydroponic medium (1.13 ± 0.03 mM). Time and salinity also were significant factors for root length; though, salinity effects cannot be grouped in two as before (Figure 3a).

Regarding salinity, those seedlings grown in 50 and 100 mM NaCl showed the longer roots, 1.3 ± 0.05 and 1.4 ± 0.05 mm respectively, in comparison with the rest of the treatments. In the first 10 days, it can be observed that 50 and 100 mM of NaCl promoted longer roots and this growth pattern was consistent for the next 10 days but showing shorter roots and this is true until 30 days (Figure 3b).

In the second set of experiments where hydroponic medium was the only one used, time had no statistical effect on root length, but associated microorganisms and salinity (Figure 4). According to the analysis of variance, those seedlings associated with C. vulgaris where different to control having longer roots (12 ± 0.4 mm) (Figure 4a). Even when the effects of NaCl concentration are not differentiated in groups as easily as with stems, in roots is noteworthy the influence of 100 mM of NaCl with longer roots (12.6 ± 0.4 mm).

Concomitantly, AW had a marked effect on root elongation after 30 days (13.2 ± 0.7 mm), similar to that observed for CV at the end of the first 10 days (13.2 ± 0.8 mm) with control showing the shorter roots after 20 and 30 days of treatment (Figure 4b).



Hydroponic culture of plants with sea water or even salted water and the use of biofertilizers is a promising technology in order to mitigate the salinity effect on food crops. In the present work we planned to analyze the effects of Azospirillum brasilense Cd wild type, A. brasilense Cd-BIF able to over accumulate the osmoprotectant trehalose and Chlorella vulgaris in the growth of tomato seedlings using two different culture media and in microbial association.

According to the observed results, Azospirillum basilense Cd-BIF followed by Chlorella vulgaris showed the better influence in seedlings promoting longer stems, with good results even at 250 mM of NaCl. Given that microbial associations showed in general better results for stem and roots elongation in tomato seedlings, this could imply that these plants could be irrigated with sea water at least partially when associated to any of this microorganisms. Seemingly, if the concentration of NaCl is increased for those seedlings growing in hydroponic medium, the effect of A. brasilense Cd-BIF will be better; meanwhile the triple association of A. brasilense Cd, C. vulgaris plus seedlings appears to be sensitive to NaCl increments. These results are in agreement with those works reported previously about Azospirillum brasilense growing up to 200 mM of NaCl, where no drop in bacterial growth rate was observed, but when 300 mM of NaCl was used, the growth rate diminished in 66% (Rivarola et al., 1998). In our results, A. brasilense Cd showed almost the same growing behavior, however we assume that A. brasilense Cd-BIF could grow with no problems even up to 300 mM.

In previous works with plants of Zea mayz under osmotic stress, Rodríguez-Salazar et al. (2009) observed an increased biomass, namely thicker roots, of those plants grown in association with A. brasilense Cd-BIF. Other works with maize and wheat report an osmoadaptative phenomena for those plant cells associated with A. brasilense (El-Baky et al., 2008)(2, 16). Some other examples in wheat and barley suggest that the association plant-microorganism are beneficial for plant's salt tolerance due to an increase in its growing capacity (Caballero-Mellado, 2006; Zawoznik et al., 2011).

On the other hand, Hiremath and Mathad et al. (2010) demonstrated that Chlorella vulgaris was positively stimulated by up to 200 mM of NaCl for chlorophyll and proline production, but when salinity concentration was increased up to 300 mM a reduction on chlorophyll was observed. Díaz et al. (1999)) reported that proline accumulates in plants in response to increased environmental salinity. These supports the evidence showed here regarding C. vulgaris and its effect on plant tolerance as mentioned above, where C. vulgaris had the second marked effect on salt tolerance over the seedlings of tomato.

In general, stems and roots lengths decreased with time, despite of the applied treatment and this could be a cause of the prolonged immersion time of the seedlings in the nutritive solutions, thus is advisable to add some kind of support for the plant. This is why for a future work we are planning to evaluate the effect of these microbial species in seedlings growing in alginate beads supports. As our results suggests, the use of A. brasilense Cd-BIF and C. vulgaris is a viable approach to increase the salt tolerance in plants and their biomass and the possible use of sea water to irrigate horticultural plants.



The main authors wish to thank the Doctoral fellowship 162578 by CONACYT to Daniel Cortés and Abril Gómez, respectively. We express our gratitude to Virginia Berenice Suarez for her English correction proof.



AW, Azospirillum brasilense Cd (wild type)

AB, Azospirillum brasilense Cd with plasmid pBBR1M:BIF

PGPB, Plant Growth Promoting Bacteria

CV, Chlorella vulgaris



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F.M.E. Escalante
Laboratory of Microbial Processes
Department of Chemistry
Autonomous University of Guadalajara
Av. Patria 1201, Lomas del Valle, Zapopan, 45110
Jalisco, Mexico

Submitted: Junho 10, 2013
Approved: September 9, 2013



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