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Introduction
Gluconacetobacter diazotrophicus is an endophytic bacterium belonging to the phylum Proteobacteria, section Alpha, order Rhodospirillales and family Acetobactereceae. It has great attractions for the preparation of biofertilizers due to its ability to fix atmospheric nitrogen, produce indoleacetic acid (IAA), solubilize mineral nutrients and exhibit antagonistic activity against phytopathogenic fungi (Reis et al., 2015). Also, the positive results obtained by the inoculation on graminaceous plants (Kumarasamy & Santhaguru, 2011; Indi et al., 2014; Patil et al., 2011, Hernández-Escareño et al., 2015) and tropical foods (Dibut et al., 2011) were interesting in this way.
Different culture media used for the growth of this bacterial species are described. The liquid medium LGIM was used by Molinari & Bioardi (2013) to evaluate the production of biomass and levana. On the other hand, Dibut et al. (2011) obtained a biopreparate for agricultural use in the SG medium. The liquid DYGS medium was used to determine the influence of gluconic acid on the antagonistic activity of the microorganism (Nieto-Peñalver et al., 2014), as well as to investigate aspects related to the endophytic colonization process (Alquéres et al., 2013). Although the culture medium used for the bacterial growth, can exert effects on its metabolism, no research was found in the consulted literature where the effect as plant growth promoting bacteria for this species is demonstrated. This aspect has great importance for the biotechnological application of G. diazotrophicus. It is also decisive for the use of this microorganism in the bioproducts obtainment for stimulate the plant growth in species of economic interest, since the effectiveness of these will depend on the composition of the medium.
The objective of this work was to determine the influence of the culture media used for the growth of G. diazotrophicus in its potential as a bacterium that promotes plant growth in vitro and in vivo.
Material and Methods
The research was conducted during 2012-2014 period
Bacterial strain and culture media
The INIFAT Gd-42 strain of Gluconacetobacter diazotrophicus conserved in the Collection of Beneficial Bacteria of the Fundamental Research Institute for Tropical Agriculture Alejandro de Humboldt (INIFAT) (Ríos et al., 2016b) was used. The microorganism was grown under fermentation conditions in an orbital shaker at 150 rpm, at 28 ± 2 °C of temperature. In the study, five culture media previously used in the study of this bacterium were used: LGI (Cavalcante & Döbereiner, 1998); SG (Döbereiner et al., 1993); SYP (Caballero-Mellado & Martínez-Romero, 1994); a modification of the DYGS medium (Siqueira et al., 2009) and the MA medium (Ortega, 2012) (Table 1). All were prepared in 500 mL Erlenmeyer flasks containing 250 mL of effective volume, which were inoculated with the pure strain. The growth of the microorganism was evaluated by counting colonies on solid medium and calculating the colony forming units (CFU mL-1) (Madigan et al., 2014). The samples were taken every two hours during the first 12 hours. Since that time, the samples were taken every four hours until the end of the 48 hours of fermentation.
Table 1 Media composition used in the study (each liter).
| LGI | SG | SYP | DYGS | MA |
|---|---|---|---|---|
| Sucrose 50 g | Glycerol 10 mL | Sucrose 10 g | Sucrose 10 g | Sucrose 50 g |
| Sugar 50 g | ||||
| Triptone 5.0 | Bacteriological Peptone 2g | |||
| Glutamic acid 1.5 | Rice cooked extract 50 g | |||
| Yeast extract 0.03 | Yeast extract 1 | Yeast extract 3 g | Yeast extract 3 g | Yeast extract 5 g |
| K2HPO4 0.1 | K2HPO4 0,1 | K2HPO4 1g | MgSO4 1.5 g | |
| KH2PO4 0.3 | KH2PO4 0,3 | KH2PO4 EG | MgSO4 1.5 g | |
| CaCl22H2O 0.01 | MgSO4 7H20 | KH2PO4 3g | K2HPO4 1.0 g | |
| MgSO47H2O 0.1 | 0.1 | KH2PO4 3g | ||
| Na2MoO4 2H2O | CaCl2 2H|2O | |||
| 0.001 | 0,001 | |||
| FeCl3 6H2O 0.005 | Na2MoO4 2H2O | |||
| 0.001 | ||||
| FeCl3 6H2O | ||||
| 0.05 | ||||
| pH 5.5 | pH 6.0 | pH 6.0 | pH 6.0 | |
| Cavalcante & Döbereiner, 1998 | Döbereiner et al., 1993 | Caballero-Mellado & Martínez-Romero, 1994 | Siqueira et al., 2009 | Ortega, 2012 |
In vitro evaluation of nitrogen fixing and phos-phorus solubilization activity
Once the bacteria had grown in the five culture media, their capacity to fix atmospheric nitrogen and to solubilize phosphorus was evaluated under in vitro conditions. For the first case, a qualitative criteria was used (Pérez et al., 2014), based on the growth of the microorganism during five successive inoculations performed by puncture in the semisolid LGI medium without combined nitrogen (Cavalcante & Döbereiner, 1998). The solubilization of phosphorus was evaluated from the subtraction between the halo of solubilization and the grouth halo of the colony formed in the NBRIP medium (Nautiyal, 1999) at 72 hours of incubation. In both determinations the bacteria was maintained at temperature of 28 ± 2 °C and three repetitions of each one were made.
Evaluation of the plant growth promotion activity
To know the effect of the culture medium on the stimulation effect of G. diazotrophicus in vivo conditions, a greenhouse experiment was carried out. 2 kg capacity pots were used, which were filled with Ferralitic Red soil (Hernández et al., 2015) without sterilization, characterized by having 2.85 % organic matter, pH 7.3, 2.09 ppm assimilable phosphorus and Ca2+, Mg2+, Na+ and K+ content of 19.1; 4.2; 0.11 and 0.38 c.mol.kg soil-1.
Unsterilized seeds of carrot (Daucus carota L.) cultivar New Kuroda and sugar beet (Beta vulgaris L.) cultivar Detroit Red were used. For the production of the bioproducts, G. diazotrophicus was cultivated in five culture media separately under submerged fermentation conditions in shaker orbital at 150 rpm and 28 ± 2 °C of temperature. Each culture was suspended in common water at 10 %, obtaining a final bacterial density of 108 CFU.mL-1. The final solution was applied by spraying the soil (10 mL per pot), after germination of the seed for both crops. The germination is considering as the emergence of the plant above the soil surface, which corresponded to a period of 15 days after sowing. At 75 days for sugar beet and 85 for carrot, several growth indicators were evaluated. The indicators were: length (cm) and diameter (cm) of the root, the widest part of the root (1 cm from the apex), the number of leaves, the height of the plants (cm), the fresh weight of the plant (g). For the first two indicators an instrument of 0.01 mm error was used, while in the height measurement of the plant a graduated rule was used and it was measured from the apex of the root to the beginning of the leaves for both plant species. The fresh weight was quantified with a semi-analytical balance (0.01 g of error). Twenty plants were used per treatment (culture medium). A control was added with the same number of plants, where only common water was applied.
Statistical tests
The obtained results in each experiment were averaged with the Microsoft Office Excel program on Windows, which was also used to prepare the graphs and to calculate the standard deviation of the mean. The statistical processing was carried out using the STATGRAPHICS version 5.0 program, which also verified the normality and homogeneity of the variances of the treatments, according to Kolmogorov-Smirnov, Cochran, Hartley and Bartlett tests. A completely randomized design was used. An Analysis of Variance (ANOVA) was performed with a Duncan test at 5 % significance.
Results
The establishment of several stages in the five culture media used is highlighted when analyzing the growth curves of the INIFAT Gd-42 strain of G. diazotrophicus (Figure 1). This behavior corresponds to a diauxic growth. The first is framed between 0 and 10 h, the second between 10 and 16 h, the third, between 16 and 24 h, the fourth between 24 and 36 hours and the last from 36 to 48 h. The last stage corresponds to the stationary phase of growth. In the previous ones, the typical characteristics of a diauxic growth can be observed, where there are two lag phases followed by their corresponding exponential phase. The concentration values of the microorganism are in the order of 1012 CFU.mL-1, and did not show significant statistical differences among the studied media when completing the 48 hours of the fermentation process.
Figure 1 Behavior of the concentration of G. diazotrophicus strain INIFAT Gd-42 growing in five culture media. Hour 48: Esx: 0.386189; CV (%): 5.08.
Although the bacterial concentration remained stable, during the study their characteristics were checked, both in vitro conditions and the effect that the application of the final fermentation product on carrots and beets in vivo, related with the culture medium used for the growth of the strain. The microorganism kept the growth during five successive inoculations in semi-solid culture medium without combined nitrogen. This result suggests that the bacterium maintained its capacity to fix atmospheric nitrogen. On the other hand, the solubilization halo produced in the NBRIP solid medium was greater when the microorganism grew previously in the SYP and MA media (Table 2), which indicates that the medium used for the growth of the microorganism affects its ability to solubilize phosphorus.
Table 2 Phosphorous solubilization in NBRIP medium by G. diazotrophicus strain INIFAT Gd-42 grew in different culture media.
| Culture media | Solubilization halo (cm) |
|---|---|
| LGI | 0.10 b |
| SYP | 0.16 a |
| DYGS | 0.09 b |
| SG | 0.11 b |
| MA | 0.16 a |
| Esx | 0.0059 |
| CV (%) | 32.49 |
Note: Different letters indicate statistically differences according ANOVA and Duncan’s test (5 %).
Differences were observed in the stimulation of the growth of the two horticultural crops studied under in vivo conditions, depending on the culture medium where the microorganism grew during the fermentation process. In the case of carrots, there are differences in the variables measured with the different treatments (Table 3). The strain of G. diazotrophicus grown in the SG medium exerted a greater effect on the variables measured in the aerial part of the plants, such as height and fresh weight of the leaves, while there is no difference in this treatment with the non-inoculated control in the variable number of leaves. When the bacteria grew in MA medium, a positive effect was observed with respect to the control and superior to the rest of the treatments in the root zone, where the root diameter and length were measured, as well as its fresh weight.
Table 3 Effect of G. diazotrophicus INIFAT Gd-42 grown in different culture media in carrot crop (Daucus carota L.).
| Culture media | Height (cm) | Number of leaves | Leaves fresh weight (g) | Root diameter (cm) | Root length (cm) | Root Fresh weight (g) |
|---|---|---|---|---|---|---|
| SG | 36 a | 8.29 a | 5.73 a | 2.925 b | 5.75 b | 0.45 b |
| LGI | 25.94 cd | 6.88 bcd | 3.85 bc | 1.2 c | 6.7 b | 0.156 c |
| SYP | 29.81 bc | 6.43 cd | 2.58cd | 3.1 b | 6.375 b | 0.30 bc |
| DYGS | 22.85 d | 6.13 d | 1.69 d | 2.475 b | 6.375 b | 0.17 c |
| MA | 31.31 b | 7.56 ab | 4.7 ab | 5.56 a | 7.9 a | 0.92 a |
| non-inoculated | 30.0 b | 7.29 abc | 4.04 b | 1.475 c | 6.175 b | 0.244 c |
| Esx | 1.3852 | 0.3557 | 0.4715 | 0.3012 | 0.3655 | 0.0543 |
| CV (%) | 18.83 | 16.52 | 48.92 | 55.79 | 14.72 | 74.32 |
Note: different letters indicate statistically differences according ANOVA and Duncan’s test (5 %).
For beet, a greater effect is shown in comparison with the non-inoculated control (Table 4). In the aerial part of the plant, greater height was obtained with the bacteria grewn in the SG medium and a greater number of leaves when inoculated with the product of the fermentation in SYP medium. The effect on the root was more evident, significant differences are shown for all media with respect to the variant not inoculated with the microorganism. In the root length the products obtained in MA, SYP, SYGS and LGI media are superior to the control and in the diameter of this the best results were obtained with the bacterium grown in MA, SYP, SYGS and SG media, although there are also differences in the effect on LGI with the witness. In the case of the variable fresh weight of the plant, the effect of the bacterium grown in MA medium is highlighted over the rest of the treatments, included in the non-inoculated control.
Table 4 Effect of G. diazotrophicus INIFAT Gd-42 grown in different culture media in sugar beet crop (Beta vulgaris L.).
| Culture media |
Height (cm) |
Number of leaves |
Root diameter (cm) |
Root length (cm) |
Root Fresh weight (g) |
|---|---|---|---|---|---|
| SG | 20.33 a | 3.33 b | 3.40 a | 2.63 bc | 3.88 abc |
| LGI | 17.8 bc | 3.67 b | 2.80 b | 2.83 b | 4.1 abc |
| SYP | 16.67 c | 4.8 a | 3.50 a | 3.0 ab | 4.38 ab |
| DYGS | 17.83 bc | 3.83 ab | 4.20 a | 2.98 ab | 3.84 bc |
| MA | 19.00 ab | 4.33 ab | 4.20 a | 3.32 a | 4.73 a |
| Testigo | 17.25 bc | 3.50 b | 2.30 c | 2.32 c | 3.27 c |
| Esx | 0.7323 | 0.3694 | 0.3574 | 0.1488 | 0.2436 |
| CV (%) | 11.02 | 24.79 | 28.83 | 16.22 | 16.05 |
Note: Different letters indicate statistically differences according ANOVA and Duncan’s test (5 %).
Discussion
Different culture media have been used for the growth of G. diazotrophicus (Eskin et al., 2014). For example, the DYGS medium (Siqueira et al., 2009) was used in fermentation trials to evaluate the effect of osmoprotective substances on this bacterial species; while the SYP medium has been used to determine the effect of different concentrations of phytohormones on the microorganism (Rojas et al., 2015). For the growth of the bacterium in order to evaluate the effect of its application on plant species such as tropical meats (Dibut et al., 2011) and grasses (Muthukumarasami et al., 2006), media such as the LGI and SG have been used, respectively.
However, in the consulted literature no papers were found where the action exerted by the culture medium on the potential as a bacterium that promotes the plant growth of G. diazotrophicus, including the effect of the final product of the fermentation of the microorganism on the stimulation produced by the application on vegetables, is evaluated. There is also no consensus on which means to use to obtain a bioproduct for agricultural use, but the use of media based on natural products are more attractive considering the economic and environmental aspects. For this reason, the potential as a plant growth promoting bacterium of the INIFAT Gd-42 strain of G. diazotrophicus was evaluated under in vitro and in vivo conditions, after growing in five culture media.
The growth curve obtained in all cases is similar to that described by Molinari & Bioardi (2013). According to these authors there are few studies about the growth of G. diazotrophicus in the presence of sucrose as a carbon source. The establishment of a diauxic growth is probably associated with the presence of sucrose as a carbon source and the impossibility of the microorganism to transform this sugar directly, as well as the gradual consumption of the different substrates that are originated by the activity of the enzyme levansucrose (Hernández et al., 1995; Molinari & Bioardi, 2013). In all used media, diauxic growth was obtained, and the two phases of adaptation or lag, and of exponential growth, typical of using more than one carbon and energy source in the medium, can be appreciated. The bacterium will first use the source of energy that allows it to grow better, and later when it is exhausted, others present in the environment (Madigan et al., 2014). In all media there is more than one compound that can have this function as can be seen in Table 1. However, regarding the values of final concentration of the bacterium reached, these were similar to each other and to those referred to in the works from Dibut et al. (2011) and Luna et al. (2010).
The fact that a formulation made from a natural product such as rice, supports the growth of the bacteria. It is an encouraging result from the technological point of view, because for the scaling of a bioproduct requires little complex means where they are incorporated natural sources. Therefore, this could be a variant to exploit in the search for a biofertilizer that has this bacterial species as an active principle. The rice medium (MA) can provide the microorganism with B vitamins, minerals, proteins, amino acids and other nutrients that are present in the rice grain (Pinciroli, 2010). These compounds can contribute to the growth of the bacteria, obtaining a medium rich in nutrients from cooking the rice and mixing with the other components of the medium.
The ability to fix nitrogen was described for G. diazotrophicus from its isolation (Cavalcante & Döbereiner, 1998). There are references where the possibility of growth of microorganisms in semi-solid media without combined nitrogen with the process of biological nitrogen fixation is associated (Pérez et al., 2014, de la Fé et al., 2015). This criteria was used in the study for determine the maintenance of this characteristic in the bacteria. However, it would be interesting to show in later research that the amount of atmospheric nitrogen that can transform the microorganism is not affected either. On the other hand, the differences in phosphorus solubilization haloes are in corresponding with previous observation of Crespo et al. (2011). They described the influence of the carbon source on the solubilization potential of G. diazotrophicus and they showed that the increase in phosphorus soluble is proportional to gluconic acid production. This semiquantitative method allows having a reference of the maintenance of the capacity for phosphates solubilization by the bacteria.
The stimulation of growth indicators by the inoculation of promotig bacteria may be associated, in addition to the contribution of nutrients made by the biological fixation of nitrogen and the solubilization of phosphorus, to other factors such as the release of phytohormones (Pazos et al., 2016 ) and colonization of the rhizospheric and endophytic environment (Jha et al., 2013). For G. diazotrophicus, the ability to release hormones, mainly indoleacetic acid, is described, which can also be affected by the culture medium used for the growth of the bacteria (Patil et al., 2011). It can be producing the particular stimulation of speciphic organ of the plant. Other authors describe the influence of the carbon source in the production of exopolysaccharides, compounds of great importance for the biofilms formation, stress tolerance and the establishment of the bacteria inside of crops (Meneses et al., 2011).
The ability of G. diazotrophicus to increase the growth of natural or occasional host crops has been demonstrated primarily for grasses (Nautiyal, 1999), food and fruit (Dibut et al., 2011). For vegetables, Hazza et al. (2014) and Kumar et al. (2013), demonstrated G. diazotrophicus colonization in carrots, and the increase of sugar beet yields due to the application of the microorganism. In Cuba, Ríos et al. (2016a) obtained encouraging results in the bacteria interaction with carrot and sugar beet. However, in the present work it is shown that the effect on growth stimulation in these crops will depend on the culture medium used for the growth of the bacteria, so attention must be paid to their composition to obtain the best agronomic results. This effect is demonstrated mainly at the root level, an organ of vital agronomic importance in these crops. For this reason in this case it is sufficient for the analysis of the stimulation of the growth of the plant the measurements of fresh weight, since just the root is what is used for human consumption.
In general, it is demonstrated that the culture medium influences in the potential plant growth promotion by G. diazotrophicus. That is why, it is an important aspect to take into account in the biotechnological production of agricultural products for the benefit of economical important plant species.
Conclusions
The culture medium used for the growth of G. diazotrophicus influences in its plant growth stimulating potential. The use of culture medium composed mainly of rice, sucrose and commercial sugar, produces greater benefits in the growth stimulation of carrot and sugar beet compared with other alternatives such as SG, SYP, DYGS and LGI.
