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Introduction
Plant growth promoting rhyzobacteria (PGPR) stimulate plant growth by direct and indirect mechanisms. Indirect promotion stems from the production of antibiotics and metabolites that enhance nutrient uptake (Glick et al., 1995; Ahmad et al., 2008). Direct promotion occurs by the production of phytohormones like auxins, cytokinins and gibberellins; as well as organic compounds and other compounds that increase plant immunity; such as jasmonic acid, salicylic acid and phytoalexins (Ahmad et al., 2008; Rojas-Solis et al., 2013).
In Solanum lycopersicum (tomato) we used Azospirillum, Azobacter, Bacillus, Agrobacterium and Streptomyces PGPRs (Alfonso, et al., 2005; Haas & Défago, 2005; Nihorimbere et al., 2010; Rentería, 2013). These PGPRs can be applied alone or bio-formulated (Berg, 2009; Choudhary & Johri, 2008; Clayet-Marcel et al., 2001). Regarding Bacillus genus, we used B. subtilis because it promotes tomato growth and acts as biological control against pathogens like Fusarium oxysporum (Airola, 2010).
Bacillus subtilis produces metabolites such as cytokinins, siderophores, auxins and antibiotics, among others. Plants can produce cytokinins endogenously, however, the exogenous addition of cytokinins increases their growth process (Arkhipova, et al., 2005). Cytokinins induce amylase and protease activity, as well as auxin synthesis (Mantilla, 2007). Meanwhile, siderophores chelate environmental iron making it more available to the plants (Abdel-Aziz, 2013). Some siderophores are considered antibiotics because they limit the iron supply that pathogens need for growing (Aguado-Santacruz et al., 2012). 3-indolic acetic acid (IAA) is one of the most important auxins; it controls the cell division process, vascular tissue differentiation, formation of the apical domain and that organ’s development (Blakeslee et al., 2005; Tsavkelova et al., 2006). Regarding antibiotics, B. subtilis can produce more than one dozen of antibiotics with a great diversity of chemistries, including surfactin, iturin A and bacillibactin (Stein, 2005), which are capable of controlling the growth of tomato’s pathogens.
Nevertheless, authors like Bharucha & Patel (2013) and Buensanteai et al. (2008) have said that growth promotion depends on the balance between auxins and phytohormones (Buensanteai, et al., 2008). The presence of tryptophan, jasmonic acid and siderophores in the culture medium can improve auxin production (Bharucha & Patel, 2013) and therefore, regulate growth promotion.
Regarding B. subtilis, there is no sufficient scientific evidence to know if all the strains have the same growth promoting effect, regardless their capacity of producing different growth promoting metabolite concentration. The goal of this research work was to assess the plant growth promoting effect of B. subtilis test strains, as well as their production of siderophores, indolic phytohormones and jasmonic acid.
Material and Methods
Test strains and seeds
We used three B. subtilis strains in this research work (BSN, BS14, and BS8). BSN strain was given by the Advanced Studies and Research Center of IPN (“Centro de Investigación y Estudios Avanzados” del Instituto Politécnico Nacional-CINVESTAV) Irapuato station; while BS8 and BS14 were provided by “Universidad Autónoma Agraria Antonio Narro”. The strains were activated in TGE medium (tryptone glucose yeast extract) at 28 °C (Calvo & Zuñiga, 2010). CINVESTAV Irapuato supplied the seeds of Arabidopsis thaliana.
B. subtilis’ fermentation broth preparation
The fermentation took place in Landy medium with tryptophan. Landy medium was prepared as follows: glutamic acid 5.0 g/L, KH2PO4 0.5 g/L, K2HPO4 0.5 g/L, MgSO4 .7 H2O 0.2 g/L, MnSO4 .H2O 0.01 g/L, NaCl 0.01 g/L, FeSO4 .7 H2O 0.01 g/L, CuSO4 .7 H2O 0.01 g/L, CaCl2 .2 H2O 0.015 g/L, and tryptophan (final concentration 5mM). We added micro-filtrated sterilized glucose solution (0.2 μm filter) until reaching a final concentration of 1 %. The Bacillus grew in 50 mL of Landy medium. We incubated them at 28 °C for 48 h for pre-inoculation. The fermentation broths grew in 1x107 CFU /mL’s medium concentration and they underwent stirring at 120 rpm, under 25 °C during 72 h (Awais et al., 2010).
Getting the cell-free supernatant
In order to obtain the cell-free supernatant, we centrifuged B. subtilis’ fermentation broths (BS8, BS14 and BSN) at 4 °C and 12,000 g (Centrifuge 5810/5810 R) during 10 min before filtering them for sterilization, using a 0.20-μm microfilter.
Detection of siderophores, using CAS
For the siderophores detection, we used Brian et al. (2011) technique. The technique uses chrome azurol S (CAS) and hexadecyltrimethylammonium bromide (HDTMA), as well as indicators (Abdel - Aziz, 2013).
Quantification of auxins and jasmonic acid by HPCL
We used UV-coupled HPLC for the auxins (IAA, tryptamine, 3-indoleacetonitrile) and jasmonic acid quantification in B. subtilis’ fermentation broth. The calibration curves required reactive standards within 1 to 1000 ppm range in mobile phase (methanol-wateracetic acid in a 60:40:1 ratio). 50 mL of the fermentation supernatant went into the sample preparation, adjusting the pH at 2.8 with HCl 1M. We did three extractions with 50 mL of ethyl acetate and we combined the organic phases before adding anhydrous sodium sulfate to remove moisture (Castillo et al., 2005). The solvent was removed with a rotary evaporator at 60 °C. Finally, the residue was re-suspended with 2 mL of the mobile phase (methanol-water-acetic acid in a 60:40:1 ratio) before micro-filtration. The chromatographic separation included a reverse phase with C18 column, using the abovementioned mobile phase (previously degassed) with 0.7 mL/min flow rate. We used UV of 296 nm to conduct the detection before the interpolation at the calibration curve (Castillo et al., 2005).
Assessment of seed’s growth promotion
The seeds of Solanum lycopersicum and Arabidopsis thaliana were treated with cell-free supernatant and with fermentation broth, in order to assess the germination percentage. In order to assess the supernatant, we mixed Murasige and Skoog (MS) medium with 1 mL of supernatant by box, before solidification and we planted the seeds previously sterilized. In order to assess the fermentation broths, the previously sterilized seeds were soaked during 30 minutes in the broths, before introducing the Petri dishes in the boxes with MS medium. The boxes were staggered in a bioclimatic chamber at 25 °C with a16/8 light-darkness photoperiod. We measured the seed germination percentage after 7 days of starting the experiment. There were nine replicates (seeds) using a fully randomized ANOVA design; a significance level of 0.05 and Tukey’s mean comparison test.
Assessment of growth promoting effect on seedlings treated with B. subtilis’ fermentation broth
We assessed the growth promoting effect by adding B. subtilis’ fermentation broth to Solanum lycopersicum seedlings. The seeds of Solanum lycopersicum germinated in trays with peat moss substrate. 21 days after germination, we transplanted the seeds into containers with peat moss, perlite and vermiculite (3:2:1). We added 3 mL of the fermentation broth previously homogenized by stirring to the seedlings (BS8, BS14 and BSN) close to the roots, with a syringe. We had plants without treatments on Landy medium as control checks. Every treatment had seven replicates (Mena-Violante et al., 2009). After the treatments, we took daily measurements along the stem and the number of leaflets on those 60 days. On day 60, we assessed root length, seedling wet weight, root wet weight, seedling dry weight, stem and leaves wet weight, seedling moisture percentage and root moisture percentage (Gómez-Luna et al., 2012; Mena-Violante et al., 2009; Airola, 2010). The experiment used a fully randomized ANOVA design, with a significance level of 0.05 and Tukey’s mean comparison test.
Results and Discussions
Siderophore synthesis
The three strains of B. subtilis were capable of producing siderophores, as demonstrated by the halos in CAS media (Figure 1). According to Bolívar-Anillo et al. (2016), the siderophores make the iron available for the plants and promote their growth. Siderophores production is important to suppress deleterious microorganisms, because they monopolize the iron available in the soil and in some cases, they seem to unleash an induced systemic resistance response in plants (Suarez-Moreno et al, 2012).
Indole production
In the quantification of indolic compounds, we observed that the test strains produce IAA and other indoles in variable concentrations. Table 1 shows the indole concentration in the strains. IAA concentration was high, as compared to the results of previous trials (Wahyudi et al., 2011; Luna et al., 2013) and that is why the strains had growth promoting effect. The presence of IAA precursors (Tryptamine and 3-indoleacetonitrile) indicate the strains’ potential to produce more IAA under specific fermentation conditions. In general, auxin bacterial production, in particular IAA, has had a significant influence on plant growth and development (Sánchez et al., 2012).
Table 1 IAA concentration, tryptamine and 3-indoleacetonitrile in B. subtilis fermentation broth quantified by HPLC and expressed in μg/L ± D.E. L.
| IAA | Tryptamine | 3-indoleacetonitrile | |
|---|---|---|---|
| BS8 | 129.53 ±3.23 | 387.64 ±29.84 | 352.64 ±19.37 |
| BS14 | 147.80 ±3.03 | 136.74 ±50.20 | 194.33 ±8.73 |
| BSN | 113.39 ±6.8 | 605.54 ±39.60 | 118.04 ±4.74 |
Jasmonic acid production
From the three strains assessed, only BS8 produced jasmonic acid at a concentration of 54.48 ppm. Some growth promoting microorganisms like Thrichoderma sp activate the plant defense mechanisms through jasmonic acid, and they can take part in the production of salicylic acid, a messenger of acquired systemic resistance (Alcedo & Reyes, 2018) that also stimulates plant’s growth.
Germination percentage
There was no increase in the germination percentage of Solanum lycopersicum seeds treated with B. subtilis and its metabolites. However, the germination percentage increased in Arabidopsis thaliana with the supernatant addition (metabolites) of strain BS14, and with the addition of germination broths BS8 and BS14. BSN caused a decrease in the germination percentages. Figure 2 shows the germination percentages of Arabidopsis thaliana.
Figure 2 Germination percentages of Arabidopsis thaliana seeds after 7 days of adding the supernatants and B. subtilis strains’ fermentation broth treatments.
Liu et al. (2013), demonstrated that auxins reduce seed dormancy by stimulating abscisic acid signaling. The test strains had auxin production capacity (IAA, tryptamine and 3-indoleacetonitrile), so the germination percentage might be associated to these metabolites. After measuring the germination rates, the control seeds were contaminated with pathogens, while B. subtilis seeds remained free from pathogens. This effect might be attributed to the strains capacity of producing siderophores, which according to previous research works, have the capacity of controlling pathogens by limiting iron availability (Jung et al., 2006; Cazorla et al., 2007; Woo & Kim, 2008; Tejera-Hernández et al., 2011; Yu et al., 2011).
Stem Length
B. subtilis test strains produced longer stems in Solanum lycopersicum seedlings. The three treatments presented significant differences according the statistical analysis. BS8 (a) increased stem length by 45 %, BS14 (b) by 33 %, and BSN (c) by 17 % in comparison to the absolute control, CA (e).
Number of leaflets
BS8 (a) produced the highest number of leaflets, followed by BS14 (b), BSN (bc) and the controls (bc) Figure 3). The increase in the number of leaflets relates to the presence of auxins in the fermentation broths. Auxins delay the abscission of organs (leafs, fruits and young fruits) by inhibiting the ethylene hormone (Van Doorn & Stead, 1997). The stem length and the leaflet number results proved that BS8 is the best treatment, maybe due to the production of other phytohormones like cytokinins. Previous research works have associated the accumulation of cytokinins to an increase in plant’s weight (30 %), combined with high levels of hormones like IAA and abscisic acid (ABA) (Arkhipova et al., 2005; Rojas- Solis et al., 2013).
Root length
Root length had no significant differences versus the control checks. However (Figure 3), BS8 produced higher quantity of secondary roots. Jordan & Cassareto (2006) mention that high concentrations of IAA promote stem elongation but inhibit root growth, while low concentrations of IAA induce root growth. Optimal concentration of IAA differs in every plant species. In this work, metabolite concentrations promoted stem length but did not promote root length.
Wet weight
Wet root weight results do not show any significant difference among treatments. Regarding seedling wet weight, BS8 (a) is better than CA (b) and BS14 (b), but it is not better than CQ (ab) and BSN (ab). The effectiveness of BS8 in increasing wet weight could be due to the increase of L-tryptophan in the culture medium. o. Ali et al. (2009) found that different isolates of Bacillus sp. Increased IAA concentration by increasing L-tryptophan concentration.
Dry weight
Root dry weight showed no significant differences. In the seedling dry weight trial, only treatment BS8 (a) gave better results than CA (b). BS14 (ab), BSN (ab), CQ (ab) did not present significant differences other than the ones mentioned before. According to Marquina et al. (2018), the addition of tryptophan to the plants can promote a dry weight increase. Therefore, a higher concentration of tryptophan in the culture media could have promoted higher dry weights in the seedlings.
Moisture percentage
We did not find any significant differences between the treatments and the control checks regarding the percentage of moisture in the roots and seedlings. These results lead to the dry and wet weights of roots and seedlings that had significant differences, with the exception of BS8 treatments.
The results show that it is possible to mix the test strains in bio-formulations to promote different agronomic parameters, other than the parameters they promote individually. The rhyzobacteria interactions with the host proved positive interaction with Bacillus spp. to promote growth (Martínez-Viveros et al., 2010).
Conclusions
The concentration of growth promoting metabolites and their interaction with other metabolites (antagonism or synergism) have an impact on the growth of any particular parameter. Therefore, it is possible to use PGPR’s metabolic profiles to predict their effect on plants. Bacteria that produce the right concentration of metabolites promote growth, whereas PGPRs that produce metabolites in too high or too low quantities, inhibit plant growth. Therefore, we conclude that in order to select a strain, it is necessary to define the parameter and the stage where we want to promote growth.
