Mitigation of osmotic stress by Serratia nematodiphila in tomato seedlings

Agriculture is the main economic activity responsible for the highest rates of water consumption worldwide. Understanding strategies that combine decreasing the amount of water available and reducing the addition of chemical fertilizers is a major challenge today. Within this context, the work aimed to evaluate the efficiency in promoting growth in tomato seedlings, inoculated with Serratia nematodiphila, submitted to different water deficit conditions. The methodology consisted of using tomato seeds cv. Santa Clara Miss Brasil previously disinfected and inoculated with the Serratia nematodiphila bacterium. The experimental design was completely randomized in the factorial scheme 2 (with bacteria and without bacteria) x 3 (irrigation levels: 100%, 50% and 25% water), making a total of 6 treatments with 6 repetitions. When evaluating the effect of the bacteria Serratia nematodiphila in the treatment with irrigation to 25% of water, it was observed that the average values of the root length of the tomato seedlings was the one that suffered the most stimulus in the root growth when compared to the other irrigation levels. These results suggest that under conditions of greater water deficit the bacteria is able to mitigate drought by promoting root growth.


Introduction
Agriculture is the activity responsible for more than 70% of current water consumption worldwide (WWF Brasil, 2013). For this reason, there are several proposals and strategies that have been studied to increase efficiency in the use of water by plants, in the search to find a balance between commercial activity and the environment.
Although there are numerous technologies available for the rational management of irrigation, the vast majority of producers still irrigate inappropriately. For 2025, the projection of scientists is that 3.3 billion people worldwide will not have water for irrigation (Coletti & Testezlaf, 2003).
The correct use of irrigation in order to efficiently manage water, fertilizers and other inputs, is essential for maintaining the food supply, in balance with its growing demand, guaranteeing the conservation of the environment (Coletti & Testezlaf, 2003).
The water content of the soil must be kept between certain specific upper and lower limits, among which water is not limited to the plant, while leaching is prevented Morgan et al. (2001), with the replacement of water to the soil by irrigation, in quantity and at the right time, decisive for the success of horticulture (Marouelli et al., 1996).
The tomato is a dicot, from the Solanaceae family, which stands out worldwide among cultivated vegetables, due to its fresh consumption, and above all, industrialized, because its production and use is considered universal (Camargo et al., 2007).
Tomato cultivation involves several cultural practices and among them is irrigation, which is present in 100% of commercial crops. Tomato is the second vegetable in economic importance in the world, being surpassed only by potatoes (Alves, 2006). According to Chaves (2009), Brazil accounts for 3% of global tomato production. In the Brazilian economy, tomato culture has stood out not only for its economic value, but also for being an activity that generates a large number of jobs.
The tomato fruit (Solanum lycopersicon L.) consists of 93% water, making it one of the vegetables most sensitive to stress due to excess or deficit of this component (Dorais et al., 2001). Alvino et al. (1986) pointed out that abundant irrigation reduces yield and fruit quality, in addition to increasing production costs. According to Pulupol et al. (1996), the reduction in plant growth, productivity, fruit size and weight, in addition to the incidence of apical rot was Research, Society and Development, v. 9, n. 10, e6109108694, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.8694 4 related to the water deficit during treatments. Thus, it becomes essential to carry out the rational management of irrigation, in order to maintain favorable soil moisture and plant health conditions (Silva & Marouelli, 1998).
One of the alternatives that has been showing beneficial effects is the application of plant growth-promoting bacteria (PGPB) or plant growth-promoting rhizobacteria (PGPR).
These bacteria facilitate the growth of plants, through any assistance in the acquisition of essential mineral resources, by modulating levels of plant hormones or even indirectly, growth can be promoted by decreasing the inhibitory effects of various pathogens that act as agents of biocontrol (Ahemad & Kibret, 2014).
The use of PGPBs, with the potential to achieve this objective, has been highlighted due to its easy applicability in treatments of seeds, roots and also in the aerial part of the plant.
The productivity efficiency of these groups of microorganisms can be applied to the planting of crops, constituting an interesting alternative, to minimize the negative effects of the water deficit. Another strong point is the fact that these bacteria are native to soils or plants, not interfering with the ecological balance and, therefore, fully fitting into the reality of organic and sustainable agriculture (Lima et al., 2020).
Sustainable agriculture requires the use of strategies that allow an increase in food production without harming the environment and health, within the economic, social and political context of each region. Thus, this study aimed to evaluate the efficiency in promoting growth in tomato seedlings, inoculated with Serratia nematodiphila, submitted to different water deficit conditions.

Material and Methods
The research was based on laboratory analysis according to the methodology proposed by Pereira, Shitsuka, Parreira, & Shitsuka (2018).
To evaluate the mitigation of osmotic stress in tomatoes, the endophytic bacterium Serratia nematodiphila was used, originally extracted from tomato seeds produced in the Horticulture sector at UFRRJ, Seropédica-RJ.
The experimental test was carried out in environmental conditions, in the city of Paty do Alferes, in the interior of the South Fluminense region of Rio de Janeiro, with Latitude: 22º 25 '43 "S Longitude: 43º 25' 07" W and altitude: 610m.
Tomato seeds, cv. Santa Clara Miss Brasil, superficially disinfected by immersion in 50% alcohol solution (30 seconds), 0.7% hypochlorite solution (three minutes) and five Research, Society and Development, v. 9, n. 10, e6109108694, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.8694 5 successive washes in sterile distilled water in a vortex shaker, with five water changes. The previously disinfected seeds were inoculated with the endophytic bacteria using the vacuum method described by Bashan & Assouline (1983). Half of the seeds were deposited in Erlenmeyer containing 20 ml of sterile distilled water and the other half deposited in a bacterial suspension of the isolate at a concentration of 10 8 CFU/mL. The flasks were placed in a desiccator coupled to the vacuum pump with the equivalent of 400 mm Hg, with slow release of air in three successive cycles lasting five minutes each cycle, interspersed with three minutes of rest. The seeds were placed to dry in a laminar flow chamber in ventilation, with a temperature of 35 o C for 1 hour. The tomato seeds were sown directly in disposable 200 ml cups containing 100 ml of commercial substrate for the production of vegetable seedlings with a composition according to the packaging: pine bark, peat, vermiculite, simple superphosphate, postassium nitrate and products formulated by third parties (HT Tropstrate).
The experimental design used was the CRD (completely randomized design), in a factorial scheme of 2 (without bacteria, with bacteria) x 3 (irrigation levels 100%, 75% and 25%), totaling 6 treatments with 6 repetitions (Figure 1). Irrigation was performed by gravity. To determine the amount of water to be applied, 3 glasses were separated, three from each treatment. Each morning these glasses received an amount of water capable of promoting drainage. The drained water was collected and the difference calculated for each glass. The amount of water applied was equivalent to the difference in the average between the applied volume and the drained volume (retained Development, v. 9, n. 10, e6109108694, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.8694 6 volume) in each treatment, multiplied by 100%, 50% or 25%, depending on the stress level of each treatment. After this procedure, the quantities of 35 ml of water for the treatment at 100%, 17.5 ml for the treatment at 50% and 8.75 ml of water for the treatment at 25% were determined.
The seeds were cultivated for 18 days, each one with its determined amount of water, until they reached a size sufficient to be measured. After these 18 days, the seedlings were removed from the cups and washed. After cleaning the seedlings, the length of the roots was measured with a digital caliper (Mitutoyo-Digimatic Calipter) (Figure 2). The data were organized in tables and the analysis of the results consisted of making basic statistics, using the Microsoft Office Excel ® spreadsheet for this purpose, and the different irrigation levels were adjusted according to the best equation for the coefficient, 2nd degree regression, tested by the corrected t test based on the residuals of the analysis of variance.
In treatment T4, irrigation at 50% water, the bacterium has a small effect on the promotion of root growth, when compared to treatment T2, although the average values recorded in table 1 do not differ significantly when compared to treatments T3 (control) and T4 (bacteria).
When evaluating the effect of the bacterium Serratia nematodiphila in the treatment with irrigation to 25% of water, it was observed that the average values of the root length of the tomato seedlings was the one that most suffered the stimulus in the root growth, reaching 118,74 mm (T6 ) when compared to other levels of irrigation. These results suggest that under conditions of greater water deficit, the bacteria is able to mitigate drought by promoting root growth ( Table 1).
The positive effect of inoculation of the Serratia bacterium on the promotion of root growth was also observed in the work described by Lima et al. (2020) who, when evaluating Development, v. 9, n. 10, e6109108694, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.8694 8 the effect of osmotic stress imposed by PEG6000 at 7%, observed that tomatoes tend to grow more when inoculated with the bacteria, thus promoting a change in root architecture.
The regression analysis of the radicular length of the tomato during the imposition of different levels of irrigation showed a significant linear trend and an R 2 = 0.9582, revealing that the greater the water deficit (25% of irrigation), the greater the growth of the main root of the seedlings inoculated in relation to seedlings free from inoculation (figure 3). These results suggest that the promotion of axial root growth observed in tomatoes may be associated with the mitigation of water stress promoted by the inoculation of the bacteria Serratia nematodiphila. The results found in the study by Araújo et al. (2012), when verifying the efficiency in promoting root growth of the bacterium Burkholderia sp. inoculated in tomato seedlings submitted to different levels of water stress, corroborate the data found, when they reveal that the bacterium was also efficient in promoting root growth, however, the effect of the bacterium Burkholderia sp. was shown to be more efficient under adequate irrigation conditions, while the bacterium used in the present study, Serratia nematodiphila, showed more significant results in conditions where water stress was greater.
In the work of Medeiros (2013) similar results were observed when the bacterium Herbaspirillum seropedicae strain HRC54 was able to mitigate the stress imposed by polyethylene glycol (PEG6000). According to the author, in the inoculated treatment + PEG, Development, v. 9, n. 10, e6109108694, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.8694 9 the growth of the axial root of the tomato was less reduced in response to the improvement of the presence of the inoculum when compared with the stressed non-inoculated plants.
The water stress mitigation process could also be associated with the availability and/or acquisition of resources essential to the plant, such as phosphorus and zinc for example. According to Rajkumar et al. (2010) the solubilization of inorganic minerals unavailable in the soil is one of the growth mechanisms promoted by bacteria of the genus Serratia. These microorganisms in situations of low iron availability in the environment are able to chelate and capture ferric ions, transporting the iron-siderophore complex into the cell.

Final Considerations
The results together allowed us to conclude that the bacterium Serratia nematodiphila is efficient in promoting root growth in plants subjected to water stress, presenting a significant result in the difference in the average length of plants inoculated with bacteria, from plants free from inoculation, with greater efficiency in conditions where water stress was greater.