The tomato (Solanum lycopersicum) is one of the most widely grown vegetables in the world. Mexico has the tenth place in production and Sinaloa is the country’s main producing state, with approximately one third of all Mexican tomatoes, acknowledged internationally (^{SAGARPA, 2018}). Therefore, current production management has focused on its safe, environmental, long-term and efficient development. Due to their importance, many researchers have focused on learning more about the microorganisms present in the phloem of plants (^{Ortiz-Galeana et al., 2018}). In modern agriculture, growth-promoting bacteria have great potential. Nowadays, the growth of most vegetables requires the production of vigorous seedlings, an important factor for an adequate development of the fruit (^{Luna-Martínez et al., 2013}). Endophytism is the mutualistic association phenomenon between a plant and a microorganism living within its tissues without causing any disease symptoms, as well as being a biological resource that participates in several crucial functions related to growth, development, tolerance and adaptation to stress (^{Gundel et al., 2012}). However, depending on the availability of nutrients and the metabolic state of the host plant, the response of the long-term association of an endophyte may be mutualistic or antagonistic (^{Eaton et al., 2011}).

This has led to the study of indirect mechanisms by competition for space and nutrients (consumption of leachates-exudates, production of siderophores, induction to the systemic response in plants with the production of phytohormones and molecular patterns) (^{Chowdhury et al., 2015}). Several investigations have proven that endophytic bacteria are capable of efficient forms of interaction with their hosts, such as growth promotion and the protection of plants against infections by phytopathogens. ^{Nawangsih et al. (2011}) isolated endophytic bacteria using asymptomatic tomato plant shoots with six strains, with an antagonistic effect on bacterial wilting (Ralstonia solanacearum) of the tomato, thus proposing an alternative control method to support sustainable agriculture of this vegetable. Currently, the interest in environmental protection, the implementation of sustainable agriculture, and different international regulations for the import/export of products without agrochemicals, demand an increase in efficiency by the study and exploitation of the beneficial effects that the endophytic microbiota can provide, and therefore, microorganisms that establish a positive interaction with plants are considered to play an important part in agricultural systems (^{Sánchez-Bautista et al., 2017}). Having stated this, the aim of this investigation was to identify the endophytic bacteria isolated from tomato plants and to characterize them based on the plant growth promoting properties.

In order to carry out the study, tomato plants of the variety Missouri were gathered during the 2017-2018 planting season in plots of the fields of the Gabriel Leyva Solano Ejido, located in the Municipality of Guasave in the state of Sinaloa, Mexico (25° 39’ 50 latitude north and 108° 38’18 longitude west). Asymptomatic plants were chosen at random from the planted surface, and sent to the laboratory for their analysis. On the other hand, based on the hypothesis that there is a large diversity of endophytes in seeds (^{Surette et al., 2003}), tomato plants were established under in vitro conditions. For this, seeds from two saladette tomato varieties (THB and SUN6366) were used, provided by a commercial company. The seeds were placed on filter paper, dampened with sterile distilled water, and they were precultured for three days under natural lighting conditions at 25 °C. Later, they were placed in ^{Murashige and Skoog} salt media, without growth regulators, and they were transferred to a growth chamber under controlled conditions (22 °C, photoperiod of 16 h day^-1 and a photosynthetic flow of 225 μmol m^-2 s^-1).

The plant root, shoot and leaf tissues gathered in the field were washed with sterile distilled water. For the roots, the adhered soil particles were carefully removed. They were then superficially sterilized by submersion in ethanol at 70% for 30 seconds and later washed with a sodium hypochlorite solution (2.5%) for 5 min, and ethanol at 70% for 30 s, to finally be washed between five and ten times with sterile distilled water. Next, the plant tissue (foliar, shoot and root) was macerated individually in 20 mL of sterile water, taking 50 µL of a dilution of 10^-4 for each tissue, which were established in nutrient agar in triplicated; for the plants obtained from seeds established in vitro, the whole plant was macerated and sown in nutrient agar to obtain and isolate the bacteria.

The Petri dishes were incubated for eight days at 27 °C (^{Yang et al., 2011}). Continuous purification rounds were carried out in the nutrient agar of the bacterial cultures, achieving strains with similar morphological characteristics to perform the culture morphology analysis, which was carried out in a stereoscope of each of the isolations with 48 h growth, where the variables considered were size, color, shape, edges, elevation, surface, aspect, light emitted, consistence and light reflected. Later, the culture-forming units (CFU) were counted per gram of fresh weight for each tissue; the Gram stain technique was carried out following the protocol on the commercial kit (Golden Bell).

For molecular identification, we began with the genomic DNA from seven bacterial strains oligonucleotides F2C (5’-AGAGTTTGATCATGGCTC-3’) and C (5’-ACGGGCGGTGTGTAC-3’) (^{Shi et al., 1997}) to amplify the gene that codifies the subunit 16S of the rDNA. The product was purified using the Wizard^® SV Gel kit and PCR Clean-Up System, and it was sent for resequencing to the National Genomics Laboratory for Biodiversity (LANGEBIO) in CINVESTAV-IPN; the homology of the sequences obtained was compared within the GenBank database, using the program Blast of the NCBI and RDP. The phylogenetic analysis of the gene 16S of the rDNA was performed using the software MEGA 5 Beta (^{Tamura, et al., 2011}). The strength of the topology of NJ was evaluated with the bootstrap test, using 1000 replications. The phylogenetic trees were created using the Neighbor-Joining (NJ) method (^{Saitou and Nei, 1987}), as well as the Tamura-Nei model. In order to identify potential plant growth-promoting microorganisms, the capabilities of each bacterial isolation were compared, using the strain (B25) Bacillus cereus as a control (^{Figueroa-López et al., 2016}); each one of the experiments was carried out in triplicate. For the analysis of the production of siderophores, we used a chrome azurol S (CAS) medium, prepared by following the method described by ^{Schwyn and Neilands (1987}). Chitinase production was evaluated with chitin as the only source of carbon, following the method by ^{Shanmugaiah et al. (2008}). The evaluation of the solubilization of phosphate was carried out on agar-^{Pikosvkaya plates, incubated at 25 °C for one week (Pikosvkaya, 1948}).

After ten weeks, 50 tomato plants were established in vitro, with an approximate growth of 5 cm. On the other hand, 20 asymptomatic tomato plants in a vegetative growth stage were gathered on the field. A total of 25 bacterial isolations were obtained, 10 from in vitro and 15 from the field (9 root, 4 shoots, and 2 foliar tissue), obtaining seven axenic isolations (Gram negative), one from in vitro and six from the field (4 root and 2 foliar tissue) (Table 2), which were used to count the culture-forming units per gram of fresh weight (log₁₀ CFU g^-1) (Table 1). The results coincide with the proposal by ^{Sørensen and Sessitsch, (2007}), who point out that the rhizosphere is a source of endophytes for plants, helping the rhizospherical bacteria penetrate the internal tissues of plants via cracks in the roots and lesions in tissues that take place as a result of plant growth.

On the other hand, there are reports of a larger diversity of phylotypes in the rhizosphere of husk tomato plants (Physalis ixocarpa), than the diversity of bacterial endophytes (^{Márquez-Santacruz et al., 2010}). The culture morphology of the isolations displayed few differences amongst each other after 48 h of growth, characteristically white or cream-colored; only one displayed bright red pigmentation, a circular shape, ruffled edges and a shiny surface. The comparison of the sequences obtained from the gene 16S rDNA of the seven endophytic isolations against the NCBI database indicated broad relations with the bacterial species identified in the gene bank, with identities above 97% representing Methylobacterium radiotolerans (NR074244.1), Shinella sp. (KF261566.1), Burkholderia cepacia (AB162427.1), Sphingobium herbicidovorans (NR113843.1), Pseudomonas sp. (KR067597.1), Achromobacter xylosoxidans (KR136349.1) and Rhizobium radiobacter (KF975413.1).

Figure 1.  Phylogenetic relations between seven endophytic bacteria (red lines) isolated from tissues (root, foliage stem) from tomato gathered on the field and under in vitro (Seed) conditions. Isolations compared with GenBank sequences. 

The data generated by the database helped tentatively identify the organisms, yet a more thorough molecular characterization is necessary to define what species they belong to. Figure 1 illustrates the phylogenetic relations of the isolations presenting seven clusters (I to VII), where the inclusion of reference sequences helped verify their identity by showing a group with those characterized and published in other papers. ^{Turner et al. (2013}) propose that plant-microorganism interactions are diverse, although those related to plant growth promotion are important for agro-biotechnological use (among many other uses).

This experiment highlights the genera Pseudomonas and Burkholderia. These have been widely studied for the production and emission of their diverse range of secondary metabolical products, including antibiotics and volatile organic antifungal compounds (^{Hernández-León et al., 2015}). Other reports show that the genera Pseudomonas and Burkholderia are dominant members in the rhizoshperic microbiot with the ability to use carbon substrates, which supports the theory that these bacteria are stimulated by the presence and composition of different root exudates (^{Marrero et al., 2015}; ^{Kumar et al., 2016}). In this study, the Pseudomonas and Burkholderia strains were isolated in tomato plant roots gathered in the field, which is consistent with other studies. ^{Madhaiyan et al. (2007}) proves that the bacteria Burkholderia sp. reduces the accumulation of cadmium and lead in tomato plant roots and shoots, as well as the metal available in the soil due to the absorption and bioaccumulation by the bacteria. An important step is the use of three commercial formulations of Pseudomonas registered in the Environmental Protection Agency of the United States for the suppression of plant diseases, respectively. These products are applied in balers to prevent fungal diseases during the storage of citrus fruits, stone fruits and potatoes (^{Stockwell et al., 2006}).

The group of bacteria called plant growth promoting rhizobacteria (PGPR) includes the genus Rhizobium. Investigations have pointed its study as the growth promoter in legume and non-legume pants (^{Piñerúa et al., 2013}). On the other hand, studies by ^{Santillana et al. (2005}) reported that strains of Rhizobium stimulate the germination of tomato seeds and promote their growth. Nowadays, sustainable agriculture suggests improving the efficiency of nitrogen fixation using legume plants and competitive rhizobia, which can be used in bioremediation and phytoremediation, thus extending the advantages of symbiosis to other crops (^{Piñerúa et al., 2013}). In this study, the strain of Methylobacterium was isolated from the plant tissue of tomato seedlings established under in vitro conditions without growth regulators; there are no reports of this genus under in vitro conditions in tomato plants. Diverse studies have reported that they are able to produce phytohormones that stimulate plant growth, favoring nitrogen fixation and protecting the plant against pathogens (^{Pérez-Montaño et al., 2014}). Another group of important bacteria isolated from the plant tissue of tomato is composed of S. herbicidovorans and A. xylosoxidans.

There are currently few investigations reported in regard to S. herbicidovorans, which is widely used against broad-leaf weeds in agriculture, grasses, lawn and industries (^{Müller et al., 2004}). Studies focusing on the evaluation of the antifungal activity of A. xylosoxidans against isolations of F. oxysporum and F. solani reported positive effects, reducing the mycelial growth of the pathogens by up to 80% in comparison with the control, suggesting a potential use as a biocontrol agent (^{Dhaouadi et al., 2018}). The inoculation of A. xylosoxidans F3B in Arabidopsis thaliana has been reported to stimulate a significant increase in root length and fresh weight, considering said bacteria as endophytic (^{Ying-Ning et al., 2009}).

In the evaluation of activities related with the promotion of plant growth, the strains Pseudomonas, Burkholderia and Rhizobium were observed to display a high phosphate solubilizing ability, in comparison with the control (B25) Bacillus cereus (Table 2). In the production of siderophores, the isolation with the greatest production in comparison with the control was Pseudomonas sp. (Table 2). This characteristic relates rhizobacteria with the increase in iron available in the soil, allowing its absorption by the plant to constitute a growth-promoting mechanism (^{Gouda et al., 2018}). Although finding quantitative evaluations of the synthesis of siderophores by the genus Pseudomonas is infrequent, there have been reports of the production of several siderophores with a strong affinity for Mo, V, Cu and Zn (^{Harrington et al., 2012}). On the other hand, endophytic bacteria can be used as biocontrol agents with the production of enzymes such as hydrolases and chitinases, considered as plant defense enzymes against the infection from pathogens (^{Perez et al., 2013}). In this investigation, the strain with the highest chitinase activity was Burkholderia cepacia (Table 2).

The endophytic bacterial species associated with tomato crop were Methylobacterium radiotolerans (in vitro), Shinella sp. and Achromobacter xylosoxidans (foliar tissue), Burkholderia cepacia, Pseudomonas sp., Sphingobium herbicidovorans and Rhizobium radiobacter (root). In general terms, under the conditions evaluated in this investigation, approximately 86% of the isolated strains were observed to have the ability to produce plant-growth promoting substances, and according to this, these results show the possible beneficial effect of the bacterial endophytes in tomato plants. This lays the foundations for future field studies to determine the effect of these strains on the production and quality of tomato fruits.