Biotechnological Potential of Endophytic Bacteria to Improve the Micropropagated Seedling of Variety RB92579 Sugarcane (<i>Saccharum officinarum L.</i>)

Endophytic bacteria may influence agricultural production in several ways, including promoting plant growth. Two experiments were conducted in order to evaluate the combination of endophytic bacteria from the Brazilian Northeast region aims at the commercial introduction of the inoculation of these bacteria in micropropagated sugarcane plants using a temporary immersion bioreactor. One experiment was done in tubes with sterile commercial substrate, and the other was done in pots with soil; both were installed in a greenhouse. A mixed inoculation was performed in six inoculated endophytic diazotrophic bacteria in micropropagated sugarcane plants, variety RB92579. In the experiment with soil, the mixed inoculation significantly increased the shoot dry matter of plants without the addition of nitrogen fertilizer. However, the accumulation of total-N in the tissues showed no significant differences between treatments with and without nitrogen fertilization. The evaluation of micropropagated seedlings showed no increases in the parameters tested. The results showed that the response of inoculation in temporary immersion bioreactor micropropagation is possible, and that the application of homologous strains may have contributed to a better response by the interaction of endophytic bacteria with sugarcane RB92579. Further studies should be conducted to improve the methodology, which indicates a great potential to optimize this process on a commercial scale.


Introduction
Brazil is the largest producer of sugarcane, with a planted area of approximately 8.6 million hectares, with an estimated production for the 2018/19 season of 62,596 million tons [1]. The production is concentrated in the South-Central and Northeast regions. This culture demands a high amount of nitrogen, the most limiting macronutrient for crop productivity. It is one of the highest costs for farmers. Since fertilizer is not subsidized in Brazil, most commercially used genotypes were chosen aiming to obtain a high productivity with low levels of soil N, favoring; even indirectly, the selection of varieties that are capable of covering part of the need for N by the association with diazotrophic bacteria [2].
The biological process of converting dinitrogen to ammonia is called nitrogen fixation (BNF), and is performed exclusively by the enzyme nitrogenase, so that it is a great support for the increase in productivity. Moreover, it is also an ecological and more economical alternative [3]. Research has shown that the key to the success of BNF processes lies in the selection of diazotrophic bacteria that can associate more efficiently. Therefore, a more detailed study on the community of diazotrophic bacteria during plant growth cycles is necessary. Studies on inoculation of micropropagated sugarcane plants with a mixture of five strains from different species showed contributions of around 30% [4]. Nitrogen fixation has a profound agronomic, economic, and ecological impact owing to the fact that the availability of fixed nitrogen represents the factor that most frequently limits agricultural production throughout the world [5].
Micropropagation is a practice widely used in many countries in Europe, Asia, United States and Brazil. This method is based on the production of more uniform and healthy plants and on a much higher growth speed within a limited physical space [6]. However, endophytic microorganisms have been mentioned in several studies as contamination sources to micropropagation. Others consider their presence as a positive factor, arguing that they are able to assist plants, since they live inside their tissues without causing symptoms of their presence, and in the case of in vitro cultivation they can favor osmotic adjustment, production of phytohormones and absorption of nutrients [7] [8]. The reintroduction of diazotrophic endophytic bacteria in micropropagated sugarcane plants has helped studies on the association between plants and diazotrophic bacteria, allowing us to evaluate the potential of BNF and growth promotion [4] [9]. In this context, the present study was performed to evaluate the micropropagated sugarcane seedlings using a temporary immersion bioreactor system aiming the

Culture Media
The culture media used for isolation of bacteria were LGI-P, JNFb, NFb and JMV according to [10]. The medium DYGS [11] was used for the growth of strains and for DNA extraction.  [10]. Then, they were diluted serially in 0.1 mL of suspension and inoculated in vials containing 5 mL of semisolid free-N, LGI-P, NFb, JNFb and JMV. According to Dobereiner et al. [10], each medium is selective for a particular genus of diazotrophic bacteria: NFb (Azospirillum spp), JNFb (Herbaspirillum spp), LGI-P (Gluconacetobacter diazotrophicus) and JMV (Burkholderia spp). After 72 -96 h of incubation, the pots with a white film on the surface were also replicated for a new source medium.

DNA Extraction
DNA bacterial extraction was conducted using phenol-chloroform. Endophytic bacterium isolates were grown in 5 mL DYGS [11] for 24 h at 28˚C. A 400 μL aliquot of the solution was transferred to a microtube and 400 μL saturated phenol solution was added. The mixture was shaken in a vortex apparatus and subjected to centrifugation at 16,000 g for 5 min. The supernatant (aqueous layer) was transferred to a new microtube and the phenolic step was repeated.
After centrifugation the supernatant was again transferred to a new microtube and 400 μL chloroform was added. The microtube was shaken in a vortex and centrifuged for 5 min at 16,000 g. The aqueous layer was transferred to another microtube, to which 1 mL cold ethanol was added. To complete the process of extracting the DNA, the microtube was centrifuged for 3 min at 16

Micropropagation of Sugarcane
Endophytic diazotrophic bacteria were used in the mixture to inoculate sugarcane plants ( Table 1).  [15] in micropropagated seedlings in rooting phase.
Two experiments were conducted at the CETENE greenhouse. One used plastic tubes filled with the sterilized commercial substrate Basaplant ® and the other used pots (8 L) with non-sterile forest soil under greenhouse conditions.
In the first experiment, the mixed inoculation was evaluated with the follow- For the second experiment, the remainder of the seedlings that did not undergo evaluation during the first experiment was transplanted into 8-liter pots containing non-sterile soil. From this soil, several single samples were collected to form a composite sample, which was then analyzed (Table 2), obtaining the fertilization recommendation. The experimental design was randomized blocks with four replications. After 120 days after planting, evaluations of accumulation of root and shoot dry mass and the determination of total nitrogen accumulated in plant tissues were made using the Kjeldahl method [16].

Statistical Analysis
Each variable studied was subjected to analysis of variance (ANOVA), F test, and Tukey's test, at 5% significance levels using the statistical software ASSISTAT version 7.7 [17].

Isolation of Diazotrophic Bacteria
Populations of nitrogen-fixing bacteria were higher in samples of roots compared with samples of culms. Previously conducted tests indicated that the bacteria used in this experiment have an ability to fix N 2 in vitro, which becomes a potential tool for the production of IAA and for inorganic phosphate solubilization ( Figure 1).

Molecular Phylogeny of Bacterial Isolates
The total DNA of the six bacterial isolates was purified and used as a model for Based on the sequence of the 16S rRNA gene, the endophytic bacterial isolates in this study were identified as Enterobacteria, Gluconacetobacter, Rhizobium, Pantoea and non-cultivable bacteria ( Figure 2).
The results of phylogenetic analyses allowed grouping the endophytic sugarcane isolates into two groups, with similarities with sequences in the GenBank public database ranging from 99% to 100% (Figure 3).
Group I was composed by the isolates 1, 5, 10, 22 and 25, which are related to Pantoea sp., Rhizobium sp., Enterobacter sp., E. asburiae, E. ludwigii and E. cloacae. This result was confirmed by the sequencing of the gene gyrβ, which enabled identifying the isolates at the species level, such as E. cloacae (Table 3).    Pantoea was found in sugarcane and soybeans [23] is at soja [24]. Studies have shown the potential of Pantoea sp. to induce a systemic resistance and protection against pathogenic microorganisms. Additionally, these bacteria may in-Advances in Microbiology duce the growth of plants, increasing the supply of nitrogen in non-symbiotic associations, solubilizing phosphorus and stimulating the production of phytochromes [25].
Although Rhizobia infect naturally legumes as host plants, some strains may form symbiotic relations with non-legume species. Besides fixing N 2 , they are also capable of contributing to the promotion of growth of these species.
Group II included the isolated 30, which is related to Gluconacetobacter diazotrophicus. G. diazotrophicus is considered the main diazotrophic endophyte in sugarcane and has been isolated from leaves, stems and roots of sugarcane plants and other economically important grasses [23]. Several studies have shown that such endophytes colonize their hosts in vast numbers and cause an increase in production [26] [27]. The possibility of replacing fertilized nitrogen for biological nitrogen fixation is a very important economic and environmental factor [28].
The results obtained in this study are essential to provide the necessary knowledge on the analysis of endophytic bacteria in micropropagated sugarcane plants and to indicate the potential for future applications of endophytes that promote plant growth.

Evaluation of Micropropagated Sugarcane Seedlings
The results of this experiment showed no significant statistical differences by Tukey test (p < 0.05) among treatments inoculated with the mixture of diazotrophic bacteria (with and without nitrogen fertilization) and the control. There was a tendency for increase for the control treatment at this micropropagated phase (Table 4). Results similar to those obtained in this study were found by [9] upon analyzing the effect of inoculation by determining the accumulation of root and shoot dry mass of sugarcane seedlings, variety SP701143, at the 65 th day. In this study, no statistically significant differences were observed among treatments inoculated with 44 strains of diazotrophic bacteria and the non-inoculated control, suggesting that at this stage the plant does not respond adequately to inoculation.

Evaluation of Sugarcane in Pots
The height of plants in pots showed no significant differences by Tukey test (p < 0.05) among treatments inoculated with the mixture of diazotrophic bacteria (with and without nitrogen fertilization) and the control. The addition of nitrogen fertilizer to the inoculated treatment resulted in a slight decrease in RDM (14.14 g). It did not differ statistically from the control treatment with the addition of nitrogen fertilizer (16.38 g), evidencing that N is a limiting factor in this experiment (Table 5). However, the inoculation without nitrogen fertilizer promoted a greater accumulation of root dry matter compared to the control treatment.
The evaluation of the SDM accumulation showed that the mixed inoculation promoted a positive effect on plant development without the addition of nitrogen fertilizer (41.7 g), differing from the other treatments (Table 4).
The results suggest that the inoculation used with the commercial variety RB92579 affected the interaction with inoculated bacteria. A better understanding of the plant-bacteria interaction, the selection of diazotrophic endophyte strains and the variety of cane needs to be further studied aiming a maximum benefit of BNF.
Microbial inoculants are an alternative method to increase crop productivity and may reduce the use of chemical fertilizers, which is one of the agricultural practices that affect the environment [30]. The positive effects of inoculating some bacteria during plant growth may be associated to the BNF process and the synthesis of growth hormones produced by bacteria. Among the effects associated with this synthesis of hormones, the growth of lateral and adventitious roots, the stimulus to cell division and the elongation of roots and stems are mentioned [31]. This may explain the accumulation of root dry matter.
Similar results were observed by some authors. Lin et al. [32], upon inoculating two strains of Enterobacter spp., observed that both strains increased the biomass content and the nitrogen of micropropagated sugarcane seedlings grown with a nitrogen fertilizer equivalent to 180 kg of urea ha −1 , a recommended nitrogen fertilization dose for the cultivation of the sugarcane ROC22 at the seedling stage. [4], upon inoculating in micropropagated sugarcane plants different species of diazotrophic bacteria, isolated and in mixtures, observed that Herbaspirillum sp., A. amazonense and a combination of five strains of different bacteria showed a significant increase in the accumulation of fresh mass in the culms of plants, evidencing a 30% contribution of BNF. However, the individual inoculation of G. diazotrophicus promoted a negative effect on the accumulation of fresh mass of culms compared to the non-inoculated control. The results presented by [9] showed that the inoculation response in micropropagated SP 701143 seedlings at the rooting stage showed variations that may have been dependent on several factors, including plant genotype and the environment. In this study, the inoculation with the strains PAL3 and CBAmC caused a significant increase in the accumulation of culm dry matter compared to the non-inoculated control treatment. However, the accumulation of N in plant tissues grown in pots after 180 days of growth showed that plants inoculated with a mixture of the strains PAL5 and HCC103 and the individual strains HRC54, Z94 and CBAmC showed a higher nitrogen content in the tissues. [33], studying potted micropropagated seedlings, observed that the total biomass increased due to inoculation with one strain or a combination of strains without nitrogen fertilization. [34] inoculated a mixture of diazotrophic bacteria and mycorrhizal fungi in micropropagated sugarcane plants and obtained an effect equivalent to half the recommended dose of nitrogen fertilizers for potted plants. [4] demonstrated that the combined inoculation of associative and endophytic bacteria promotes a synergistic effect compared to the individual inoculation of bacteria in micropropagated sugarcane plants. Increases of 30% in the accumulation of N in plants via BNF were observed for these plants.

Effects on Growth
The contribution of endophytic bacteria for the nutrition of legume plants by BNF is well known. Among non-legume species, BNF is still subject of much discussion. The contributions observed are varied and depend on specific interactions among bacterial and plant genotypes [35].
The inoculation of micropropagated sugarcane plants has already been per- with the strain PAL-5 associated with small doses of nitrogen, as shown by [36].
When the colonization of the plant is established, one result arising from the association is the promotion of plant growth by direct and indirect mechanisms.
In addition to fixing N, endophytic diazotrophic bacteria produce plant growth hormones such as auxin and gibberellic acid [37], Improvements in nutrient absorption are also reported [38].
Several experiments demonstrated that endophytic bacteria may indirectly benefit the development of the plant, increasing the plant's tolerance to biotic and abiotic stresses [39] [40] [41]. Beneficial results from such associations in sugarcane plants include a significant increase in plant height and biomass, root length and production of dry matter [42] [43].
Current evidence indicates that the BNF process performed by diazotrophic bacteria may contribute up to 60% to the sugarcane's N uptake [2], and that it depends on the plant genotype and on its interaction with various associative bacteria genera [44].
Quantitative analyses of BNF and the promotion of plant growth evidenced that plant and bacterial genotypes are important factors to the control of association efficiency [45]. In this context, the determination of the best combination between diazotrophic bacteria and plant varieties to obtain the maximum benefit of such association in agriculture is a challenge in this area.

Conclusion
This study showed that inoculation using a temporary immersion bioreactor is possible. This is the first inoculation report for seedlings using this system. The use of homologous strains may also have contributed to the benefit of the interaction with the plant (sugarcane variety RB92579). The results suggest a high response potential to inoculation and optimization of the process on a commercial scale.