Seed Microbiolization Associate With Nitrogen Doses Increase the Nutrition of Tomato Fruits

Endophytic bacteria can promote growth and improve the quality of plant production. The objective of this work was to evaluate the efficiency of inoculation of a mix of non-host endophytic bacteria isolates in the nutrition of tomato fruits cultivated, fertilized with rock powder and different doses of nitrogen. The experimental design was in randomized blocks, in a factorial arrangement (2 × 6 + 3), with four replications. The treatments consisted of two methods of inoculation of the mix of endophytic bacteria: seed microbiolization and post-emergence inoculation; six doses of nitrogen fertilization: 0, 129, 258, 387, 516 and 645 kg ha -1 ; and 3 controls (without inoculation of the bacterial mix). The average export of macro and micronutrients in tomato fruits was, in descending order: K > N > P > Ca > Mg > S (averages of 1.33; 0.46; 0.07; 0.06; 0.05 and 0.05 g plant -1 , respectively) and Mn > Cu > Fe > Zn (means of 2.67; 1.98; 1.71 and 0.79 mg plant -1 , respectively). The inoculation method by seed microbiolization associated with nitrogen doses promoted significant increment in the dry mass of the fruits and in the content of the nutrients P, Ca, Zn, Fe and Mn.


Introduction
Tomato is one of the most important vegetable species in the world, both from an economic and social point of view, due to the value of production and the generation of direct and indirect jobs; and health, due to the nutraceutical benefits.World tomato production in the year 2019 was 181 million tons.Brazil, which is the 10th largest producer, represents about 2.2% of total production (FAO, 2020).
It is currently the most consumed vegetable in the country (IBGE, 2020), and also one of the most demanding in nutritional terms.About 91% of the minerals accumulated in tomato fruits are made up of potassium (K), nitrogen (N) and phosphorus (P).In highly weathered soils, N and P are the main limiting nutrients for increasing the productivity and nutritional quality of crops (Baldotto et al., 2012).phosphorus (P) (Purquerio et al., 2016).In highly weathered soils, N and P are the main limiting nutrients for increasing the productivity and nutritional quality of crops (Baldotto et al., 2012).
In recent years, the use of bioinoculants capable of promoting growth and increasing agricultural quality and production has been described by several authors.Among the microorganisms that promote plant growth, endophytic bacteria have shown enormous potential in improving nutritional quality, including tomato fruits (Tommonaro et al., 2021).
Endophytic bacteria are beneficial organisms that colonize the interior of healthy plant tissues, without causing symptoms or apparent damage to them.They act in the promotion of plant growth through the production of growth regulators, antagonistic effect to pathogens with the production of antibiotics and in the increment of mineral nutrition, through the fixation, solubilization and availability of nutrients (Rosenblueth & Martínez-Romero, 2006).
Despite the many known benefits, there is still much to be studied, mainly in agricultural crops under stressful climatic conditions, in the use of bacteria selected from another species, as well as on the most efficient inoculation methods and their interaction with conventional fertilization.Therefore, the objective of this work was to evaluate the inoculation efficiency of a mix of non-host endophytic bacteria isolates in the nutrition of tomato fruits, fertilized with rock powder and different doses of nitrogen.
The experimental design was in randomized blocks, in a factorial arrangement (2 × 6 + 3), with four replications and one plant per plot.The treatments consisted of two methods of inoculation of the mix of endophytic bacteria (IMB): seed microbiolization (SM) and post-emergence inoculation (PEI), six doses of nitrogen fertilization (DN): 0, 129, 258, 387 , 516 and 645 kg ha -1 , and three controls (without inoculation of the bacteria mix): (1) absence of nitrogen fertilization and application of rock dust-Natural phosphate of sedimentary origin-Itafós; (2) 100% nitrogen fertilization and application of rock dust; and (3) 100% nitrogen fertilization and application of simple superphosphate (conventional control, based on the current model of chemical fertilization).Among all treatments, only the third control did not receive rock dust as a phosphorus source.
The hybrid tomato 'Dominador F1', salad type, from the Persimmon group, with indeterminate growth, high vigor, good leafing, firm fruits with excellent color and shape, high resistance to TYLCV (Geminivirus), cycle 120 days was used.The soil, classified as eutroferric red latosol, was collected at a depth of 0-20 cm and sterilized in an autoclave.Fertilization was performed according to soil analysis and crop recommendations (Ribeiro et al., 1999).
The seeds were disinfected and dried in a laminar flow chamber on sterile filter paper for two hours.At SM, the disinfected seeds were immersed in bacterial suspensions and kept in an automatic shaker for one hour, at 150 rpm, 27 °C.After microbiolization, the seeds were kept for two hours in trays with filter paper and then sown in polystyrene trays, with 128 cells (40 cm 3 ), containing commercial substrate (Bioplant-HT ® ), sterilized in an autoclave.
In PEI, the disinfected seeds were immersed in sterile water and kept in an automatic shaker for one hour, at 150 rpm, 27 °C.Subsequently, they were kept for two hours to dry on filter paper and seeded according to the methodology described above.Seven inoculations were performed: the first, 10 days after seedling emergence and the following at 15, 30, 45, 60, 75 and 90 days after transplanting.In the first inoculation, 2.6 mL of the mix was applied per cell of the tray, while in the inoculations carried out after transplanting, 100 mL of the mix was applied per pot.Increases in DN resulted in gains in FDM in both IM (Figure 1).However, the highest averages were observed for seed microbiolization (SM), which presented a quadratic effect for this variable, with a maximum point at 51.1 g, estimated at a dose of 650 kg ha -1 of N. linear effect with an increment of 0.0483 g for each kilogram of N added.Halo et al. (2020) when evaluating the use of an endophytic, non-host fungus obtained from a plant in the desert observed, among multiple advantages, gains of 27% in the increase in weight of tomato fruit grown under water stress.SM also showed a quadratic effect for Ca, Zn and Fe contents, as a function of DN.The estimated doses as a function of the maximum points for these nutrients were 500; 486 and 410 kg ha -1 of N, respectively.Only the Mn content showed linear adjustment for both inoculation methods.Even so, the increments from the SM were higher than those from the PEI.For each kilogram of N added to the soil, there was an increment of 0.0081 and 0.0053 mg of Mn for these methods, respectively.These results suggest that SM may result in higher yield and nutritional quality of tomato fruits.According to Joe et al. (2012) and Souza et al. (2017) the inoculation of endophytic bacteria by the seed microbilization method is the most efficient, besides being economical, safe, reliable and fast (Hallmann et al., 1997).
In the study of the IM splitting within each DN level it can be observed that at the dose of 387 kg ha -1 of N, regardless of the variable, the SM presented higher, significant means, in comparison with the PEI.The gains were of the order of 65; 50; 47; 31 and 62 % for FDM and for the nutritional contents of Ca, Zn, Fe and Mn, respectively.The higher nutritional contents may be related to the release of siderophores, responsible for the chelation of Fe and Mn, and compounds that solubilize phosphate and zinc by the bacterial mix, thus improving the absorption of these nutrients by plants (Firdous et al., 2019;Zecchin & Mógor, 2017).Other studies, in tomato cultivation, also emphasize the efficiency of endophytic bacteria in the supply of macro and micronutrients (Barretti et al., 2008) and in improving the quality of the vegetable in a greenhouse (Mena-Violante & Olalde-Portugal, 2007).
The IM, independently of the DN, influenced the phosphorus content of the tomato fruit.Plants resulting from SM showed fruits with higher P content compared to those that received PEI.This result suggests that the mix of endophytic bacteria has the potential to solubilize low-solubility phosphate forms.This is probably due to the production of organic acids that reduce soil pH by increasing phosphate solubilization (He et al., 1996).
Research that addresses the use of endophytic, non-host bacteria and their different inoculation methods, associated with the use of fertilizers in agricultural crops, sensitive to stressful climatic conditions, is necessary to improve the use of resources and technologies in cropping systems, especially in cost reduction.The information obtained in this work can help researchers to understand the interactions between plant, endophyte and climate, where the best inoculation method and its relationship with the use of fertilizers was determined.

Conclusions
Endophytic, non-host bacteria present nutritional benefits in tomato crops even under heat stress conditions.The inoculation method by seed microbiolization associated with different doses of nitrogen in the tomato crop 'Dominador F1' promoted significant gains in the dry mass of the fruits and in the content of the nutrients P, Ca, Zn, Fe and Mn.

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Figure 2. N Figure 8