Production of Lettuce Seedlings with Association of Diazotrophic Bacteria and Humic Acids

The objective of this work was to verify the responses in agronomic and phy-siological characters of lettuce seedlings when submitted to different doses of humic acids and the presence or absence of inoculation of the bacterium Azospirillum brasilense. The experiment was carried out at the Protected Cultivation Station and Biological Control Teacher Mário César Lopes, belonging to the Experimental Stations Nucleus of the State University of the West of Paraná. The experimental design was a randomized block design, in a 5 × 2 factorial scheme, with four repetitions, totaling 40 plots. The first factor con-sisted of five solutions containing humic acid (100%, 75%, 50%, 25% and 0%) and the second factor was the presence or absence of the diazotrophic bacterium Azospirillum brasilense obtained by commercial product. Germination tests were carried out in addition to the determination of agronomic characters such as the number of fully expanded leaves, plant height, stem diameter, main root length,


Introduction
The lettuce (Lactuca sativa L.) is one of the most important leafy vegetables in Brazil, belonging to the Asteraceae family, being cultivated and consumed throughout the country [1]. As a result, the production of quality seedlings is a determining factor for the success of any enterprise related to cultivation.
The production of seedlings is a technique used by the producer to reduce the harvest time, thus increasing the number of productive cycles per year, consequently allowing better utilization of the area and greater financial return [2].
However, malformation of seedlings reduces the initial growth of plants in the field, affecting the crop yield and limiting the genetic potential of cultivars [3].
Some techniques favor the production of quality seedlings, such as water availability with good quality, cultivation in appropriate places or in greenhouses, use of substrates and containers for this purpose. In addition, other ecological and economic alternatives can be adopted for the quality of seedlings, such as the use of humic substances and growth promoting bacteria [4]. The use of growth promoting and nitrogen fixing bacteria that may benefit the initial development of plants by several factors, such as nitrogen fixation, root development improvement among others are good techniques for lettuce culture [5].
Some researchers have demonstrated the influence of humic acids on plants, such as the promotion of rooting [6], the growth and development of root and aerial structures, as well as crop productivity and quality [1], with potential gain in economic income for the producer [4]. Its use as a biostimulant can attenuate the effect of potentially toxic substances, promoting improvements in the physical and biological conditions of the medium, improving the absorption of nutrients, and inducing changes in several metabolic processes, such as in the proton pump activity affecting the potential of membranes, on glycolysis and Krebs cycle, the release of molecules with hormonal activities, such as auxin, cytokinin, gibberellin and polyamines [7] [8].
The increase in crop production can be optimized through the use of a biofertilizer with prokaryotic microorganisms, capable of fixing atmospheric nitrogen in the soil and make it available to plants [9]. The mixture of humic substances associated with growth promoting bacteria shows positive results in the development of various crops such as corn [7], wheat [10], tomato [11] and lettuce [4].
However, the success in the combined use of these compounds is dependent on several factors, such as the crop in which the substance is used, the dilution applied and the origin of the humic substances used [11] [12]. Therefore, studies

Material and Methods
The experiment was carried out at the Protected Cultivation Station and Biolog- with rains well distributed throughout the year and hot summers [13]. The annual mean temperature is between 295.15 to 296.15 K and the mean annual precipitation total of 1600 to 1800 millimeters [14]. Regarding the temperature inside the greenhouse during the experiment period are presented in Figure 1.
The experimental design was a randomized block design, in a 5 × 2 factorial scheme, with four replications, totaling 40 plots and being evaluated the results of the separated factors and the interaction between them. The first factor consisted of five solutions containing humic acid (100%, 75%, 50%, 25% and 0%), the dilutions were obtained by proportionally using the content in milliliters of humic acid with distilled water, extracted from an oxisol cultivated oat and handled in livestock farming systems integration, obtained through the differential   [15] and adapted by Benites et al. [16]. The second factor was the presence or absence of bacteria diazotrophic A. brasilense, obtained by commercial product the basis of strains AbV5 and AbV6, containing 2.0 × 10 8  to determine the emergency rate (ER) [17], mean emergence time (MET) [17], emergence speed index (ESI) [18] and mean speed of emergency (MSE) [17].
where: ER is the emergency rate; N is the number of emerged seeds; and A is the total number of seeds placed to germinate.
The data obtained between the treatments were submitted to analysis of variance (ANOVA) and the means compared by the Tukey test (P ≤ 0.05), using the statistical program SISVAR 5.3 [20].

Results and Discussion
For the variables ER, MET, ESI, MSE, CR and H there were no significant differences by variance analysis for factors doses of humic acids and the absence or presence of diazotrophic bacteria, in addition to the combined factors (Table 1).
It can be observed in Table 1 the significant absence for characteristics of ER, MET, ESI and MSE, where the dilution of humic acid and the presence or absence of A. brasilense caused interference in the germination and emergence of lettuce. Cotrim et al. [10] found that inoculation of substances, such as humic acid, and/or, A. brasilense, in the early stages of germination and emergence of seeds tend not confer differences in these variables, since these phases, the embryonic axis depends on the accumulated resources throughout the process of seed maturation.
The results found, corroborate with those found by Venâncio et al. [21] that did not identify significant differences in the variables related to germination and emergence for pepper seeds, except in the ESI under some dosages of humic acids tested. These results of ER corroborate with those obtained in some studies of germination of different crops, such as corn [12] and chili [21].
Catão et al. [22] observed that high temperatures (308.15 K) negatively influ- Regarding the characteristics of LR and H, it can be observed that there was no difference between the dilutions of humic acid nor between the presence or absence of inoculation. In contrast, Santos et al. [23], evaluating the dilutions of humic acid and nitrogen in the production of seedlings of Lactuca sativa L., found significant effects. These divergences occur mainly due to the presence of nitrogen, which assists in the growth process of the evaluated characteristics and due to the essential that is this nutrient for the initial development of the seedlings.
For the dry matter of roots (RDM) and total dry matter (TDM), a significant interaction was found for both the absence and the presence of bacteria with dilutions of humic acids (P ≤ 0.05), and for leaf dry matter (DSM) only obtained interaction in the absence of inoculation with the dilutions of humic acids ( Figure 2). These results indicate that inoculated seedlings presented variable responses to the doses used.
It can be observed in Figure 2 that the inoculation for TDM and RDM showed cubic behavior, which was observed a peak point at a dilution of 50% humic acid, leading to a decrease in their mass. For the absence of bacteria showed an increasing linear behavior with the increase of the dilutions of humic acid employed, causing an increase in total dry mass and root. According to Façanha et al. [24], the application of humic substances promotes an increase in root development due to its stimulating effect similar to plant hormones, highlighting the ability of humic acids to provide an increase in an enzyme that   the use of humic substances in other crops, such as cucumber [26], tomato [27] and wheat [10]. Borcioni et al. [1] observed higher lettuce NL with the application of these humic substances up to the dose of 4 ml L −1 , from this concentration, the number of leaves reduced.
For the SD, significant differences were found for inoculation with the bacterium. The inoculated plants presented a SD average of 0.24 cm and those not inoculated 0.22 cm. Differences were also observed for the dilutions of humic acids, being detected significance for the quadratic adjustment, with coefficient of determination explaining 76% of the observed data. For this variable under the conditions of the study, a reduction in stem diameter averages occurred with the increase of the dilution of humic acids up to approximately 66% and, from this dilution, there was an increase in the means of this variable. Besides the presence of bacteria influenced to a larger diameter when compared to non-inoculated plants.
Cavalcante et al. [6] observed reduction in stem diameter in passion fruit seedlings with applications of humic substances. Nomura et al. [28] found a decrease in banana SD in winter when they tested two commercial biofertilizers.
Some authors argue that seedlings with larger diameter tend to favor the survival American Journal of Plant Sciences rate of these seedlings after transplanting [28] [29] [30]. This tendency is due to the fact that this variable is strongly influenced by the number and vigor of the root system, which allow plants more tolerant to biotic stresses, thus favoring initial development as well as increasing production [28].
In relation to the IQD, significant differences were found only for the dilutions of humic acid, which the best fit was the quadratic one due to the different dilutions tested and with a correlation coefficient explaining 53% of the observed data. The behavior of the obtained curve pointed to increase in IQD up to approximately 50% of humic acid dilution, with IQD of 0.0054, with decrease in the highest dilutions. The IQD is obtained by the ratio between several variables, TDM, H, SD, DSM and RDM, and from its result it is possible to infer about the quality by means of its equilibrium, thus, the higher the IQD value obtained, the higher the quality of seedlings produced. Santos et al. [30] observed improvement in the quality of lettuce cv. "Verônica" with the application of humic acids, obtaining a IQD up to 0.0123.
The increase in the dilution of humic acid negatively affected the SD, which has negative consequences for the IQD of the lettuce seedlings. However, due to the fact that this index takes into account several parameters of seedling growth [29], it was still possible to verify IQD higher than those observed in other studies involving this index in lettuce seedlings [31] [32].
In general, the results presented here indicate that the seedlings produced were of good quality. It is possible to emphasize that the use of humic acid up to the 50% dilution applied in seed was beneficial for the quality of the seedlings produced.

Conclusions
The main conclusions are: The association of diazotrophic bacteria and humic acids did not influence the emergency rate, mean emergence time, mean speed of emergency, emergence speed index, plant height, and root length of lettuce seedlings.
The dilutions of humic acids, regardless of inoculation with bacteria diazotrophic favored the development of the number of leaves, stem diameter and Dickson quality index.
Inoculation of lettuce seeds diazotrophic bacteria associated with the addition of humic acids favored the development of the dry mass of root and total dry matter.