Chlorophyll concentration and production of Urochloa decumbens treated with diazotrophic bacteria and thiamine in the Brazilian Cerrado Concentración de clorofila y producción del pasto Urochloa decumbens tratado con bacterias diazotróficas y tiamina en el Cerrado brasileño

The effects of application of Azospirillum brasilense and thiamine on chlorophyll concentration and forage mass of Urochloa decumbens were evaluated in a small plot experiment conducted in Goiânia, Goiás, Brazil. The treatments were applications of: A. brasilense (concentration of 10 mL/L); thiamine at 2 concentrations: 50 and 100 mg/L; combinations of A. brasilense and thiamine at the 2 concentrations; and a Control treatment (untreated grass). At the first harvest there was a trend for applying A. brasilense, either alone or in combination with thiamine, to increase the concentrations of chlorophyll, but differences were not always significant at P<0.05. Dry mass of forage from applying A. brasilense plus thiamine at 100 mg/L was greater than that for Control and thiamine at both concentrations. At the second harvest, chlorophyll concentrations were not affected by treatment (P>0.05), while dry matter production of forage from applying A. brasilense alone and thiamine at 100 mg/L was greater (P<0.05) than that of Control and thiamine at 50 mg/L. Larger-scale and longer-term studies to validate these preliminary findings are needed.


Introduction
Brazil has the largest cattle herd in the world, with about 218 million animals which represent approximately 15% of the world's total cattle population (FAO 2017). Most of these animals are raised on pasture, generating a product in great global demand (Jacinto et al. 2005). The production of cattle on pasture is dependent on both forage quantity and quality. As for other grasses, applying N fertilizer has a significant impact on the production of species of the genus Urochloa (Duarte et al. 2020). Economically, applying N fertilizer is a significant component of the costs of grass production (Costa et al. 2015).
Although N is commonly applied as chemical fertilizer, biological fixation through diazotrophic bacteria of the Azospirillum genus has been studied as a technique for the provision of atmospheric N to grasses (Hungria 2011), e.g. maize (Longhini et al. 2016). Nitrogen plays a major role in plant development, participating in the synthesis of several compounds, such as nucleic acids and proteins, as well as photosynthetic activity (Taiz et al. 2017). Chlorophyll concentration in a grass is generally accepted as a suitable indicator of a plant's N status and consequently crude protein (N × 6.25) concentration (Rocha et al. 2010;Rincón Castillo et al. 2019).
In addition to N fertilizer, other compounds with biostimulating and protective effects, such as vitamins, can improve physiological and morphological characteristics of species of commercial interest. Among the vitamins studied, thiamine (vitamin B1) has the capacity to stimulate the production of secondary metabolites, which exert antioxidant activity and avoid the degradation of the photosynthetic system (Goyer 2010;Kaya et al. 2015). This vitamin is also related to the ability to stimulate the accumulation of energy reserves in plant tissues (Barakat 2003); its application has been shown to provide productive gains in beans (Phaseolus vulgaris) (Vendruscolo et al. 2018).
The present study aimed to evaluate the effect of inoculation with Azospirillum brasilense and treatment with thiamine, either alone or in combination, on forage mass production and chlorophyll concentration of Urochloa decumbens, an economically important grass in the Brazilian Cerrado.

Materials and Methods
The study was conducted in Goiânia, Goiás, central Brazil (16°40' S, 49°15' W; 750 masl). The climate in the experimental area is tropical with a rainy season from October to April and a dry season from May to September. The monthly average temperatures range from 20.8 °C in June and July to 25.3 °C in October (Cardoso et al. 2014).
The experiment was conducted in a pasture of U. decumbens cv. Basilisk using a randomized complete block design with 5 replicates. The 6 treatments used in the study were as follows: 1 -Control; 2 -Azospirillum brasilense (NITRO 1000 Gramineae, NITRO 1000, Cascavél, PR, Brazil) at a concentration of 10 mL/L of water; 3 -Thiamine (Neon, Suzano, SP, Brazil) at a concentration of 50 mg/L; 4 -Thiamine at a concentration of 100 mg/L; 5 -Thiamine at a concentration of 50 mg/L + A. brasilense; 6 -Thiamine at a concentration of 100 mg/L + A. brasilense. The plots were established in an area of existing 5-year-old pasture, and each plot was 1 × 1 m. Products were applied via a manual sprayer at a volume equivalent to 200 L/ha.
The pasture had previously been mown quarterly. Prior to the application of the treatments, on 29 November 2016, the area was mown, leaving a stubble height of approximately 10 cm. Treatments were applied 5 days after mowing. For treatments that combined A. brasilense and thiamine, the products were mixed before application.
The evaluations were carried out when plants started flowering, on 2 occasions, the first at 50 days after the initial mowing (first harvest, 18 January 2017) and the second after a further 45 days (second harvest, 4 March 2017). At this time, the concentrations of chlorophyll a, chlorophyll b and total chlorophyll were evaluated using a digital chlorophyllometer (CFL1030; Falker, Porto Alegre, RS, Brazil) on 5 flag leaves per plot. All forage on each plot was harvested at about 10 cm above ground level and weighed immediately. Samples (200 g) of green forage were selected, packed in brown paper bags and placed in a forcedventilation oven at 65 °C until constant weight and weighed with a digital scale (ML 600, Marte, São Paulo, SP, Brazil) to determine dry matter yields. The data obtained for each variable were submitted to analysis of variance and compared by the Tukey test at P<0.05. For data analysis the computer program System for Variance Analysis -SISVAR was used (Ferreira 2014).

Results
At the first harvest there was a trend for treatments that included A. brasilense, either alone or in combination with thiamine, to increase the relative concentrations of chlorophyll, but differences were not always significant at P<0.05 (Table 1). Green mass of forage was greater (P<0.05) for A. brasilense plus thiamine at 100 mg/L than for Control and thiamine at 100 mg/L. Dry mass of forage for A. brasilense plus thiamine at 100 mg/L was greater than that for Control and thiamine at both concentrations.
At the second harvest, chlorophyll concentrations were totally independent of treatment. Application of A. brasilense alone produced more green mass than the Control and thiamine at 100 mg/L (Table 1). However, dry matter production of forage was greater (P<0.05) for A. brasilense alone, and for thiamine at 100 mg/L was greater than that of Control and thiamine at 50 mg/L.

Discussion
The positive response to the inoculation with Azospirillum is due to the bacteria's capacity to biologically fix atmospheric N through the enzyme nitrogenase (Hungria 2011), leading to increased synthesis of nucleic acids, proteins and hormones, which are essential to plant development, as well as to increased photosynthetic activity (Taiz et al. 2017). This is shown by the increase in the chlorophyll concentrations at the first harvest, since these concentrations are directly related to the N concentrations in the leaves (Rocha et al. 2010).
The superior results obtained at the first harvest for the combined application of A. brasilense and thiamine are also related to the ability of the vitamin to improve the conditions of the photosystem by protecting it, avoiding its degradation by oxidation (Goyer 2010;Kaya et al. 2015). Consequently, with the improvement of physiological conditions, there is an increase in the accumulation of energy reserves of tissues (Barakat 2003), which can be used for plant development and tissue maintenance during periods of stress (Taiz et al. 2017).
Although treatments had no effects on chlorophyll concentration at the second harvest, applying A. brasilense and thiamine at 100 mg/L either alone or in combination significantly increased dry mass yields over that of Control. This response may be related to the priming effect of both the bacteria and the vitamin. Both are recognized for their ability to mitigate stress caused by water restriction (Leite et al. 2019;Vendruscolo et al. 2020) and improve the nutritional condition of plants (Kaya et al. 2015;Galindo et al. 2020). However, under adequate growth conditions, including e.g. good availability of water, their effect is reduced (Goyer 2010;Naoe et al. 2020). In the present study, this became evident, since the second harvest occurred at the end of the rainy season, while the first harvest was on plant regrowth that occurred under conditions of erratic rainfall.
Inoculation with A. brasilense and application of thiamine, alone or in combination, seemed to have some potential for the maintenance and improvement of physiological and productive characteristics of U. decumbens. Larger-scale studies to test the repeatability of these findings seem warranted as well as longer-term studies to endeavor to clarify the mediumand long-term effectiveness of these treatments.