Predominantly in the agricultural production system, areas with pasture are occupied with plants from the Poaceae family, which require high doses of nitrogen fertilizers to enhance their yield. However, the high cost compromises the supply of this input, leading to low availability of nitrogen (N) for cultivation, which leads to degradation of pastures (Canto et al. 2016 and Vasques et al. 2019). The yield recovery in these areas requires sustainable alternatives (Dias-Filho 2017). In this sense, the introduction of forage legumes associated with biostimulants can be adopted, envisioning productive systems with less impact on the environment.
Fodder legumes, such as forage peanuts (A. pintoi cv. Amarillo MG 100), are efficient in biological nitrogen fixation (BNF), have better nutritional quality, and can reduce production costs due to the acquisition and availability of N (Olivo et al. 2019). Also, this stoloniferous legume has a good adaptation to acid soils, resistance, and tolerance to pests and diseases (Simeão et al. 2017). Inoculation with diazotrophic bacteria of the Bradyrhizobium (BD) genus ensures more efficient nodulation and fixation, contributing to the yield and stability of pastures (Terra et al. 2019).
Complementary to BD effects in association with forage peanut, humic substances (HS) appear to be promising in stimulating plant growth. This benefit may be due to hormonal signaling that results in more structured root systems and thus greater nutrient uptake and final yield (dos Santos et al. 2021). In addition, they also act in osmotic regulation and promote better photosynthesis rates (Du Jardin 2015).
This study is based on the premise that the application of HS benefits the development of forage peanut when grown in soil at a stage of degradation. Considering the above, the authors aimed to evaluate the yield, nutritional and physiological parameters in these plants when subjected to applications of different concentrations of HS.
Material and Methods
The soil used was representative of a degraded pasture situation in the region of Jerônimo Monteiro-ES, collected in the 0-20 cm layer and classified as Ultisol (dystrophic Red-Yellow Argisol, Brazilian Classification System) (Santos et al. 2013). Subsequently, the soil was air-dried and sieved through a 2.0 mm mesh sieve to obtain the air-dried fine soil (ADFS) as well as the physical and chemical characterization (Teixeira et al. 2017) (table 1).
pH | P | K | Ca | Mg | Al | H+Al | SB | CEC | ECE | OM |
---|---|---|---|---|---|---|---|---|---|---|
(H2O) | ppm | cmolc kg-1 | % | |||||||
4.10 | 0.25 | 0.06 | 0.23 | 0.08 | 0.8 | 5.5 | 0.38 | 5.88 | 1.18 | 0.74 |
BS | m | S | B | Fe | Cu | Mn | Zn | Sand | Silt | Clay |
% | ppm | % | ||||||||
6.46 | 67.8 | 24.8 | 0.55 | 48.73 | 0.15 | 1.74 | 0.66 | 54.36 | 2.96 | 42.68 |
SB - sum of bases (SB = Ca +Mg + K); CEC - cation exchange capacity (at pH 7.0); ECEC - effective cation exchange capacity; OM - organic matter; BS - base saturation; m - aluminum saturation.
The experiment was conducted in a greenhouse at the Federal University of Espírito Santo (20° 45’ S and 41°29’ O) in the period January-March 2019. Daily maximum and minimum temperature values were recorded inside the greenhouse. Daily maximum temperatures reached 38.0 °C, with an average of 34.7 °C, and the daily minimum temperatures averaged 20.9 °C. The average humidity in this same period ranged from 57 % (minimum) to 78 % (maximum).
The experimental design was in randomized blocks, with five HS concentrations (0, 20, 40, 60, and 80 mg L-1 of C) and five replications, totaling 25 experimental units. Seedlings were obtained by seeds germinated in a tray. Ten days after emergence (DAE), six seedlings were transplanted, which presented greater homogeneity in force and size for each definitive pot (with a capacity of 5 dm3). Soil moisture was maintained at 60 % of maximum storage capacity (pre-determined by gravimetric analysis method), based on weight difference by precision balance and deionized water was applied to the pots daily.
Seeds were disinfected according to Hungria et al. (2010) after removing the pericarp. For inoculation with Bradyrhizobium spp. BR1433 (SEMIA 6440) performed in all treatments, the authors followed the recommendation of Purcino et al. (2000) to better adherence of the inoculants to the seeds, which were subsequently dried in the shade for 15 minutes.
The HS were extracted from vermicompost (Canellas et al. 2002) and presented pH (H2O): 6.29; C: 60.4 g kg-1; P: 987.50 mg dm-3; for K, Ca, Mg, Al, and H + Al, 2.57, 7.71, 8.25, 0.0, 1.80 cmolc dm-3 were found, respectively; SB: 18.52 cmolc dm-3 and CEC: 20.32 cmolc dm-3. The concentrations of HS were based on the carbon content (C). The chemical fractionation of humic substances was carried out according to the method adapted from Swif (1996). The percentages found through the chemical fractionation of vermicompost humic substances corresponded to 67% and 33% in the fraction fulvic acids and humic acids, respectively.
The first HS application was performed at 20 DAE and the second, at 40 DAE, between 6 and 7 p.m. A manual pressure sprayer with an adjustable nozzle was used. The plants received 2.0 mL in the first application and 4.0 mL in the second (largest shoot dry matter) per plant. This quantity was standardized, aiming at the total covering of the leaves during the application periods.
At 60 DAE, the total chlorophyll (Chl t), chlorophyll a (Chl a), and chlorophyll b (Chl b) content in fully expanded leaves (2 leaves/plant) was estimated by the portable chlorophyll meter, ClorofiLOG (Falker Agricultural Automation, Brazil), between 8 and 11 a.m. The total height (cm) was determined, and after root collection and washing, the active nodules (pinkish colors) were counted in each pot (NN). No high frequency of inactive nodules was observed.
Determinations of the chlorophyll a fluorescence were quantified using a portable modulated light fluorometer (PSI FluorPen, FP 100 model, Drasov, Czech Republic), from which the maximum quantum yield of PSII (FV/FM) was obtained after adaptation of the leaves to the dark for 15 minutes. The measurements were made simultaneously and on the same leaves in which the chlorophyll content was read.
The plants were separated into roots and shoots, stored in paper bags, and placed in an oven at 65° C for 72 hours (until reaching constant weight) to determine the shoot dry matter (SDM) and root dry matter (RDM). The samples were subjected to grinding in a knife mill for later determination of nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), and magnesium (Mg).
For chemical analysis of leaf N, sulfuric digestion was performed, followed by Kjeldahl distillation, according to Carmo et al. (2000). For foliar chemical analysis of the other nutrients, nitric digestion was performed, and the reading was performed according to Malavolta et al. (1997).
The data were analyzed for normality (Shapiro-Wilk) and homogeneity of variance (ANOVA). Regression analysis was performed for the quantitative factors, where the models were chosen based on the significance of the regression coefficients using student t-test at the 5 % significance level and the coefficient of determination (R2). For plotting the graphics, SigmaPlot® software, 10 version (Systat Software Inc 2006) was used.
Results and Discussion
The application of different concentrations of HS in forage peanuts increased the SDM by around 10.98 % (p≤ 0.05). It was estimated that the maximum yield of SDM would be obtained with the concentration of 31 mg L-1 of C (figure 1).
Humic substances act similarly to hormones or alkamides. They also stimulate a greater activity of H+-ATPases in the plasma membrane, which generates an electrochemical gradient with the extrusion of H+, therefore greater permeability of the cell wall (Canellas et al. 2015, Zandonadi et al. 2019). Besides, they promote greater emission of radicular hair and thin lateral roots, increasing the root system surface area and exuding low molecular weight organic acids (dos Santos et al. 2021). This activity improves the rhizosphere environment and increases nutrient absorption (Du Jardin 2015).
The regression was not significant (p>0.05) for RDM. Rosa et al. (2009) evaluated the same species and found the root system growth when applying HS concentrations from mineral coal in the nutrient solution. It can be inferred that the composition, as well as the HS concentrations, induce different stimuli. The presence of root hair has been observed with HS application; however, such modification is difficult to measure from the dry matter.
There was an increase in NN (figure 2) up to the C concentration of 40 mg L-1 (52.8 nodules pot-1), about 25.41 to 45.85 % compared to the control treatment (36.2 nodules pot-1). Note that the variation obtained in this parameter is highly related to HS concentrations (R2=0.968). Capstaff et al. (2020) demonstrated, employing transcriptional RNA analysis, that leguminous forages treated with humic acid have positive regulation of many important genes in nodulation.
The quadratic model (R2 = 0.90; p ≤ 0.01) (figure 2) reveals that the concentration of 55 mg L-1 of C of HS provides the best result of plant height. This result can be associated with the hormonal effect of HS when applied in low concentration. A reduction in plant height was observed at the HS concentration of 80 mg L-1 of C, classifying this concentration as inhibitory to plant growth.
Rosa et al. (2009), when applying 11 mg L-1 of C in Phaseolus vulgaris L. (Fabaceae), observed an increase in SDM. For Pinheiro et al. (2018), there was an increase of 44 % in plant height and 196 % in the production of SDM from Urochloa decumbens (Poaceae) when 60 mg L-1 of C was applied. According to Gao et al. (2015), HS stimulates cell metabolism and nutrient transport, generating increased cell density of Bradyrhizobium spp., in addition to promoting the expression of BNF-related proteins.
The concentrations of HS were significant in the regression analysis for N and Ca (p ≤ 0.01) and K and Mg (p ≤ 0.05) (figure 3). For P, there was no adjustment to the linear and quadratic models.
The applied HS concentrations increased the nutrient content in the shoot of the forage peanut plants compared to the control. Increases of 12 % N (37.1 to 41.5 mg pot-1), 8.6% K (35.3 to 38.3 mg pot-1), 13.5 % Ca (21.8 to 24.7 mg pot-1), and 7.5 % Mg (9.8 to 10.53 mg pot-1) were found (figure 3). The largest increments in N and K accumulation based on the regression adjustment would be obtained at C concentrations of 43 and 41 mg L-1, respectively. The concentration of 38 mg L-1 of C would be the best for Ca and Mg accumulation.
HS maximize nutrient uptake, even at low concentrations in the soil. Pinheiro et al. (2010) found that N accumulation showed a quadratic performance in response to different humic acid concentrations application in eucalyptus seedlings (Myrtaceae). The increase in the macronutrient content was also reported by Savita and Girijesh (2018), studying the soybean (Glycine max L.) (Fabaceae) and, Júnior et al. (2019) in mangosteen plants (Garcinia mangostana) (Clusiaceae) when humic acid is applied.
Humic acids act in the soil-microbiota-plant interaction, mainly in the availability and nutrient assimilation (Hayes and Swift, 2020). The BNF contribution to forage peanut can reach almost the totality of their N nutritional demand (Longhini et al. 2021). The higher N accumulation and nodulation rate may be correlated. This is due to the stimulation in root hair emission resulting from the addition of HS. Furthermore, Quaggiotti (2004) shows that application of humic acids increases NO3- uptake in maize due to mRNA regulation of its principal H+-ATPase.
K accumulation was elevated with increasing concentrations of HS up to 40 mg L-1 of C, in contrast to Rosa et al. (2009), who observed a decrease in the uptake rates of this element in beans. Although the Ca and Mg content was low in the soil, HS application raised their uptake rate, according to Pinheiro et al. (2010).
There was a slight decrease in P accumulation compared to the control, varying from 2.71 to 7.42 % depending on HS concentration application. These reductions were more expressive in the C concentrations of 20 and 40 mg L-1 and rising in the C concentration of 80 mg L-1. These findings can be attributed to these nutrients’ dilutions due to higher SDM in the referred concentrations, besides the low P content available in the soil.
There was an effect of HS concentrations only for FV/FM, among the evaluated physiological parameters (figure 4). Values that run out of the 0.75-0.85 range indicate photoinhibition damage in the PSII reaction center (Guidi et al. 2019).
The values of FV/FM ranged from 0.802 to 0.834, and the HS concentrations promoted an average increase of 2.99 % compared to the control (0.802), highlighting the applications of 80 mg L-1 of C (0.834). Only about 10 % of the FV/FM variability was not attributed to the HS concentrations effect (R2=0.89; p ≤ 0.01) (figure 4). Regardless of the treatment, there was no damage to the PSII reaction center.
Additionally, we showed in the course of the study, higher SDM results in improved nutrient uptake. This enhanced nutritional status may culminate in plants that are less susceptible to stresses (> Fv/Fm) (Fan et al. 2014). Perhaps antioxidant enzyme analyses could explore this point better, than chlorophyll contents.
The values of the Chl t, Chl a, Chl b, and Chl a/Chl b ratio did not fit into the regression models evaluated (p>0.05), showing a slight increase due to the HS applications (3.53; 3.91 and 3.58 %, respectively) compared to the control. The chlorophyll content was positively correlated with N content (r = 0.75), showing that the increase in HS concentrations led to a greater contribution of this element and higher chlorophyll levels.
Conclusions
The application of humic substances extracted from vermicompost increases the shoot dry matter, plant height, and the number of nodules, as well as the maximum quantum yield of the plants of Arachis pintoi cv. Amarillo MG 100, indicating improvements in the PSII reaction center.
The application of 40 mg L-1 of humic substances is recommended to obtain maximum N, K, Ca, and Mg absorption in forage peanut plants.