Vermicompost alkaline extract mitigates water stress in soybean plants¹

Extrato alcalino de vermicomposto mitiga o estresse hídrico em plantas de soja

ABSTRACT

Soybean is an intensive care crop that overexploits the soil and requires great amounts of water and fertilizers. The objective of the study was to assess the potential of the vermicompost alkaline extract in mitigating water stress in soybean cultivated in greenhouse. Soybean plants were subjected to different water availability values - 50, 70 and 90% of the pot holding capacity, and presence/absence of vermicompost extract at the optimal concentration of 135 mg L^-1. At the end of the experiment, biometric variables of plants and enzymatic activities in root tissues were measured. The optimal extract dose of 135 mg L^-1, under control (90% of pot holding capacity) and mild stress (70% of pot holding capacity) conditions increased stem and root biomass. Under severe stress (50% of pot holding capacity), it positively influenced root architecture. The biomass decreased compared to plants that did not receive the alkaline extract, whereas enzymatic activities were depleted under all the conditions.

Key words:
Glycine max; abiotic stress; humus; antioxidants; humic substances

HIGHLIGHTS:

Vermicompost alkaline extract stimulates soybean growth depending on optimal concentration.

Optimal vermicompost alkaline extract concentration minimizes the negative effects of water stress.

The optimal concentration of vermicompost alkaline extract decreases the activity of enzymes related to oxidative stress.

RESUMO

A soja é uma cultura de tratos intensivos que superexplora os solos e requer grande quantidade de água e fertilizantes. O objetivo do estudo foi verificar o potencial do extrato alcalino de vermicomposto na mitigação do estresse hídrico no cultivo de soja em casa de vegetação. As plantas de soja foram submetidas a diferentes disponibilidades hídricas - 50, 70 e 90% da capacidade de retenção do vaso, e presença/ausência de extrato de vermicomposto na concentração ótima de 135 mg L^-1. No final do experimento, as variáveis biométricas das plantas e as atividades enzimáticas nos tecidos radiculares foram medidos. A dose ótima de extrato de 135 mg L^-1, nas condições controle (90% da capacidade de retenção do vaso) e estresse leve (70% da capacidade de retenção do vaso) aumentou a biomassa do caule e da raiz. Em estresse severo (50% da capacidade de retenção do vaso), influenciou positivamente a arquitetura da raiz. A biomassa diminuiu em relação às plantas que não receberam o extrato alcalino, enquanto as atividades enzimáticas foram reduzidas em todas as condições.

Palavras-chave:
Glycine max; estresse abiótico; húmus; antioxidantes; substâncias húmicas

Introduction

Soybean (Glycine max L.) is one of the most cultivated grains in the world, with Brazil being a major producer and exporter of the oilseed (Beutler, 2024). Crop yield is strongly influenced by climatic conditions, soil quality and management, adapted cultivars and water availability (Agovino et al., 2019; Pittarello et al., 2021).

According to the Sixth Assessment Report of the IPCC (2023), climate change is influencing the rainfall pattern and affecting the dynamics of the hydrological cycle worldwide, modifying the available water content in cultivated soils. In soybean, drought hastens grain maturation and depletes daily dry matter accumulation rate. Moreover, water deficiency causes leaf area reduction, abortion of flowers and pods and the development of empty pods (Kulundžić et al., 2022).

To achieve a more efficient and sustainable agriculture, crops must be helped to exploit soil resources and water under both favorable and unfavorable environmental conditions. One of the potential solutions is the use of biostimulants, in particular extracts of humic substances (HS) (Carletti et al., 2021). HS are biomolecules resulting from the decomposition and polycondensation of animal and plant residues, through soil chemical and microbial activities. They are organized in a super molecular net that can be dismantled by root organic acids, releasing entrapped products of soil microbial activities, like auxins (Souza et al., 2022).

HS positively influence root architecture and nutrient uptake, increasing plant yield (Nardi et al., 2021). Furthermore, HS bioactivity enhances antioxidant enzymatic activities, increasing plant tolerance to abiotic stresses (Santos et al., 2022). These plant responses allow HS to be considered as plant biostimulants (Mackiewicz-Walec & Olszewska, 2023). The objective of the study was to assess the potential of the vermicompost alkaline extract in mitigating water stress in soybean cultivated in greenhouse.

Material and Methods

The experiment was conducted in a greenhouse in the Olericulture Sector of Universidade Federal dos Vales do Jequitinhonha e Mucuri (UFVJM) in Diamantina - MG, Brazil (Campus JK - MGT 367 - Km 583, 5000, Alto do Jacuba), at the geographical coordinates 18° 12’ 06.8” S and 43° 34’ 24.9” W and 1280 m altitude approximately, from June 25 to November 11, 2022, for a total of 139 days, until the soybean reached grain harvest stage (R8). During the experiment, temperatures ranged from 11.02 to 28.34 °C, with averages between 17.69 and 22.25 °C. Relative humidity ranged from 59 to 76% according to National Institute of Meteorology - INMET. The greenhouse used was air-conditioned and the average temperatures inside varied from 24 to 28 °C and the relative humidity was maintained at around 75% during the experiment.

The experiment was set up in a completely randomized design in a 2 × 3 factorial scheme, referring to 0 and a previously defined optimal concentration of 135 mg L^-1 of the vermicompost alkaline extract (VAE), and three water reduction levels of 50, 70 and 90% of the pot holding capacity (PHC) with four replicates, except for the enzymatic analyses, for which three replicates were made.

Bovine manure was used as a substrate for vermicomposting, to obtain the VAE. The manure was first composted for 40 days with mechanical turning every 10 days. Subsequently, the material was vermicomposted for 120 days more using 50 earthworms (Eisenia foetida) per kilogram of bovine manure. The moisture content was maintained at 60-70% throughout the process.

Humic substances were then extracted using 0.1 mol L^-1 NaOH solution (1:20, vermicompost: solution ratio), stirring the suspension (125 rpm, 24 hours) at room temperature. The material then remained unstirred for a period of 12 hours, and the solution supernatant (VAE) was separated from the precipitated material by siphoning and centrifugation (6000 rpm, 5 min). The supernatant solution was adjusted to pH 7.0 with 0.1 mol L^-1 HCl and then dialyzed against deionized water using a 12-14 kDa-cutoff dialysis membrane (Thomas Scientific, Inc.) until the electrical conductivity was less than 1.5 μS cm^-1 and later lyophilized (L101, Liotop, São Paulo, Brazil) (Santos et al., 2022).

The carbon, hydrogen and nitrogen percentages were determined in triplicate in a Perkin Elmer 2400 automatic elemental analyzer with 4 mg VAE samples, while the oxygen content was obtained by difference.

Total acidity was determined by the barium hydroxide Ba(OH)₂ method, through excess titration with HCl. Carboxylic acidity was determined by the Calcium acetate (Ca(C₂H₃O₂)₂) method; free acetate was determined through NaOH. Phenolic acidity was calculated by difference (total - carboxylic).

VAE was characterized by Fourier transform infrared spectroscopy (FT-IR), which was obtained within the range from 400 to 4000 cm^-1, using tablets with 1 mg of VAE in 100 mg of KBr, in a Perkin Elmer 1420 spectrometer (Perkin-Elmer, Waltham, MA, USA).

Fluorescence emission intensity of the fixed blue wave spectrum at 465 nm (FI465) was determined by a F-4500 spectrophotometer (Hitachi, Tokyo, Japan) in aqueous solutions of VAE at concentration of 50 mg of lyophilized material L^-1 equilibrated at 25 °C and pH 8.

VAE characterization showed the following characteristics described in Table 1.

Table 1
Vermicompost alkaline extract characterization

VAE	C	H	N	O	H/C	O/C	COOH	OH Phenolic	Total acidity	FI (u.a.)
					(%)		(cmolc Kg-1)
	52.172.42	4.840.26	6.300.41	36.693.82	0.0920.3	0.700.4	183.354.2	1195.630.3	1378.923.7	91.33.1

• The values are the average of three repetitions. Values in superscript represent the standard error of the mean. FI -Fluorescence emission intensity

The elemental compositions observed was: C - 52.17%, H - 4.84%, N - 6.30% and O - 36.69%. All variables reported are within a range typical for humic materials. The VAE spectral infra-red showed the following characteristics described in Figure 1.

Figure 1
IR Spectrum of vermicompost alkaline extract from bovine manure

The VAE infrared spectrum (Figure 1) showed a wide band at 3400 cm^-1, attributed to OH stretching of alcohols and/or phenols and NH stretching of amines and/or amides (Esteves et al., 2009). The small band close to 2900 cm^-1 reveals symmetrical stretching of C-H bonds, especially aliphatic methyl (CH₃) and methylene (CH₂) groups. The band at 1640 cm^-1 can be attributed to the C=C vibrations of aromatic rings and symmetrical stretching of C=O bonds belonging to amides (the Amide II band) and quinones. The band at 1400 cm^-1 indicated stretching and vibrations of C-H of CH₃, O-H and C-O belonging to phenols. Other two weak peaks at 1260 and in 1240 cm^-1 revealed vibrations of C-O of ethers (Shen et al., 2016). The region between 1150 and 1000 cm^-1 showed several peaks of vibrations of polysaccharides and similar structures (García et al., 2014).

A preliminary test was conducted to evaluate the best VAE concentration to be applied to soybean seedlings. This step was carried out in the Departamento de Agronomia of the UFVJM, Diamantina, MG.

The experimental units consisted of 500 ml plastic pots filled with 0.4 kg of an Oxisol (United States, 2014) that corresponds to a Latossolo Vermelho-Amarelo in the Brazilian Soil Classification System (EMBRAPA, 2018). Soybean seedlings were evaluated for initial growth, from June 1 to 21, 2022, and each experimental unit had two seedlings per pot.

At this stage, six treatments were evaluated, carried out by adding VAE in aqueous solution concentrations of 0, 50, 100, 150, 200 and 250 mg L^-1, respectively. The growth and development of soybean seedlings were evaluated at 20 days after sowing. At the end, root images of 10 plants per treatment were captured by EZ-Rhizo software (Laboratory of Plant Physiology and Biophysics - Glasgow University) to measure root system and evaluate its architecture. Images were analyzed and total root area, total root length, lateral root area and lateral root length were calculated.

After defining the recommended optimal dose (through regression analysis) at which there was higher seedling growth, the main experiment was started. The experimental units consisted of two plants per pot, and 10 dm³ plastic pots were perforated at the bottom and filled with approximately 7 kg of the same soil used in the preliminary dose test described above, fertilized as follows: 0.1 g dm^-3 of P₂O₅, 0.075 g dm^-3 of K₂O, and 1 g dm^-3 of CaCl₂, according to Ribeiro et al. (1999). Physicochemical and textural analysis of the soil used in the experiment is described below. Thirty-eight soil samples were collected from area 13 at the Rio Manso experimental farm in Couto Magalhães de Minas, Minas Gerais, Brazil. The soil water pH was 5.57, and the chemical characterization showed the following contents: phosphorus of 2.16 and potassium of 49.64 mg dm^-³, calcium of 1.38, magnesium of 0.80 and aluminum of 0.15 cmol_c dm^-³. Potential acidity (H+Al) was 2.02, and the sum of bases (SB) was 2.31 cmol_c dm^-³. Effective cation exchange capacity - CEC (t) and potential CEC (T) showed values of 2.46 and 4.33 cmol_c dm^-³, respectively. Aluminum saturation (M) and base saturation (V) were 6.14 and 53.37%, respectively. The organic matter content found was 0.76 dag kg^-1. Regarding the physical characterization, the soil had 51.1% sand, 31% clay and 17.9% silt, and was characterized texturally as a sandy clay loam soil. The methods and types of extractants are described as follows: pH in water - relation 1 cmol_c dm^-³ = meq 100 cm^-³, P and K - Mehlich-1 extractant, Ca, Mg and Al KCl extractant, H+Al - 0.5 mol L^-1 calcium acetate extractant, OM - Organic Matter = C. org × 1.724, Gypsum - m (%) greater than or equal to 30 and Ca less than 0.4.

The soil was moistened up to saturation and distributed in pots sealed with a plastic bag for incubation of mineral fertilizers. After 15 days of incubation, the pots were unsealed, and the soil was dried in open air for 3 days. After that, the soil water content was determined by drying in an oven at 60 ºC for 24 hours; then, the soil weight to be maintained in each pot was recorded, in order to maintain the three levels of water availability (50, 70 and 90% of pot holding capacity - PHC) during the entire experimental period. PHC was determined according to Eq. 1 (Girardi et al., 2016; Silva, 2022):

$$PHC=PW-\left(P{W}_{whc}-P{W}_{dry}\right)\times WRD\times P{W}_{dry}$$ (1)

where:

PW - pot weight (kg^-1);

PW_whc - water holding capacity (L^-1);

PW_dry - pot weight filled with completely dry substrate (kg^-1); and,

WRD - water replacement depth (cm^-1).

The soybean seeds were previously treated with liquid solution containing Rhizobium spp., and the experiment was conducted with two soybean plants per pot. The plants were treated as follows: T1, -VAE at 50% of PHC; T2, +VAE best dose of 135 mg L^-1 at 50% of PHC; T3, -VAE at 70% of PHC; T4, +VAE best dose of 135 mg L^-1 at 70% of PHC; T5, -VAE at 90% of PHC assumed as control conditions; T6, +VAE best dose of 135 mg L^-1 at 90% of PHC.

To maintain the water availability at the given percentages, the pots were weighed on a manual scale twice a day and topped up with water whenever necessary. To minimize the variations among different weighing times, weight was recorded always at 12:00 and at 16:30 p.m. During the experiment in the greenhouse, chemical control was carried out for the whitefly (Bemisia tabaci) with acephate at a dose of 0.3 kg c.p. ha^-1 and manual control was carried out for the soybean caterpillar (Anticarsia gemmatalis).

At the end of the experimental period, on November 11, 2022, 300 dpi pictures of fresh roots were taken and processed with EZ-Rhizo software to calculate total root area, total root length, lateral root area and lateral root length. Stem and root samples from each treatment were dried at 60 ºC for 48 hours in an oven to determine the dry mass.

For enzyme evaluations, 500 mg of fresh root samples per treatment were collected and grinded in a mortar with liquid nitrogen. Afterwards, 2.8 mg of polyvinylpyrrolidone (PVPP) were added to 40 mg of grinded tissue per treatment, which was resuspended in 800 μL of extraction buffer composed of 0.1 M potassium phosphate buffer, pH 6.8, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM phenylmethylsulfonic fluoride (PMSF) and 1% (w/v) polyvinylpolypyrrolidone (PVPP) (Peixoto et al., 1999). The homogenate, after being filtered through four layers of gauze, was centrifuged at 12.000 g for 15 min at 4 ºC, and the supernatant was used as crude enzyme extract.

Superoxide dismutase reaction mixture contained 0.1 mM riboflavin, 50 mM methionine, 5 mM EDTA and 1 mM nitroblue tetrazolium (NBT) in 100 mM phosphate buffer (pH 7.5), and 50 μL of enzyme extract in a final volume of 3 mL. SOD activity was assayed by measuring the ability of the enzyme extract to inhibit the photochemical reduction of NBT. Glass test tubes containing the mixture were immersed in a thermostatic bath at 25 °C and illuminated with a fluorescent lamp. Identical tubes not illuminated served as blanks. After illumination for 15 min, absorbance was measured at 560 nm. One unit of SOD was defined as the enzyme activity which inhibited the photoreduction of NBT to blue formazan by 50%, and SOD activity of the extracts was expressed as SOD units mg^-1 of protein min^-1 (Goel & Sheoran, 2003).

Catalase activity was measured at 28 °C. The enzyme assay contained 3.125 mM H₂O₂ in 50 mM phosphate buffer (pH 7.0) and 25 μL of enzyme extract in total volume of 3,800 mL. Catalase activity was estimated by the decrease in absorbance of red H₂O₂ every 15 s for 180 s at 240 nm and was expressed as nmol H₂O₂ decomposed (mg protein)^-1 min^-1. The H₂O₂ extinction coefficient considered was 36 mM^-1 cm^-1 (Nakano & Asada, 1981).

Ascorbate peroxidase reaction mixture contained 50 µl of enzyme extract, 0.2 mL of 0.5 mM ascorbic acid in 200 mM potassium phosphate buffer (pH 7.0) and 2 mL of potassium phosphate buffer (pH 7.0) previously incubated at 28 °C. The reaction was started by the addition of 200 μL of 1 mM H₂O₂ and absorbance decrease was recorded at 290 nm after every 15 s for a period of 180 s. APX was calculated using the H₂O₂ extinction coefficient equal to 2.8 mM^-1 cm^-1 (Nakano & Asada, 1981).

In the regression models for determining the optimal application dose, on the x axis are the representations as a function of VAE concentrations in mg L^-1, and on the y axis, the variables of total root area and lateral root area expressed in mm², root length and lateral root length expressed in cm. A two-way analysis of variance was performed. Differences between means were compared by Tukey test (p ≤ 0.05). All statistical analysis was performed using R software.

Results and Discussion

Figure 2 shows the polynomial regression model effect, typical of hormone-like activity of humic substances on plant metabolism. Based on the fitted quadratic regression models all the biometric variables recorded the peak of positive stimulation at an average concentration of 135 mg L^-1.

Figure 2
Biometric variables of soybean roots subjected to concentrations of vermicompost alkaline extract (VAE)

The dose-response results (Figures 2A, B, C and D) are consistent with those of many studies showing positive effects on root architecture, in terms of root length and area, and plant metabolism by the employment of humic acids extracted and purified following the entire IHSS protocol (Castro et al., 2021), a genuine mix of humic and fulvic acid extract (Sá et al., 2023), humic and fulvic acid separated and then remixed (Araújo et al., 2021) and VAE (Santos et al., 2022) on maize, black mangrove, Enterolobium contortisiliquum and maize, respectively. The linear fit for all variables was not adequate, yielding the following equations and determination coefficients for total root area (Figure 2A), root length (Figure 2B), lateral root area (Figure 2C), and lateral root length (Figure 2D), respectively: y = 7.0241 + 0.0411^nsx and R² = 0.07; y = 24.28 + 0.0191^nsx and R² = 0.001; y = 3.3907 - 0.0071^nsx and R² = 0.008; y = 14.587 + 0.0181^nsx and R² = 0.002.

At 90% PHC, root and stem biomass of treated plants (+VAE) were +20 and +8% higher than those of the Control, although both values were not significantly different. At 70% PHC, treated plants increased root biomass by up to +57% and stem biomass by up to +29.7% compared to Control, whereas untreated plants showed a +18% in root biomass and a -3.4% in stem biomass compared to Control, both values being not statistically different. At 50% PHC, +VAE group slightly decreased root and stem biomass by -1.5 and -19.2%, respectively, compared to Control, whereas -VAE group increased root biomass by +71% and decreased stem biomass by 11% (Table 2).

Table 2
Seedling biomass at different values of pot holding capacity (PHC), with or without vermicompost alkaline extract (VAE) addition

PHC	+VAE	-VAE	PHC × VAE p value
	G
	Root dry biomass (g)
90%/Control	1.80 bB	1.54 bB	≤0.001*
70%	2.37 aA	1.78 bB
50%	1.52 bB	2.65 aA
CV=16.44%
	Stem dry biomass (g)
90%/Control	5.22 bB	4.89 bB	≤0.001*
70%	6.36 aA	4.73 bB
50%	3.96 bC	4.36 Bb
CV=12.07%

• Different lowercase letters mean differences within PHC groups. Different capital letters indicate differences within +VAE and -VAE groups. *p value means interaction between factors VAE and PHC

At 90 and 70% PHC, total root length (TRL) and lateral root length (LRL) datasets showed a trend similar to that of biomass, with the highest increase at 70% PHC in +VAE group compared to Control (+75% and +116%, respectively) and, in -VAE group, a significant decrease (-20.7%) and a slight increase (+9%), respectively (Table 3).

Table 3
Seedling root variables at different values of pot holding capacity (PHC), with or without vermicompost alkaline extract (VAE) addition

PHC	+VAE	-VAE	PHC average	PHC × VAE p value
	TRL (cm)
90%/Control	30.10 aB	25.65 bA	-	≤0.001*
70%	45.59 aA	20.62 bB	-
50%	15.45 bC	20.88 aB	-
CV=15.05%
	LRL (cm)
90%/Control	16.80 aB	14.00 bB	-	≤0.018*
70%	30.34 aA	15.29 bA	-
50%	14.33 aB	13.76 aB	-
CV=11.18%
	TRA (mm2)
90%/Control	-	-	9.63 b	0.624
70%	-	-	13.50 a
50%	-	-	9.55 b
+VAE /-VAE average	12.14 A	9.6 B
CV=16.62%
	LRA (mm2)
90%/Control	-	-	6.05 a	0.057
70%	-	-	6.61 a
50%	-	-	5.79 a
+VAE /-VAE average	6.58 A	5.72 B	-
CV=15.08%

• Total root length (TRL); Lateral root length (LRL); Total root area (TRA); Lateral root area (LRA). Different lowercase letters mean differences within PHC groups. Different capital letters indicate differences within +VAE and -VAE groups. *p value means interaction between factors VAE and PHC

Total root area (TRA) and lateral root area (LRA) do not show any significant difference at each drought stress level (Table 3). The LRL/TRL ratio dataset (Table 4) shows in +VAE group increasing values from 90%PHC (0.56 ± 0.05) to 50%PHC (0.93 ± 0.11), being the latter, the highest value compared both to Control (0.55 ± 0.05) and to 50%PHC untreated plants (0.66 ± 0.08).

Table 4
Lateral root length/Total root length ratio (LRL/TRL) at different levels of pot holding capacity (PHC), with or without vermicompost alkaline extract (VAE) addition

PHC	+VAE	-VAE	VAE × PHC p value
LRL/TRL
90%/Control	0.56 aB	0.55 aB	≤0.001*
70%	0.67 aB	0.74 aA
50%	0.93 aA	0.66 bA
CV=14.81%

• Different lowercase letters mean differences within PHC groups. Different capital letters indicate differences within +VAE and -VAE groups. *p value means interaction between factors VAE and PHC

Catalase and ascorbate peroxidase activities show in +VAE group a significant decrease at all drought stress levels compared to -VAE plants. The highest increases compared to Control, both in treated and in untreated plants, are recorded at 50%PHC: +16 and +51%, respectively, for catalase, and +48 and +135%, respectively, for ascorbate peroxidase. Superoxide dismutase activities show the same behavior of first two antioxidant enzymes. The only noticeable difference is the no statistically significant difference between treated and untreated plants at each water availability level, although the raw data and the significant difference between the group averages suggest the opposite.

Root dry biomass (Table 2), TRL, LRL, and TRA and LRA (Table 3) show different situations in which VAE effect or water availability is prevalent or both factors cause a combined effect on plant growth and metabolism. At highest drought stress level (50% PHC), untreated plants (-VAE) show total root dry biomass and length higher than VAE treated plants (Tables 2 and 3). Although apparently VAE absence causes a higher biomass production at PHC 50%, LRL/TRL ratio is higher in VAE treated plants (Table 4) compared to untreated plants, evidencing the VAE ability to favor the development of thinner lateral absorbing roots, enhancing root system efficiency (Tavares et al., 2021). This lets us question on the real positive effect of VAE on root architecture: when comparing the root dry biomass at -VAE 50% PHC and +VAE 70% PHC, it is evidenced that VAE effect is prevalent and effective on plant parameters at mild stress (70% PHC) whereas, at severe drought stress (50% PHC), it is reduced but not absent, acting on the efficiency of a reduced root system, through the balance among the biomass and length of main and lateral roots. This is confirmed by TRA (Table 3), which shows the highest value at 70% PHC in VAE treated plant, not significantly different from 70% PHC untreated plants, whereas at 50% PHC, there are no differences between VAE treated and untreated seedlings. LRA shows the same behavior confirming the VAE highest effectiveness at 70% PHC.

The data seem to be in agreement with the drought avoidance strategy reported by Fang & Xiong (2015), consisting in an enhanced water uptake through a well-developed root system. In this experimental scenario, VAE improves root development (Table 3) and stem biomass (Table 2) at mild drought stress, while inducing a more efficient root system despite the depletion in biomass under the most severe drought conditions.

Humic acids do not necessarily improve crop production in terms of biomass. Indeed, humic substances belong to the class of biostimulants, not fertilizers, improving plant ability in stress resistance. Antioxidant enzyme activity in soybean confirms this role of VAE: CAT, APX and SOD (Table 5) activities increase significantly both in +VAE and -VAE following the drought stress increase, but showing values always lower in +VAE group. SOD activity linearly increases with drought stress evidencing, as confirmed by WA × VAE p-value (0.149), no interaction between PHC and VAE, which act separately in increasing and decreasing SOD activity, respectively. On the other hand, CAT and APX show their lowest activity at 70% PHC, with a significant difference between untreated plants and treated plants, evidencing a synergistic effect of PHC and VAE at 70% PHC. This suggests that a moderate stress together with humic substances treatment can enhance plant health.

Table 5
Enzymatic activities in roots at different values of pot holding capacity (PHC), with or without vermicompost alkaline extract (VAE) addition

PHC	+VAE	-VAE	PHC average	PHC x VAE p value
	CAT (mol min-1 mg-1 of protein)
90%/Control	9.63 bB	11.80 aB	-	≤0.001*
70%	8.96 bC	10.83 aC	-
50%	13.76 bA	17.93 aA	-
CV=13.21%
	APX (mol min-1 mg-1 of protein)
90%/Control	5.87 bB	13.40 aB	-	≤0.001*
70%	4.44 bB	12.80 aB	-
50%	19.93 bA	31.60 aA	-
CV=16.50%
	SOD (units mg-1 of protein min-1)
90%/Control	-	-	0.253 b	0.149
70%	-	-	0.301 b
50%	-	-	0.640 a
+VAE /-VAE average	0.268 B	0.524 A
CV=11.61%

• Catalase (CAT), Ascorbate peroxidase (APX), and Superoxide dismutase (SOD). Different lowercase letters mean differences within PHC groups. Different capital letters indicate differences within +VAE and -VAE groups. *p value means interaction between factors VAE and PHC

It is noteworthy that SOD is the first enzyme that acts in the antioxidant system, performing the dismutation of the superoxide radical (O₂ ^•-) to hydrogen peroxide (H₂O₂). The toxic H₂O₂ generated by SOD must be detoxified (Meloni et al., 2003) by APX and/or CAT. Thus, while APX would be responsible for the refined modulation of reactive oxygen species (ROS) for signaling, CAT would be responsible for the removal of excess ROS generated during stress (Mittler, 2002). This sequence of detoxification chain could also explain the different APX and CAT behavior from SOD, due to their acting on a different substrate.

In comparison to several studies on soybean, focused on short-period (3 to 9 days) drought stress (Akitha & Giridhar, 2015) and/or young seedlings (Kausar et al., 2012), the drought stress application during the entire soybean life cycle causes lower enzymatic activities both in +VAE and -VAE. This is consistent with Jumrani & Bhatia (2018), who applied drought stress at R5 developmental stage, at the beginning of seed filling, although the drought stress was applied for a limited time till the water potential was reduced to -2.0 MPa.

Conclusions

1. Under continuous mild (70% of PHC) and severe (50% of PHC) drought stress conditions, VAE were able to deplete abiotic stress severity, increasing the whole root system development under mild drought conditions in terms of dry biomass, length and area.

2. At 50% of PHC, although VAE could not avoid significant biomass decrease, it changed the ratio between main and lateral roots, which were more efficient in water uptake. The decrease of antioxidant enzymes activity confirms the positive role of VAE.

Acknowledgements

The authors would like to thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) and Postgraduate Program in Plant Production at UFVJM.

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• ¹ Research developed at Universidade Federal dos Vales do Jequitinhonha e Mucuri, Programa de Pós-graduação em Produção Vegetal, Departamento de Agronomia, Diamantina, Minas Gerais, Brazil

Supplementary documents

• The authors declare that there are no supplementary documents.

Financing statement

• This study was funded in part by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) under process no. 305255/2020-7, Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), process no. APQ-00437-21 and APQ-00422-23, and by the Financiadora de Estudos e Projetos - (FINEP), process 01.22.0118.00.

Edited by

• Editors: Lauriane Almeida dos Anjos Soares & Walter Esfrain Pereira

Data availability

The authors declare that there are no supplementary documents.

Publication Dates

• Publication in this collection
  10 Mar 2025
• Date of issue
  July 2025

History

• Received
  07 Jan 2024
• Accepted
  27 Jan 2025
• Published
  30 Jan 2025