Isolated and combined application of bioinputs and humic and fulvic substances on lettuce crop performance

Aplicação isolada e combinada de bioinsumos e substâncias húmicas e fúlvicas no desempenho da cultura da alface

ABSTRACT

This study aimed to evaluate the effects of isolated and combined applications of humic and fulvic substances and liquid bacterial inoculants on the agronomic development and productivity of lettuce varietal groups. Two greenhouse experiments were conducted simultaneously, one using crisphead lettuce and the other using loose-leaf lettuce, in a randomized blocks design with six treatments: T1: control (no application); T2: humic and fulvic substances + Bacillus megaterium and Bacillus subtilis; T3: B. megaterium and B. subtilis; T4: humic and fulvic substances + B. megaterium, B. subtilis, and Bacillus aryabhattai; T5: B. megaterium, B. subtilis, and B. aryabhattai; and T6: B. aryabhattai; with 18 replicates. Agronomic variables evaluated included stem diameter (SD), head height (HH), head diameter (HD), head circumference (HC), head fresh mass (HFM), and head dry mass (HDM). Foliar macro- and micronutrient accumulation were also assessed. Data were analyzed using multivariate statistical techniques. For both varietal groups, canonical discriminant analysis (CDA) indicated that treatments T3 and T6 were more closely associated to each other and with yield-related responses, such as SD, HH, HD, and HFM. Greater similarity was also observed between T1 and T5, which were generally associated with higher accumulations of S, Zn, N, B, and Fe. Principal component analysis (PCA) and hierarchical clustering identified three distinct groups: the first comprising T4, the second T2, and the third T1, T5, T3, and T6. Overall, multivariate analysis combined with box-and-whisker plots confirmed the superior performance of treatments T1, T3, T5, and T6 in both nutrient accumulation and plant development.

Keywords:
Lactuca sativa L.; Bacillus megaterium; Bacillus subtilis; Bacillus aryabhattai; humic and fulvic substances

RESUMO

Neste estudo, objetivou-se avaliar os efeitos da aplicação de substâncias húmicas, fúlvicas e inoculantes líquidos à base de bactérias isoladas ou combinadas no desenvolvimento agronômico e na produtividade de grupos varietais de alface. Dois experimentos foram conduzidos simultaneamente, sob casa de vegetação, sendo um com alface crespa e outro com americana, no delineamento em blocos ao acaso, com seis tratamentos: T1: sem aplicação (controle); T2 - Substâncias húmicas e fúlvicas + Bacillus megaterium e Bacillus subtilis; T3: B. megaterium e B. subtilis; T4: Substâncias húmicas e fúlvicas + B. megaterium e B. subtilis + Bacillus aryabhattai; T5: B. megaterium e B. subtilis + B. aryabhattai e; T6: B. aryabhattai; com 18 repetições. Foi avaliado o diâmetro do caule (DC), altura da cabeça (AC), diâmetro da cabeça (HD), circunferência (CC), massa fresca (MFC) e seca da cabeça (MSC). Também foram avaliados o acúmulo de macronutrientes e micronutrientes foliares. Os dados foram submetidos à análise multivariada. Para ambos os grupos varietais a análise discriminante canônica evidenciou que os tratamentos T3 e T6 são mais próximos entre si, e mais associados a respostas produtivas como DC, AC, HD e MFC. Também foi possível verificar uma maior proximidade entre T1 e T5, e que, em geral, estes tratamentos estão associados a maiores acúmulos de S, Zn, N, B e Fe. A análise de componentes principais e de agrupamento hierárquico evidenciou a formação de três grupos, o primeiro formado por T4, o segundo por T2 e o terceiro formado pelos tratamentos T1, T5, T3, T6. A análise multivariada dos dados em conjunto com o diagrama de extremos e quartis, confirmaram o desempenho superior dos tratamentos T1, T3, T5 e T6, tanto com relação ao acúmulo de nutrientes, quanto ao desenvolvimento das plantas.

Palavras-chave:
Lactuca sativa L.; Bacillus megaterium; Bacillus subtilis; Bacillus aryabhattai; substâncias húmicas e fúlvicas

Lettuce (Lactuca sativa L.) is among the most widely cultivated and consumed leafy vegetables worldwide and in Brazil, valued for its nutritional quality, pleasant flavor, and ease of preparation (Santos et al., 2025). As a crop with high nutrient demands, lettuce benefits from soils rich in organic matter to achieve high yields (Meireles et al., 2017). In Brazil, crisphead and loose-leaf lettuce are the most commonly produced types, each with distinct growth cycles and nutritional requirements, with crisphead lettuce generally exhibiting a longer production cycle and greater nutrient demand.

In response to the challenges of conventional production systems and the growing demand for more sustainable foods, alternative practices such as the use of liquid inoculants and humic substances have gained prominence (Luz & Brito, 2022; Alsudays et al., 2024). Derived from biomass decomposition, humic and fulvic substances are known to improve soil physical, chemical, and biological properties, enhance nutrient availability and water retention, stimulate root development, and increase plant tolerance to abiotic stresses such as drought and salinity, thereby contributing to improved crop productivity (Olivares et al., 2017; Quin & Leskovar, 2020; Ribeiro et al., 2022; Alsudays et al., 2024).

Microbial bioinputs, particularly those based on Bacillus spp., are widely used in crop production due to their ability to promote plant growth through mechanisms such as phytohormone synthesis, phosphate solubilization, and induced systemic resistance (Calvo et al., 2014). Liquid inoculants formulated with isolated bacterial strains (Bacillus aryabhattai) or bacterial consortia (Bacillus megaterium + Bacillus subtilis) have been applied to various crops to improve nutrient use efficiency and agronomic performance, even under adverse environmental conditions, including low soil nutrient availability (particularly nitrogen and phosphorus), high temperatures, drought, and salinity (Rouphael & Colla, 2020).

The combined use of humic substances and microbial biostimulants is a promising strategy to enhance agronomic performance in vegetables such as lettuce. Previous studies suggest that this association can amplify beneficial physiological effects on plant growth and development, leading to gains in productivity and product quality (Du Jardin, 2015; Borcioni et al., 2016; Colla et al., 2017). However, further research is needed to better understand the interactions between these technologies and their effects on different lettuce varieties.

In this context, the hypothesis of the present study is that the combined application of humic substances and bioinputs promotes microbial establishment in the soil, thereby intensifying the beneficial effects of these inputs and improving lettuce productivity and quality. Accordingly, this study aimed to evaluate the effects of humic and fulvic substances and liquid bacterial inoculants based on isolated or combined bacterial strains on the agronomic development and productivity of lettuce cultivars from different varietal groups.

MATERIAL AND METHODS

The study was conducted in an arch-type greenhouse covered with a 150 µm light-diffusing polyethylene film and sidewalls enclosed with 50% shade cloth, located in Uberaba, Minas Gerais state, Brazil (19°45′26″S, 47°55′27″W; 800 m altitude). The experiment was carried out during late autumn and early winter, a period characterized by milder temperatures and no rainfall.

The regional climate is Aw, with hot, rainy summers and cool, dry winters, according to the updated Köppen classification (Beck et al., 2018). The annual mean temperature and relative humidity are 22.6°C and 68%, respectively (Inmet, 2024).

Two experiments were conducted simultaneously, one using loose-leaf lettuce (cv. Vanda) and the other crisphead lettuce (cv. Lucy Brown). Sowing for both experiments was performed on May 1, 2023, in polyethylene trays with 12.5 cm³ cells filled with Bioplant Plus® substrate, using one pelleted seed per cell. Seedlings were grown for 30 days and transplanted on May 31, 2023. Loose-leaf lettuce was harvested on July 17, 2023 (47 days after transplanting (DAT)), and crisphead lettuce on July 30, 2023 (60 DAT).

A randomized block design was used, with six treatments and 18 replicates, totaling 108 plots. Each experimental unit consisted of one pot containing a single plant. Treatments were: T1: control (no application); T2: humic and fulvic substances + Bacillus megaterium and Bacillus subtilis; T3: B. megaterium and B. subtilis; T4: humic and fulvic substances + B. megaterium and B. subtilis + Bacillus aryabhattai; T5: B. megaterium and B. subtilis + B. aryabhattai; and T6: B. aryabhattai

Treatments consisted of combinations of commercial products, including humic and fulvic substances (Bioplant H25®; 11% N, 2% K₂O, and 6% total organic carbon), liquid bacterial inoculants containing B. megaterium and B. subtilis (Biomaphós®), and a liquid inoculant based on B. aryabhattai (Auras®).

Each experimental unit comprised a 6 dm³ pot (24 × 20 × 16.5 cm) filled with 6 kg of dystrophic Red Latosol (Oxisol) of sandy clay loam texture (Santos et al., 2018). Prior to pot filling, soil fertility was analyzed, and results are shown in Table 1.

Liming was performed 45 days before planting using a motorized concrete mixture, with dolomitic limestone (PRNT 90%) applied at a rate of 1.2 t/ha, to raise base saturation to 70%. The dose per pot was calculated based on the soil volume equivalent to one hectare and adjusted to pot size. After liming, pots were filled with soil, placed on wooden stands 15 cm above the ground, and incubated for 45 days with weekly irrigation.

Table 1. Chemical
characterization of the soil used in the experiments, based on samples collected from the 0-20 cm depth layer in the greenhouse. Uberaba, IFTM, 2025.

pH	Ca	Mg	Al	H+Al	CEC	V(%)	P	K	SB	TC	OM
(CaCl2)	(mmolc /dm3)						(mg/dm3)			(g/dm3)
5.7	14	6.1	0.1	24	45	46.63	8.8	34	21	0.78	0.78
Micronutrients					Relationship between nutrients
B	Cu	Fe	Mn	Zn	Ca/Mg			Ca/K		Mg/K
(mg/dm3)
0.04	0.8	97.8	11.1	1.2	2.3			16.10		7.02

• Ca, Mg, Al = KCl solution (1 mol/L); H+Al = SMP buffer solution (pH: 7.5); CEC = cátion Exchange capacity at pH 7; V = base saturation; P = resin; K = 0.05 mol/L HCl + H₂SO₄ 0.0125 mol/L; SB = sum of bases; S-SO₄ = calcium phosphate; TC = total carbon; OM = organic matter (colorimetric method); B, Cu, Fe, Mn, Zn = DTPA (diethylene triamine penta acetic acid).

At planting, all treatments received basal fertilization of 52.8 kg N/ha, 240 kg P₂O₅/ha, and 60 kg K₂O/ha, applied in surface furrows in the pots. Top-dressing fertilization consisted of 150 kg N/ha and 90 kg K₂O/ha, in three split applications at 15, 30, and 40 DAT, following the recommendations of Ribeiro et al. (1999). Fertilization practices were identical for all treatments within each experiment.

Treatments were applied on the day of transplanting. Spray solutions were prepared using the respective commercial products. The inoculants Biomaphós® and Auras® were applied at 200 mL each, while Bioplant H25®, the source of humic and fulvic substances, was used at 2 L/ha.

An application volume of 20 L/ha was adopted, corresponding to the estimated volume required to treat one hectar of lettuce seedlings by root immersion. Accordingly, 2 L of solution were prepared for each treatment, containing 0.2 L of Bioplant H25®, 20 mL of Biomaphós®, and 20 mL of Auras®, depending on treatment composition.

For treatment application, the root system of each seedling, along with its substrate plug, was immersed for 30 seconds in the respective solution before transplanting into the pots. Thus, treatments T2, T3, T4, T5, and T6 were applied pre-planting through a single root immersion.

This application method was selected to ensure efficient treatment of transplanted crops without the need for specialized machinery, although the manufacturer also recommends the product for seed treatment or directed application at sowing.

Irrigation was managed using an automated drip irrigation system, scheduled to apply five irrigation events per day, thereby maintaining the soil close to field capacity (container capacity) throughout the entire crop cycle. Other crop management practices, including spraying and weeding, were performed uniformly across treatments as needed.

At 47 DAT for loose-leaf lettuce and 60 DAT for crisphead lettuce, plants reached commercial maturity and were harvested for evaluation. Plants were cut at the soil surface, and the following agronomic variables were evaluated: stem diameter (SD), measured with a digital caliper; head height (HH), and head diameter (HD), with a graduated ruler; head circumference (HC), with a measuring tape; and head fresh mass (HFM) and head dry mass (HDM).

The aerial parts (heads) were prepared for chemical analysis by washing with a detergent solution, rinsing in running water followed by distilled water, and drying in paper bags in a forced-air circulation oven at 65°C for 72 h until constant mass, following Embrapa (2009) recommendations. After drying, head dry mass was determined, and the material was ground for chemical determination of leaf nutrient contents (N, P, K, Ca, Mg, S, B, Cu, Fe, Mn, and Zn), following Embrapa (2009), with subsequent calculation of nutrient accumulation per plant.

To evaluate treatment effects, data were subjected to multivariate analysis of variance (MANOVA), given that multiple response variables were assessed in the study (n = 17). The Wilks’ Lambda statistic was adopted due to its robustness to moderate departures from the assumptions of normality and homoscedasticity (Ates et al., 2019).

Prior to conducting MANOVA, missing values in the response variables were imputed to prevent parameter estimation bias and ensure a nonsingular covariance matrix. Imputation was performed using principal component analysis (PCA), following the methodology proposed by Josse & Husson (2016), with the number of retained components determined by cross-validation. This approach preserves multivariate correlations among variables and minimizes potential distortions in hypothesis testing.

Following the confirmation of significant treatment effects by MANOVA, canonical discriminant analysis (CDA) was applied to assess multivariate separation among treatments and to identify the variables with the greatest contribution to group discrimination. In addition, exploratory analyses were conducted using PCA and hierarchical cluster analysis, applying the UPGMA clustering method and Euclidean distance as the dissimilarity measure, to visualize clustering patterns. Box-and-whisker plots were also used to examine the distribution of agronomic traits and nutrient accumulation across treatments.

All statistical analyses were performed using R software (R Core Team, 2025), adopting a 5% significance level where applicable.

RESULTS AND DISCUSSION

Crisphead lettuce

Multivariate analysis of variance (MANOVA) using Wilks’ Lambda test indicated a significant treatment effect (Λ = 0.0634; F(85,338) = 3.06; p<0.001) on the set of evaluated variables. The low Wilks’ Lambda value (0.0634) demonstrates that a substantial proportion of the total variability was explained by differences among treatments.

CDA showed that the first two canonical functions were significant according to the Wilks test (p<0.001), and together explained approximately 85% of the total treatment variability, with 56% attributed to canonical variable 1 and 29% to canonical variable 2 (Figure 1). The CDA indicated that treatments T3 and T6 were closely associated with each other and primarily related to higher values of K, SD, HH, and HFM. Treatments T1 and T5 also showed strong similarity and were generally associated with higher accumulations of S, Zn, N, B, and Fe. Conversely, treatments T2 and T4 were clearly separated from the others and showed weaker associations with nutrient accumulation and agronomic development (Figure 1). The CDA also suggested a high degree of correlation among the evaluated variables (Figure 1).

Pearson's linear correlation analysis confirmed strong linear relationships among the evaluated variables (Figure 2). Except for the correlations between Cu and HD (r = 0.69) and between Cu and SD (r = 0.56), all correlations were statistically significant according to the Student’s t-test. These results indicate that agronomic variables related to plant development are strongly interconnected and closely aligned with patterns of nutrient accumulation, such that higher nutrient accumulation is associated with enhanced plant growth and development (Figure 2).

Figure 1
Canonical discriminant analysis (CDA) of agronomic traits and nutrient accumulation in crisphead lettuce (Lactuca sativa L.) subjected to different combinations of humic substances and liquid bacterial inoculants. Treatments were: T1: control (no application); T2: humic and fulvic substances + Bacillus megaterium and Bacillus subtilis; T3: Bacillus megaterium and Bacillus subtilis; T4: humic and fulvic substances + Bacillus megaterium and Bacillus subtilis, + Bacillus aryabhattai; T5: Bacillus megaterium and Bacillus subtilis + Bacillus aryabhattai; and T6: Bacillus aryabhattai; Agronomic traits included head fresh mass (HFM), head dry mass (HDM), head diameter (HD), head circumference (HC), head height (HH), and stem diameter (SD). Nutrients evaluated were nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), sulfur (S), boron (B), copper (Cu), iron (Fe), manganese (Mn), and zinc (Zn). Uberaba, IFTM, 2025.

Figure 2
Correlation matrix of agronomic traits and nutrient accumulation in crisphead lettuce subjected to different combinations of humic substances and liquid bacterial inoculants. Treatments were T1: no application (control); T2: humic and fulvic substances + Bacillus megaterium and Bacillus subtilis; T3: Bacillus megaterium and Bacillus subtilis; T4: humic and fulvic substances + Bacillus megaterium and Bacillus subtilis + Bacillus aryabhattai; T5: Bacillus megaterium and Bacillus subtilis + Bacillus aryabhattai; and T6: Bacillus aryabhattai. Agronomic traits included head fresh mass (HFM), head dry mass (HDM), head diameter (HD), head circumference (HC), head height (HH), and stem diameter (SD). Nutrients evaluated were nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), sulfur (S), boron (B), copper (Cu), iron (Fe), manganese (Mn), and zinc (Zn). Uberaba, IFTM, 2025.

PCA corroborated the treatment differentiation observed in CDA. PCA indicated that treatments T1, T5, T6, and T3 promoted the greatest nutrient accumulation. T1 and T5 were linked to higher levels of Cu, Zn, N, Fe, B, S, and Mg, whereas T3 and T6 were associated with higher means for SD, HH, K, HD, and Mn (Figure 3). By contrast, treatments T2 and T4 were characterized by lower nutrient accumulation and reduced plant development (Figure 3).

Figure 3
Principal component analysis (PCA) of agronomic traits and nutrient accumulation in crisphead lettuce subjected to different combinations of humic substances and liquid bacterial inoculants. Treatments were T1: no application (control); T2: humic and fulvic substances + Bacillus megaterium and Bacillus subtilis; T3: Bacillus megaterium and Bacillus subtilis; T4: humic and fulvic substances + Bacillus megaterium and Bacillus subtilis + Bacillus aryabhattai; T5: Bacillus megaterium and Bacillus subtilis + Bacillus aryabhattai; and T6: Bacillus aryabhattai. Agronomic traits included head fresh mass (HFM), head dry mass (HDM), head diameter (HD), head circumference (HC), head height (HH), and stem diameter (SD). Macronutrients evaluated were nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), sulfur (S), boron (B), copper (Cu), iron (Fe), manganese (Mn), and zinc (Zn). Uberaba, IFTM, 2025.

Consistent with the CDA and PCA results, hierarchical clustering analysis identified three main groups: one consisting solely of treatment T4, the second of treatment T2, and the third of treatments T1, T5, T3, and T6. This last group was further divided into two subgroups, one including T1 and T5 and the other T3 and T6 (Figure 4). As previously noted, this group (T1, T5, T3, and T6) was associated with superior nutrient accumulation and agronomic performance.

Figure 4
Hierarchical cluster analysis of six treatments comprising different combinations of humic substances and liquid bacterial inoculants applied to crisphead lettuce. Treatments were T1: no application (control); T2: humic and fulvic substances + Bacillus megaterium and Bacillus subtilis; T3: Bacillus megaterium and Bacillus subtilis; T4: humic and fulvic substances + Bacillus megaterium and Bacillus subtilis + Bacillus aryabhattai; T5: Bacillus megaterium and Bacillus subtilis + Bacillus aryabhattai; and T6: Bacillus aryabhattai. Uberaba, IFTM, 2025.

These multivariate patterns were also evident in the distribution of individual variables. Overall, the results confirm the superior performance of T1, T3, T5, and T6, which exhibited higher mean values for both nutrient accumulation and plant development variables (Figures 5, 6, and 7).

Figure 5
Boxplots showing the distribution and mean values (red dots) of biometric traits on crisphead lettuce subjected to different combinations of humic substances and liquid bacterial inoculants. Treatments were T1: no application (control); T2: humic and fulvic substances + Bacillus megaterium and Bacillus subtilis; T3: Bacillus megaterium and Bacillus subtilis; T4: humic and fulvic substances + Bacillus megaterium and Bacillus subtilis + Bacillus aryabhattai; T5: Bacillus megaterium and Bacillus subtilis + Bacillus aryabhattai; and T6: Bacillus aryabhattai. Agronomic variables included head fresh mass (HFM), head dry mass (HDM), head diameter (HD), head circumference (HC), head height (HH), and stem diameter (SD). Uberaba, IFTM, 2025.

Figure 6
Boxplots showing the distribution and mean values (red dots) of macronutrient accumulation in crisphead lettuce subjected to different combinations of humic substances and liquid inoculants. Treatments were T1: no application (control); T2: humic and fulvic substances + Bacillus megaterium and Bacillus subtilis; T3: Bacillus megaterium and Bacillus subtilis; T4: humic and fulvic substances + Bacillus megaterium and Bacillus subtilis + Bacillus aryabhattai; T5: Bacillus megaterium and Bacillus subtilis + Bacillus aryabhattai; and T6: Bacillus aryabhattai. Macronutrients evaluated were nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), and sulfur (S). Uberaba, IFTM, 2025.

Figure 7
Boxplots showing the distribution and mean values (red dots) of micronutrient accumulation in crisphead lettuce subjected to different combinations of humic substances and liquid bacterial inoculants. Treatments were T1: no application (control); T2: humic and fulvic substances + Bacillus megaterium and Bacillus subtilis; T3: Bacillus megaterium and Bacillus subtilis; T4: humic and fulvic substances + Bacillus megaterium and Bacillus subtilis + Bacillus aryabhattai; T5: Bacillus megaterium and Bacillus subtilis + Bacillus aryabhattai; and T6: Bacillus aryabhattai. Micronutrients evaluated were boron (B), copper (Cu), iron (Fe), manganese (Mn), and zinc (Zn). Uberaba, IFTM, 2025.

Loose-leaf lettuce

Consistent with the results observed for crisphead lettuce, MANOVA using Wilks’ Lambda test revealed a significant treatment effect (Λ = 0.2067; F(85,338) = 1.53; p<0.01) on the set of variables evaluated for loose-leaf lettuce. The low Wilks’ Lambda value (0.2067) suggests that a substantial proportion of the total variability was explained by differences among treatments.

CDA showed that the first two canonical functions were significant according to the Wilks’ test (p<0.01) and together explained 75% of the total treatment variability, with 57% attributed to canonical variable 1 and 18% to canonical variable 2 (Figure 8).

The CDA indicated that T3 and T6 were closely associated and primarily related to higher values of productive traits such as HH, HD, and HFM (Figure 8). Likewise, T1 and T5 also showed strong similarity and were associated with greater nutrient accumulation, particularly Zn, K, B, Fe, Mn, and Cu. By contrast, T2 and T4 were clearly separated from the others and exhibited weaker associations with nutrient accumulation and agronomic development (Figure 8). The CDA also indicated a high degree of correlation among the evaluated variables. Overall, the CDA patterns observed for loose-leaf lettuce closely resembled those obtained for crisphead lettuce (Figures 1 and 8).

Figure 8
Canonical discriminant analysis (CDA) of agronomic traits and nutrient accumulation in loose-leaf lettuce subjected to different combinations of humic substances and liquid inoculants. Treatments were T1: no application (control); T2: humic and fulvic substances + Bacillus megaterium and Bacillus subtilis; T3: Bacillus megaterium and Bacillus subtilis; T4: humic and fulvic substances + Bacillus megaterium and Bacillus subtilis + Bacillus aryabhattai; T5: Bacillus megaterium and Bacillus subtilis + Bacillus aryabhattai; and T6: Bacillus aryabhattai. Agronomic traits were head fresh mass (HFM), head dry mass (HDM), head diameter (HD), head circumference (HC), head height (HH), and stem diameter (SD). Nutrients evaluated were nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), sulfur (S), boron (B), copper (Cu), iron (Fe), manganese (Mn), and zinc (Zn). Uberaba, IFTM, 2025.

Correlation analysis confirmed strong linear relationships among most evaluated variables, with all correlations being statistically significant according to the Student’s t-test (Figure 9). The only exception was head height (HH), which showed significant positive correlations only with HDM (r = 0.76) and P (r = 0.74).

These findings demonstrate that, regardless of the varietal group, most agronomic variables are closely interconnected, except for HH, and follow the same trend as nutrient accumulation. In other words, higher nutrient accumulation is consistently associated with greater plant growth and development (Figure 9).

Figure 9
Correlation matrix of agronomic traits and nutrient accumulation in loose-leaf lettuce subjected to different combinations of humic substances and liquid bacterial inoculants. Treatments were T1: no application (control); T2: humic and fulvic substances + Bacillus megaterium and Bacillus subtilis; T3: Bacillus megaterium and Bacillus subtilis; T4: humic and fulvic substances + Bacillus megaterium and Bacillus subtilis + Bacillus aryabhattai; T5: Bacillus megaterium and Bacillus subtilis + Bacillus aryabhattai; and T6: Bacillus aryabhattai. Agronomic traits included head fresh mass (HFM), head dry mass (HDM), head diameter (HD), head circumference (HC), head height (HH), and stem diameter (SD). Nutrients evaluated were nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), sulfur (S), boron (B), copper (Cu), iron (Fe), manganese (Mn), and zinc (Zn). Uberaba, IFTM, 2025.

PCA further supported the treatment differentiation observed in CDA (Figure 10). Treatments T1, T5, T6, and T3 were associated with the highest nutrient accumulation, with T1 and T5 linked to increased levels of Zn, B, K, Mn, and Fe. T3 and T6 were generally associated with higher mean values of agronomic variables, with T6 primarily related to higher values of HH, HD, HFM, HC, HDM, and SD (Figure 10). By contrast, T2 and T4 were characterized by lower nutrient accumulation and reduced plant development (Figure 10). As observed for crisphead lettuce, the response patterns in loose-leaf lettuce indicate that treatment effects were largely independent of the varietal group evaluated.

Figure 10
Principal component analysis (PCA) of agronomic traits and nutrient accumulation of loose-leaf lettuce subjected to different combinations of humic substances and liquid bacterial inoculants. Treatments were T1: no application (control); T2: humic and fulvic substances + Bacillus megaterium and Bacillus subtilis; T3: Bacillus megaterium and Bacillus subtilis; T4: humic and fulvic substances + Bacillus megaterium and Bacillus subtilis + Bacillus aryabhattai; T5: Bacillus megaterium and Bacillus subtilis + Bacillus aryabhattai; and T6: Bacillus aryabhattai. Agronomic traits included head fresh mass (HFM), head dry mass (HDM), head diameter (HD), head circumference (HC), head height (HH), and stem diameter (SD). Nutrients evaluated were nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), sulfur (S), boron (B), copper (Cu), iron (Fe), manganese (Mn), and zinc (Zn). Uberaba, IFTM, 2025.

In agreement with the CDA and PCA results, hierarchical clustering identified three main groups:one consisting of T4, a second of T2, and a third of T1, T5, T3, and T6. This last group was further divided into three subgroups, with T1 and T5 forming one subgroup, T3 another, and T6 a third (Figure 11). As indicated by the multivariate analyses, this group (T1, T5, T3, and T6) was associated with the highest nutrient accumulation and overall agronomic performance (Figures 8, 9, and 10).

Figure 11
Hierarchical cluster analysis of six treatments comprising different combinations of humic substances and liquid bacterial inoculants applied to loose-leaf lettuce. Treatments were T1: no application (control); T2: humic and fulvic substances + Bacillus megaterium and Bacillus subtilis; T3: Bacillus megaterium and Bacillus subtilis; T4: humic and fulvic substances + Bacillus megaterium and Bacillus subtilis + Bacillus aryabhattai; T5: Bacillus megaterium and Bacillus subtilis + Bacillus aryabhattai; and T6: Bacillus aryabhattai. Uberaba, IFTM, 2025.

These multivariate patterns were also evident in the distribution of individual variables, confirming the superior performance of T1, T3, T5, and T6 in terms of nutrient accumulation and plant development (Figures 12, 13 and 14).

Importantly, the similarity between the loose-leaf and crisphead lettuce experiments reinforces the consistency of treatments across different lettuce varietal groups (Figures 5,6,7 for crisphead lettuce and Figures 12, 13; 14)for loose-leaf lettuce).

Figure 12
Boxplots showing the distribution and mean values (red dots) of agronomic variables in loose-leaf lettuce, subjected to different combinations of humic substances and liquid bacterial inoculants. Treatments were T1: no application (control); T2: humic and fulvic substances + Bacillus megaterium and Bacillus subtilis; T3: Bacillus megaterium and Bacillus subtilis; T4 - humic and fulvic substances + Bacillus megaterium and Bacillus subtilis + Bacillus aryabhattai; T5: Bacillus megaterium and Bacillus subtilis + Bacillus aryabhattai; and T6: Bacillus aryabhattai. Agronomic variables included head fresh mass (HFM), head dry mass (HDM), head diameter (HD), head circumference (HC), head height (HH), and stem diameter (SD). Uberaba, IFTM, 2025.

Figure 13
Boxplots showing the distribution and mean values (red dots) of macronutrient accumulation in loose-leaf lettuce subjected to different combinations of humic substances and liquid bacterial inoculants. Treatments were T1: no application (control); T2: humic and fulvic substances + Bacillus megaterium and Bacillus subtilis; T3: Bacillus megaterium and Bacillus subtilis; T4: humic and fulvic substances + Bacillus megaterium and Bacillus subtilis + Bacillus aryabhattai; T5: Bacillus megaterium and Bacillus subtilis + Bacillus aryabhattai; and T6: Bacillus aryabhattai. Macronutrients evaluated were nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), and sulfur (S). Uberaba, IFTM, 2025.

Figure 14
Boxplots showing the distribution and mean values (red dots) of micronutrient accumulation in loose-leaf lettuce subjected to different combinations of humic substances and liquid bacterial inoculants. Treatments were T1: no application (control); T2: humic and fulvic substances + Bacillus megaterium and Bacillus subtilis; T3: Bacillus megaterium and Bacillus subtilis; T4: humic and fulvic substances + Bacillus megaterium and Bacillus subtilis + Bacillus aryabhattai; T5: Bacillus megaterium and Bacillus subtilis + Bacillus aryabhattai; and T6: Bacillus aryabhattai. Micronutrients evaluated were boron (B), copper (Cu), iron (Fe), manganese (Mn), and zinc (Zn). Uberaba, IFTM, 2025.

The results indicate that T2 and T4, which included humic and fulvic substances, generally had a negative effect on the performance of both lettuce varietal groups.

For crisphead lettuce, these treatments reduced all evaluated agronomic variables. T2 (humic and fulvic substances +Bacillus megaterium and Bacillus subtilis) decreased HFM by 33% and HDM by 44% compared with the control. Similarly, T4 (humic and fulvic substances +B. megaterium, B. subtilis, and B. aryabhattai) resulted in HFM values 2.1 times lower and HDM values 1.7 times lower than the control (Figure 5).

Comparable patterns were observed for loose-leaf lettuce, where T2 reduced HFM by 21% and HDM by 44%, and T4 decreased HFM by 5% and HDM by 21% compared with the control (Figure 12).

By contrast, treatments T6 (Bacillus aryabhattai) and T3 (Bacillus megaterium and Bacillus subtilis) consistently performed best across both lettuce varieties, showing the highest production-related values (Figures 5 and 12). For instance, T6 increased HFM by 12% and HDM by 4% in loose-leaf lettuce and HFM by 10% and HDM by 5% in crisphead lettuce, relative to the control (Figures 5 and 12).

In terms of macro and micronutrient accumulation, treatments T1, T3, T5, and T6 exhibited similar values (Figures 6, 7, 13, and 14), which were consistently higher than those observed in T2 and T4, the only treatments containing humic and fulvic substances. These results suggest that, under the application conditions adopted in this study, these substances negatively affected nutrient accumulation and, consequently, crop yield.

The literature reports contrasting responses to humic and fulvic substances and to microbial bioinputs, including Bacillus spp., suggesting that their effects depend strongly on source, application method, and dose. For instance, Hernandez et al. (2015) reported increased lettuce yield and a shortened crop cycle after leaf humin application 15 days after transplanting, differing from the present results.

Likewise, Borcioni et al. (2016) reported improved growth and increased head fresh and dry mass in crisphead lettuce growth following field-applied fulvic acid doses, particularly due to enhanced root development. However, they applied only fulvic substances, which are costly and differ compositionally from the commercial humic product used here. In the present study, a complex mixture of humic acids, fulvic acids, and humin was applied, while Borcioni et al. (2016) used only low-molecular-weight fulvic acids, which are often associated with improved nutrient uptake and translocation.

Quin & Leskovar (2020) reported positive effects of humic substances on lettuce seedling establishment and post-transplant performance. However, they applied solid humic material rather than the liquid formulation adopted here. Rodrigues et al. (2018) found that foliar application of liquid humic substances improved lettuce growth at low concentrations (≤3 mg/L), whereas higher doses exerted negative effects. These studies highlight that both application method and dosage are critical determinants of agronomic response.

In this study, treatments T2 and T4, which included humic substances, yielded lower values across all assessed variables. The relatively high concentration applied (100 mL of commercial product per liter of solution) likely disrupted rhizosphere conditions and induced phytotoxicity.

The application method may have further exacerbated these effects, since full root immersion was used rather than the foliar or soil application methods adopted by Borcioni et al. (2016), Rodrigues et al. (2018), and Quin & Leskovar (2020). Immersion of the entire root system in a concentrated solution may have caused excessive exposure and potential pH-related stress. Given the lack of standardized protocols exist for seedling immersion, these findings provide valuable insights for defining appropriate doses and application strategies in future studies.

Conversely, the present study demonstrated the feasibility of applying Bacillus sp.-based bioinputs via seedling immersion, particularly for treatments T6 and T3, which consistently outperformed the control.

Commercial liquid inoculants containing B. aryabhattai or B. megaterium + B. subtilis are increasingly recognized as sustainable technologies for enhancing lettuce productivity by improving nutrient uptake, increasing tolerance to abiotic stress, and providing biocontrol against phytopathogens (Silva, 2022).

Among the Bacillus species, B. subtilis is one of the most extensively researched in agricultural systems. Its application in lettuce has been associated with agronomic benefits. Sarti et al. (2023), for instance, reported a 30% increase in shoot biomass and a 37% rise in root biomass, via seed biofilm, linked to enhanced phosphate solubilization and phytohormone synthesis. Similarly, Oliveira et al. (2023) found that B. subtilis inoculation in hydroponic systems increased leaf number by 20% and fresh head mass by 22-25%, while the net photosynthetic rate rose by nearly 95%. The same study reported that the bacteria improved the uptake of essential nutrients such as N, K, Ca, and Mg, indicating greater nutritional efficiency.

Likewise, B. megaterium is widely recognized for its phosphate-solubilizing capacity. Petkova & Dimova (2024) demonstrated that B. megaterium inoculation reduced root disease incidence and improved the uptake of P and Zn, essential for lettuce growth. In the present study, treatment T3, which included B. megaterium, resulted in the highest mean P accumulation in crisphead lettuce, corroborating previous findings and highlighting the potential of microbial inputs to reduce mineral fertilizer requirements.

In this regard, Ikiz et al. (2024) reported that the combined application of B. megaterium and B. subtilis reduced mineral fertilizer use by up to 40% without affecting hydroponic lettuce yield. Although B. aryabhattai is a growth promoter in crops such as wheat and soybean, its effects on lettuce remain poorly documented. The present study is therefore among the first to evaluate its role in lettuce cultivation. The positive response observed in treatment T6, where the isolated application of B. aryabhattai produced the highest mean values for most productive variables in both lettuce types, confirms its potential agronomic value.

Overall, the results indicate that isolated application of commercial bioinputs, particularly those based on Bacillus aryabhattai (T6) or Bacillus megaterium + Bacillus subtilis (T3), enhances lettuce growth and productivity. By contrast, the high dose of humic substances tested in this study was detrimental to crop performance.

ACKNOWLEDGMENTS

The authors would like to thank the Federal Institute of Triângulo Mineiro, Uberaba Campus, for providing the infrastructure to conduct this project. We also thank the Research Support Foundation of Minas Gerais State (FAPEMIG) and the National Council for Scientific and Technological Development (CNPq) (Process 305703/2025-30) for partial funding and research scholarships provided to the students involved in this study.

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• Agreement for Publication
  All authors have read and agreed to the published version of the manuscript.
• Conflicts of Interest
  The authors declare that there are no conflicts of interest.
• Data Availability
  The datasets generated and analyzed during the study are available from the corresponding author upon reasonable request.

• Responsible editor:
  José Magno Queiroz Luz

Data availability

The datasets generated and analyzed during the study are available from the corresponding author upon reasonable request.

Publication Dates

• Publication in this collection
  27 Feb 2026
• Date of issue
  2026

History

• Received
  16 July 2025
• Accepted
  21 Jan 2026