Bioinoculation with Bacillus spp. and its physiological effects on the performance of maize seedlings

Bioinoculação com Bacillus spp. e seus efeitos fisiológicos no desempenho de mudas de milho

Abstract

The growing demand for sustainable alternatives in agriculture has stimulated the use of plant growth-promoting microorganisms as a strategy to improve the early performance of crops. In this context, the objective of this study was to evaluate the initial physiological effects of a commercial bioinoculant containing Bacillus spp. on maize (Zea mays L.) seedlings grown in sterile sand under controlled conditions. The experiment was conducted using five bioinoculant doses (0, 50, 100, 150, and 200 µL), arranged in a completely randomized design with four replicates. Morphological variables (seedling emergence, root and shoot length, and dry mass), physiological parameters (chlorophyll a fluorescence and gas exchange), as well as anatomical and histochemical characteristics, were evaluated. The data were analyzed by analysis of variance, principal component analysis (PCA), and Spearman's rank correlation (ρ). The 100 µL treatment was associated with the highest seedling emergence (85%), greater root development, increased root biomass accumulation, and higher potential quantum efficiency of photosystem II (Fv/Fm). In contrast, the highest bioinoculant dose (200 µL) was associated with anatomical alterations and increased non-regulated energy dissipation, suggesting greater physiological stress under the experimental conditions. Principal component analysis indicated that the 100 µL treatment was associated with variables related to early seedling growth and photochemical efficiency, whereas Spearman's correlation revealed positive associations between root growth and photosynthetic performance. Overall, the results suggest that the 100 µL dose promoted the most favorable morphophysiological responses during the initial development of maize seedlings. These findings indicate that the appropriate use of Bacillus spp., based bioinoculants represents a promising strategy for improving early crop establishment and may contribute to more efficient and sustainable production systems.

Keywords:
root development; photosynthetic efficiency; seedling emergence; Zea mays L

Resumo

A crescente demanda por alternativas sustentáveis na agricultura tem estimulado o uso de microrganismos promotores de crescimento vegetal como estratégia para melhorar o desempenho inicial das culturas. Nesse contexto, objetivou-se avaliar os efeitos fisiológicos iniciais da aplicação de um bioinoculante comercial contendo Bacillus spp. em plântulas de milho (Zea mays L.) cultivadas em areia estéril sob condições controladas. O experimento foi conduzido em delineamento inteiramente casualizado, utilizando cinco doses do bioinoculante (0, 50, 100, 150 e 200 µL), com quatro repetições. Foram avaliadas variáveis morfológicas (emergência de plântulas, comprimento e massa seca da raiz e da parte aérea), parâmetros fisiológicos (fluorescência da clorofila a e trocas gasosas) e características anatômicas e histoquímicas. Os dados foram submetidos à análise de variância, análise de componentes principais (PCA) e correlação de postos de Spearman (ρ). A dose de 100 µL esteve associada à maior emergência de plântulas (85%), ao maior desenvolvimento do sistema radicular, ao maior acúmulo de massa seca radicular e à maior eficiência quântica potencial do fotossistema II (Fv/Fm). Em contrapartida, a dose de 200 µL foi associada a alterações anatômicas e ao aumento da dissipação não regulada de energia (ΦNO), sugerindo maior estresse fisiológico nas condições experimentais avaliadas. A análise de componentes principais indicou associação da dose de 100 µL com variáveis relacionadas ao crescimento inicial e à eficiência fotoquímica das plântulas, enquanto a correlação de Spearman revelou associações positivas entre o desenvolvimento radicular e o desempenho fotossintético. Em conjunto, os resultados sugerem que a dose de 100 µL promoveu as respostas morfofisiológicas mais favoráveis durante o desenvolvimento inicial de plântulas de milho. Esses achados indicam que a aplicação adequada de bioinoculantes à base de Bacillus spp. representa uma estratégia promissora para otimizar o estabelecimento inicial da cultura, podendo contribuir para sistemas de produção mais eficientes e sustentáveis.

Palavras-chave:
desenvolvimento radicular; eficiência fotossintética; emergência de plântulas; Zea mays L

1. Introduction

Maize (Zea mays L.) is one of the most widely cultivated cereal crops worldwide, playing a fundamental role in food, feed, and industrial production. Owing to its high productive potential and broad adaptability, maize has become one of the main commodities of global agriculture. Brazil is currently among the world's leading producers and exporters, contributing substantially to global grain supply (Brasil, 2024).

In the 2023/24 growing season, national maize production was estimated at 115.72 million tons, representing a 12.3% reduction compared with the previous cycle (Brasil, 2024). This reduction has been associated with adverse climatic conditions and the occurrence of biotic and abiotic stresses, including high temperatures, irregular rainfall patterns, and pest outbreaks in major producing regions. These stress factors frequently occur simultaneously, impairing seed germination, seedling establishment, and plant physiological performance by affecting photosynthetic activity, gas exchange, and other biochemical processes essential for early plant development (Gentil Jacinto Junior and Lucena, 2022; Silva et al., 2022).

The initial establishment of maize seedlings is a key determinant of crop productivity. Seeds with high physiological vigor generally produce seedlings with greater root and shoot growth, higher dry matter accumulation, and more efficient reserve mobilization during germination, resulting in rapid and uniform establishment in the field. These characteristics enhance water and nutrient acquisition during the early stages of development and contribute to improved crop performance under field conditions (Tetreault et al., 2023; Andrade et al., 2019).

The physiological quality of maize seeds is considered a key determinant of successful crop establishment, as seed vigor influences the speed and uniformity of seedling emergence and the initial performance of seedlings under field conditions (Sommer et al., 2024). Therefore, maintaining high seed quality is essential to meet the increasing global demand for food while promoting more efficient and environmentally sustainable agricultural systems (Reed et al., 2022). In this context, bioinputs have emerged as promising biotechnological tools capable of improving seedling vigor, plant growth, and stress tolerance through beneficial plant–microorganism interactions (Sommer et al., 2024; Diniz et al., 2025).

Bioinputs used in agriculture can be classified as macrobiological, microbiological, and phytochemical/bioactive products. Among microbial bioinputs, bioinoculants based on Bacillus spp. have received considerable attention because these bacteria promote plant growth through multiple mechanisms, including phosphate solubilization, phytohormone production, nutrient-use enhancement, and the induction of systemic resistance, thereby contributing to improved plant establishment and development (Jiao et al., 2021; Diniz et al., 2025).

Studies in maize using bacteria of the species B. aryabhattai have demonstrated significant increases in shoot length and root system development of seedlings, in addition to positively modulating the rhizospheric microbiota (Deng et al., 2022; Fuga et al., 2023). Singh et al. (2024) also reported that B. aryabhattai promoted root elongation and seedling height through the production of indole-3-acetic acid (IAA), being particularly effective during the early stages of development. Research involving B. subtilis, such as the study by Song et al. (2023), showed improvements in germination, root growth, and biomass of seedlings exposed to saline stress. Although still scarcely studied in maize, B. haynesii also stands out for its promising potential in promoting plant growth and providing biological control of phytopathogens in other crops (Mishra et al., 2023)

The combined application of these species may result in synergistic effects, leading to substantial improvements in seedling emergence, morphophysiological development, and photosynthetic efficiency, in addition to contributing to physiological vigor and overall crop productivity (Santoyo et al., 2021; Tsotetsi et al., 2022).

In this context, the significant reduction in maize productivity observed in the 2023/24 season, combined with the climatic and phytosanitary challenges faced by producers, highlights the need for more efficient and sustainable agronomic strategies. It is hypothesized that the application of a commercial bioinoculant based on Bacillus spp. is capable of promoting positive effects on the initial physiological performance of seedlings, resulting in greater vigor, enhanced root growth, and improved photosynthetic efficiency.

It is assumed that increasing the doses of bioinoculants may modify the morphophysiological responses of maize seedlings due to different interaction patterns between the plant and the bacterial consortium.

In light of this, the objective of this study was to evaluate the effects of different doses of a Bacillus spp., based bioinoculant on the physiological performance of maize seedlings grown in sterile sand under controlled conditions.

2. Material and Methods

2.1. Plant material and treatment application

The experiments were conducted in the Seed Analysis, Plant Anatomy, Tissue Culture, and Plant Ecophysiology Laboratories of the Federal Institute of Education, Science and Technology – IF Goiano, Rio Verde Campus, Goiás, Brazil. The study was carried out using the maize cultivar BRS 284, 2023/2024 growing season. Prior to the establishment of the experiment, seed moisture content was determined and the germination test was performed according to Brasil (2025).

Sand was used as the substrate; it was sieved, sterilized, and kept in an oven at 200 °C for 2 hours. After cooling, the sand was placed into 10 Styrofoam trays (21 × 18 × 1.8 cm); for each dosage, two trays were used, totaling 4 kg of sand in each tray.

Subsequently, the commercial product based on Bacillus spp. was diluted in distilled water to obtain the following concentrations: 0 (distilled water), 50, 100, 150, and 200 µL. The dosage levels were defined according to the recommendations of the commercial product. The solution was then applied directly to the sand until reaching its field capacity (60%), following the methodology described in the Rules for Seed Testing (Brasil, 2025).

After preparing the inoculated substrate, 200 maize seeds were sown at a depth of 1 cm. The treatments were arranged in four replicates and conducted in two trays. The trays were lined with plastic bags to prevent moisture loss through evaporation and kept in a germination chamber at a controlled temperature of 25 ± 0.5 °C.

2.1.2. Physiological analyses of seeds

-Emergence Test: On the seventh day after sowing, using the previously inoculated sand substrate, the trays were maintained in a germination chamber at 25 ± 0.5 °C. The evaluation consisted of counting emerged seedlings, defined as those whose cotyledon was visible above the substrate surface, according to the criteria described by Nakagawa (1999).

-Seedling Length: On the twelfth day after sowing, ten seedlings per replicate were randomly selected. These seedlings were washed under running water to ensure the complete removal of sand and preservation of the entire root system. The lengths of the root system and shoot were measured using a millimeter ruler, and the results were expressed in centimeters (Brasil, 2025).

-Dry Mass: After length measurements, the same seedlings were used to determine dry mass. The plant material was separated into shoot and root system and placed in a forced-air circulation oven at 80 °C until reaching constant weight. Subsequently, the samples were weighed on a precision analytical balance, following the methodology described by Nakagawa (1999). The results were expressed in grams per seedling.

2.1.3. Gas exchange

Gas exchange was measured on four fully expanded leaves per treatment from maize seedlings on the twelfth day after sowing. The following parameters were analyzed: net photosynthetic rate (A, µmol m⁻² s⁻¹), transpiration (E, mmol H₂O m⁻² s⁻¹), stomatal conductance (Gs, mol H₂O m⁻² s⁻¹), and the ratio between internal (Ci) and external (Ca) CO₂ concentration (Ci/Ca). Measurements were performed using an open-system infrared gas analyzer (IRGA, LI-6800, LI-COR Inc., Lincoln, NE, USA). Data were collected between 8:00 and 11:00 a.m. under photosynthetically active radiation (PAR, 1000 µmol photons m⁻² s⁻¹) and a temperature of 25 °C.

2.1.4. Chlorophyll a fluorescence imaging

Chlorophyll a fluorescence images were obtained using a modulated fluorometer, Imaging-PAM (MAXI-Standard version, Heinz Walz). Initially, four seedlings per replicate were randomly selected and placed in the fluorometer in the dark for at least 40 minutes. During this dark adaptation period, initial fluorescence (F₀) and maximum fluorescence (Fm) were determined, where F₀ corresponds to the minimum fluorescence yield elicited by low-intensity modulated red light (3 μmol m⁻² s⁻¹), and Fm represents the maximum fluorescence obtained through the application of a saturating light pulse with a duration of 0.8 s and intensity above 6000 μmol m⁻² s⁻¹. From these values, the potential quantum yield of photosystem II (PSII) was calculated using the Equation 1:

$$Fv/Fm=\left(Fm-F_0\right)/Fm$$ (1)

Variables associated with the slow phase of fluorescence induction were determined sequentially by applying constant illumination for 30 s, followed by a saturating light pulse. From this procedure, the fluorescence of light-adapted leaves before the pulse (F) and the maximum fluorescence of light-adapted leaves (Fm’) were obtained. The minimum fluorescence in illuminated tissue was calculated using the Equation 2

$$F_0/\left[\left(\left(Fm-F_0\right)/Fm\right)+\left(F_0/Fm\right)\right]$$ (2)

as described by Oxborough and Baker (1997). Based on these parameters, the following were determined: the effective quantum yield of photochemical energy conversion in PSII ($\Phi II=(Fm\text{'}-F)/Fm\text{'}$); the quantum yield of regulated energy dissipation ($\Phi NPQ=(F/Fm\text{'})-(F/Fm)$); and the quantum yield of non-regulated energy dissipation ($\Phi NO=F/Fm$), following Genty et al. (1989) and Hendrickson et al. (2004).

2.1.5. Anatomical and histochemical characterization

Plant material was collected on the twelfth day, and anatomical sections were obtained from the leaf blade region of four fully expanded leaves from each treatment. The material was then fixed in Karnovsky’s solution (Karnovsky, 1965) for 24 hours. After fixation, samples were dehydrated in an ascending ethanol series, pre-infiltrated and infiltrated in historesin (Leica, Nussloch, Germany), and subsequently embedded. Using a rotary microtome (Model 1508R, Logen Scientific, China), the samples were sectioned transversely at a thickness of 5 μm and stained with toluidine blue (O'Brien et al., 1964). For histochemical analysis, the sections were stained with Xylidine Ponceau (XP) to identify total proteins (O'Brien and McCully, 1981). Finally, the leaf slides were examined under an Olympus microscope (model BX61, Tokyo, Japan), equipped with a DP-72 camera for image capture, using bright-field settings.

2.1.6. Statistical analysis

The experiment was conducted using a completely randomized design (CRD) with four replicates of 50 seeds each. Data were subjected to analysis of variance (ANOVA), and means were compared using Tukey's test at a 5% probability level. Variables related to physiological potential, chlorophyll fluorescence, and gas exchange were subjected to principal component analysis (PCA) and Spearman's rank correlation (ρ). The correlation matrix was calculated using 20 individual observations—corresponding to the five treatments with four replicates—and the significance of the coefficients was assessed at a 5% probability level. Statistical analyses were performed using R software (version 4.3.2), and graphs were generated using SigmaPlot® (version 11.0).

3. Results

3.1. Effect of different bioinoculant doses on emergence, root length, shoot length, and root and shoot dry mass of maize seedlings

The assay showed statistical differences among the bioinoculant doses, with an increase in seedling emergence percentage as evaluated by Tukey’s test (p < 0.05) (Figure 1).

Figure 1
Mean percentage of maize seedling emergence (%) subjected to different doses: 0, 50, 100, 150, and 200 µL of bioinoculant. Means followed by the same lowercase letter do not differ statistically according to Tukey’s test at the 5% probability level. Bars represent mean ± standard error.

Application of the bioinoculant significantly influenced seedling emergence, exhibiting a dose-dependent response. The control (0.00 µL) showed the lowest emergence rate (67%). Emergence increased progressively, reaching its highest value at the 100 µL dose, with 85% emergence, which was statistically superior to the other treatments. The 50, 150, and 200 µL doses also promoted higher emergence compared with the control (73%, 75%, and 75%, respectively), but did not differ significantly from one another.

For root length (cm), all doses resulted in a significant increase compared with the control (Figure 2).

Figure 2
Root length (cm) of maize seedlings subjected to different doses: water (0) and concentrations of 50, 100, 150, and 200 µL of bioinoculant. Means followed by the same lowercase letter do not differ statistically according to Tukey’s test at the 5% probability level. Bars represent mean ± standard error.

Control seedlings (0.00 µL) exhibited the shortest root length, averaging 19 cm, and were statistically inferior to all other treatments. A positive response was observed with the application of doses ranging from 50 to 200 µL. The 100 µL dose resulted in the greatest mean root length, with approximately 68% longer roots compared with the control (Figure 2), and was statistically superior to the other treatments (p < 0.05). The 150 and 200 µL doses did not differ from each other but showed higher values than those observed in the 0 and 50 µL treatments.

Shoot length (cm) was also influenced by the application of the bioinoculant. The control with distilled water (0.00 µL) showed the lowest shoot length, while all inoculated doses differed from the control but not from one another, according to Tukey’s test (p < 0.05). Thus, regardless of the dose applied, no significant differences were observed in shoot length among the bioinoculant treatments, indicating that lower doses are already sufficient to promote maximum seedling growth (Figure 3).

Figure 3
Shoot length (cm) of maize seedlings subjected to different doses: water (0) and concentrations of 50, 100, 150, and 200 µL of bioinoculant. Means followed by the same lowercase letter do not differ statistically according to Tukey’s test at the 5% probability level. Bars represent mean ± standard error.

Root dry mass (g) was also influenced by the bioinoculant doses, as shown in Figure 4. A significant treatment effect was observed according to Tukey's test (p < 0.05), with the greatest root biomass accumulation occurring at the 100 µL dose.

Figure 4
Root dry mass (g) of maize seedlings subjected to different doses: water (0) and concentrations of 50, 100, 150, and 200 µL of bioinoculant. Means followed by the same lowercase letter do not differ statistically according to Tukey’s test at the 5% probability level. Bars represent mean ± standard error.

A significant effect of the bioinoculant dosages was observed on root dry mass. Among the inoculated treatments, the 50 μL dosage resulted in the smallest increase in root biomass, although it differed significantly from the control. The 100 μL dosage promoted the greatest accumulation of root dry mass, differing from the other dosages evaluated. The 150 and 200 μL treatments showed intermediate values; they did not differ from each other and indicated a reduced response compared to the 100 μL dosage.

The shoot dry mass was not influenced by the bioinoculant doses, and no statistically significant differences were observed among the treatments (Figure 5).

Figure 5
Shoot dry mass (g) of maize seedlings subjected to different doses: water (0) and concentrations of 50, 100, 150, and 200 µL of bioinoculant. Means followed by the same lowercase letter do not differ statistically according to Tukey’s test at the 5% probability level. Bars represent mean ± standard error.

3.2. Effect of different bioinoculant doses on physiological analysis by Chlorophyll fluorescence imaging in maize seedlings

Chlorophyll fluorescence imaging was used to assess the impact of the bioinoculant doses on the photosynthetic physiology of the seedlings, with emphasis on parameters of photosystem II (PSII) (Figure 6).

Figure 6
Potential quantum yield of PSII (Fv/Fm), maximum fluorescence (Fm), initial fluorescence (F₀), effective quantum yield of PSII (ΦII), and quantum yield of non-regulated energy dissipation of PSII (ΦNO) in maize seedlings treated with different doses: water (0) and concentrations of 50, 100, 150, and 200 µL of bioinoculant. Asterisks indicate differences at the 5% (*) probability level compared with the control according to Tukey’s test. Means followed by the same lowercase letter do not differ statistically according to Tukey’s test at the 5% probability level. Bars represent mean ± standard error.

There was a significant effect on the potential quantum yield of PSII (Fv/Fm) and on the quantum yield of non-regulated energy dissipation (ΦNO). The 100 µL dose stood out, presenting the highest Fv/Fm value, which was statistically superior to the results obtained with the other evaluated doses. For non-regulated energy dissipation, only the 200 µL dose showed a significant difference. Thus, the bioinoculant did not alter photochemical dynamics, except for the increase in Fv/Fm at the 100 µL dose.

3.3. Effect of different bioinoculant doses on gas exchange physiology

Statistical analysis of gas exchange variables, conducted on the twelfth day, showed no differences among the parameters according to Tukey’s test at the 5% probability level. The mean values (± standard error) were homogeneous across all concentrations (Table 1).

Table 1
Transpiration rate (E), photosynthetic rate (A), ratio between internal and external CO₂ concentration (Ci/Ca), and stomatal conductance (gs) in maize seedlings at 12 days after emergence.

Doses	E (mmol m−2 s−1)	A (μmol m−2 s−1)	Ci/Ca	gs (mol m−2 s−1)
0 (água)	0.0025±0.0011	1.73±1.04	1.15±0.07	0.12±0.06
50	0.0024±0.0015	3.06±1.02	1.43±0.39	0.11±0.07
100	0.0025±0.0023	1.96±1.53	1.35±0.40	0.12±0.11
150	0.0008±0.0005	2.44±0.57	1.64±0.21	0.04±0.02
200	0.0033±0.0024	3.39±1.47	1.74±0.77	0.16±0.12
CV (%)	91.44	61.3	40.3	95.82

• Data are presented as means ± standard error. No statistically significant differences were observed among treatments according to Tukey's test (p > 0.05).

3.4. Effect of different doses on anatomical and histochemical alterations induced by the bioinoculant

Maize (Zea mays L.) is a C4 species whose leaf anatomy is characterized by the presence of bundle sheath cells surrounding the vascular tissues, forming the so-called Kranz anatomy, in addition to a well-developed chlorenchyma and bulliform cells (Sage, 2016). Anatomical analysis revealed differences in seedling morphology, as shown in figure 7.

Figure 7
Cross sections of maize (Zea mays L.) seedling leaves observed via light microscopy. (A) Control treatment (0 µL), stained with toluidine blue; (B) treatment with 200 µL of the bioinoculant, stained with toluidine blue; (C) control treatment (0 µL), stained with xylidine ponceau for protein identification; (D) treatment with 200 µL of the bioinoculant, stained with xylidine ponceau for protein identification. AdEp = adaxial epidermis; AbEp = abaxial epidermis; CB = bulliform cell; PC = chlorenchyma; BK = Kranz sheath; X = xylem; FL = phloem. Bars = 100 µm.

Anatomical analysis revealed structural differences between the extreme treatments evaluated. Representative images of the control treatment (0 µL; Figures 7A and 7C) showed preserved leaf tissue organization, with well-defined epidermal layers, intact bulliform cells, a bundle sheath adequately enclosing the vascular tissues, organized chlorenchyma, and a vascular system with clearly delimited xylem and phloem. In contrast, representative images of the 200 µL treatment (Figures 7B and 7D) showed greater parenchyma disorganization, increased intercellular spaces, and reduced definition of vascular tissues, suggesting structural alterations associated with the higher dose of the bioinoculant. The images presented correspond to the extreme treatments (0 and 200 µL), selected because they represent the conditions that most clearly demonstrated the observed anatomical differences.

3.5. Principal Component Analysis (PCA) of morphophysiological responses to bioinoculant doses in maize seedlings

Principal Component Analysis (PCA) analyzed the treatments and variables across three principal components (PC1, PC2, and PC3), which jointly explained 67.5% of the total data variance. PC1 explained 29.30% of the total variance, PC2 20.00%, and PC3 18.20% (Table 2; Figure 8). The three components exhibited eigenvalues greater than 1 (4.40, 3.00, and 2.72, respectively) and were retained for interpretation based on the Kaiser criterion (Kaiser, 1960), as they adequately represented the multivariate structure of the dataset. The contributions of the variables to each principal component are presented in Table 3.

Table 2
Eigenvalues, explained variance and cumulative variance of the principal components obtained by principal component analysis (PCA).

Principal Component	Eigenvalue	Explained variance (%)	Cumulative variance (%)
PC1	4.393	29.29	29.29
PC2	2.995	19.97	49.26
PC3	2.725	18.17	67.43

• The principal components were retained based on the Kaiser criterion (eigenvalues > 1). The first three components jointly explained 67.5% of the total data variance.

Figure 8
Principal component analysis (PCA) biplots showing PC1 × PC2 (A) and PC2 × PC3 (B) axes for physiological quality, growth, and vigor variables of maize seedlings: emergence (EM), root length (CR), shoot length (CP), root dry mass (MSR), shoot dry mass (MSP), initial fluorescence (F₀), maximum fluorescence (Fm), potential quantum yield of PSII (Fv/Fm), effective quantum yield of PSII (ΦII), non-photochemical quenching yield (YNPQ), quantum yield of non-regulated energy dissipation of PSII (ΦNO), photosynthetic rate (A), transpiration rate (E), stomatal conductance (gs), and the ratio of internal to external CO₂ concentration (Ci/Ca) in maize seedlings subjected to water (D0) and bioinoculant (D50, D100, D150, and D200) doses. Points represent individual replicates grouped by 95% confidence ellipses, while vectors indicate the direction and magnitude of each variable's contribution to the principal components.

Table 3
Contribution (%) of the variables to the first three principal components (PC1, PC2, and PC3).

Variable	PC1 (%)	PC2 (%)	PC3 (%)
Emergence (EM)	16.22	1.71	6.71
Root length (RL)	15.62	1.87	6.94
Shoot length (SL)	6.87	2.97	2.26
Root dry mass (RDM)	14.95	1.63	5.43
Shoot dry mass (SDM)	8.65	0.00	6.98
Transpiration rate (E)	0.06	26.53	4.22
Net photosynthetic rate (A)	0.16	4.16	2.53
Intercellular to ambient CO2 concentration ratio (Ci/Ca)	0.01	11.49	10.85
Stomatal conductance (gs)	0.04	26.04	4.35
Initial fluorescence (F0)	7.06	12.62	4.68
Maximum fluorescence (Fm)	6.30	6.72	3.40
Maximum quantum efficiency of PSII (Fv/Fm)	11.27	1.34	0.07
Effective quantum yield of PSII (ΦII)	5.73	1.41	19.39
Non-photochemical quenching yield (YNPQ)	6.43	0.13	1.00
Quantum yield of non-regulated energy dissipation of PSII (ΦNO)	0.63	1.37	21.19

• Percentage contribution of the variables to the first three principal components (PC1, PC2, and PC3) of the principal component analysis (PCA). Higher values indicate a greater contribution of the variable to the formation of the respective principal component.

Principal component analysis showed that the 100 µL dose (D100) was associated with variables related to early seedling growth specifically emergence, root length, shoot length, root dry mass, and the potential quantum yield of photosystem II (Fv/Fm) which contributed most significantly to PC1 (Table 3). Conversely, the 200 µL dose (D200) was primarily associated with gas exchange variables specifically transpiration rate (E) and stomatal conductance (gs) which contributed predominantly to PC2 (Table 3). The D0, D50, and D150 treatments were distributed across different regions of the multivariate space, reflecting distinct patterns of association with the evaluated variables, without demonstrating, in isolation, any biological superiority or inferiority among the treatments.

3.6. Spearman correlations among growth, chlorophyll fluorescence, and gas exchange variables

Spearman correlation analysis, conducted with 20 individual observations, revealed significant associations among variables related to early seedling growth, chlorophyll fluorescence, and gas exchange (Figure 9). Emergence showed a strong positive correlation with root length (ρ = 0.86) and root dry mass (ρ = 0.81), and a moderate correlation with shoot length (ρ = 0.55). Root length also showed a strong association with root dry mass (ρ = 0.88) and a positive correlation with shoot length (ρ = 0.52).

Figure 9
Spearman correlation matrix (ρ) among growth, chlorophyll fluorescence, and gas exchange variables in maize seedlings, calculated using 20 individual observations. Only statistically significant correlations (p < 0.05) are shown. The intensity and direction of the associations are represented by the color scale, ranging from −1 (red) to +1 (blue).

Among the fluorescence parameters, shoot dry mass was positively correlated with Fv/Fm (ρ = 0.45) and ΦII (ρ = 0.58), and negatively correlated with YNPQ (ρ = −0.61). Furthermore, ΦII showed a negative correlation with ΦNO (ρ = −0.63), whereas F₀ showed a positive correlation with Fm (ρ = 0.79) and a negative correlation with ΦII (ρ = −0.55). Among the gas exchange variables, positive correlations were observed between transpiration rate and photosynthetic rate (ρ = 0.54), as well as between photosynthetic rate and stomatal conductance (ρ = 0.52), while the Ci/Ca ratio showed a negative correlation with transpiration rate (ρ = −0.67) and stomatal conductance (ρ = −0.66). Correlations of low magnitude or lacking statistical significance were not used to support biological interpretations.

4. Discussion

Plant growth-promoting bacteria (PGPB) comprise a group of free-living microorganisms capable of stimulating plant development through direct or indirect mechanisms. They act by solubilizing nutrients, producing phytohormones, and activating defense mechanisms such as the induction of systemic resistance. In addition, these bacteria colonize the rhizosphere or even internal plant tissues, establishing beneficial interactions for both the host and the microorganisms (Dias and Santos, 2022; Diniz et al., 2025).

The results obtained in this study indicate a positive effect of the bioinoculant on seedling emergence. A higher emergence rate was observed at the 100 µL dosage, surpassing the control treatment (67%). This result suggests that inoculation with Bacillus spp. may have promoted the seedling emergence process. Similar behavior was reported by Deng et al. (2022), who observed increased emergence in maize seedlings inoculated with Bacillus aryabhattai an effect the authors attributed to phytohormone production and enhanced nutrient solubilization in the substrate. In maize, Gao et al. (2020) reported that a biofertilizer mixture containing Bacillus circulans, Azotobacter chroococcum, and arbuscular mycorrhizal fungi promoted plant growth and nutrient uptake, supporting the potential use of beneficial microorganisms in maize production.

The observed increase in growth and biomass may be related to the ability of these bacteria to promote plant growth through multiple mechanisms, such as phosphate solubilization, the biosynthesis of phytohormones like indole-3-acetic acid (IAA), siderophore production, and biological nitrogen fixation (Patani et al., 2024; Vey et al., 2025). Studies conducted by Radhakrishnan et al. (2017) suggest that these mechanisms can contribute to root development by stimulating plant metabolism and increasing nutrient availability.

At the 100 µL dosage of the Bacillus spp.-based bioinoculant, a more favorable response was observed in the bacterium-plant interaction; this may be linked to a more efficient interaction compared to higher dosages, thereby promoting early seedling development. Principal Component Analysis (PCA) revealed an association with the 100 µL dosage, positioning it distinctly along the PC1 axis in conjunction with the variables root length (RL), root dry mass (RDM), and potential quantum yield of PSII (Fv/Fm), indicating an association among these variables within the multivariate space, corroborating reports that bacterial inoculation can simultaneously improve root development and physiological performance in maize (Müller et al., 2025).

Studies with Bacillus aryabhattai in maize have demonstrated that plant growth responses may vary according to bacterial inoculum density. Deng et al. (2022) observed that the stimulation of maize seedling growth varied according to bacterial cell density, indicating that plant responses may depend on inoculum concentration. This pattern was also observed in the present study, where doses of 150 and 200 µL resulted in lower root dry mass compared to the 100 µL dose, corroborating the findings of Egamberdieva et al. (2018), who suggest that over-inoculation can lead to competition among bacteria in the rhizosphere for space and nutrients, as well as promote abiotic stress in the root zone.

The use of the Bacillus spp., based bioinoculant resulted in a significant increase in shoot length at all tested dosages compared to the control treatment. However, this trend was not observed regarding biomass, as there was no proportional increase, suggesting a predominance of cell elongation. This pattern may be related to the action of phytohormones produced by microorganisms, as described in the literature.

This dissociation between elongation and biomass accumulation was also observed by Alonazi et al. (2025), who found an increase in shoot length without a corresponding increase in dry mass in maize inoculated with Bacillus haynesii under salt stress. Previous studies suggest that this type of response may be linked to the production of gibberellins by plant growth-promoting bacteria; these hormones promote cell elongation without necessarily increasing tissue density (Song et al., 2023). Similarly, Liu et al. (2022) observed that inoculating wheat with Bacillus velezensis promoted an increase in root system development and plant biomass, indicating that this species can favor resource allocation toward root growth, especially under salt stress conditions.

Fluorescence imaging analysis revealed a higher potential quantum yield of photosystem II (Fv/Fm) at the 100 µL dose of the bioinoculant, suggesting greater potential photochemical efficiency of PSII. Similar results were reported by Li et al. (2024), who, in a study on cucumber (Cucumis sativus), observed that the application of Bacillus subtilis combined with potassium promoted increases in root length, plant height, and Fv/Fm values, indicating improved photosynthetic performance in the plants.

Fluorescence imaging analysis revealed a higher potential quantum yield of photosystem II (Fv/Fm) at the 100 µL dose of the bioinoculant, suggesting greater potential photochemical efficiency of PSII. Similar results were reported by Li et al. (2024), who, in a study on cucumber (Cucumis sativus), observed that the application of Bacillus subtilis combined with potassium promoted increases in root length, plant height, and Fv/Fm values, indicating improved photosynthetic performance in the plants. These findings are consistent with the growing body of evidence demonstrating that inoculation with plant growth-promoting bacteria enhances photosynthetic performance by improving PSII efficiency and chlorophyll fluorescence parameters (Diniz et al., 2025).

Furthermore, the increase in Fv/Fm may be associated with the production of phytohormones, such as indole-3-acetic acid (IAA), by Bacillus species, which can promote root growth. Studies show that inoculation with Bacillus increased the Fv/Fm ratio in pepper plants, accompanied by auxin production (Samaniego-Gámez et al., 2016). In maize, it has been demonstrated that the IAA biosynthesis pathway in a Bacillus strain is essential for the growth-promoting effect (Figueredo et al., 2023). Taken together, these findings suggest that the improvement in photosynthetic efficiency observed in the present study may be partially related to hormonal mechanisms described for Bacillus species, particularly those involving IAA production and root growth stimulation, although these mechanisms were not directly investigated.

According to Galicia-Campos et al. (2023), in a study conducted with olive trees (Olea europaea) under salt stress, inoculation with Bacillus spp. also increased Fv/Fm values. Collectively, these findings indicate that Bacillus species can help maintain the photochemical efficiency of PSII across different crops, particularly under stress conditions, although the underlying mechanisms may vary depending on the plant species, bacterial strain, and environmental conditions.

Regarding the quantum yield of non-regulated energy dissipation (YNO), the highest value was observed at the 200 µL dose, suggesting a reduced capacity for regulated dissipation of excess excitation energy. This condition may increase the risk of photochemical damage to photosystem II (PSII) and indicates greater physiological stress associated with higher bioinoculant doses. Consistently, anatomical and histochemical analyses of seedlings treated with the 200 µL dose revealed visible alterations indicative of greater cellular disorganization compared with the control treatment.

In this study, no significant differences were observed in the gas exchange variables. Although no direct effects were detected at this early stage of seedling development, the possibility that physiological responses may emerge during later stages of crop development cannot be ruled out. Likewise, histochemical analyses revealed relevant alterations in cellular structures, highlighting the need for further investigations. Additional studies are required to better elucidate the influence of Bacillus spp. on the physiological mechanisms associated with gas exchange and on histochemical modifications in plant tissues.

Principal Component Analysis (PCA) effectively synthesized the combined effects of the morphological, physiological, and biochemical variables evaluated. The 100 µL dose was clearly associated with the positive side of the first principal component (PC1), being primarily related to root length, shoot length, root dry mass, and the maximum quantum yield of PSII (Fv/Fm). These findings indicate that the 100 µL dose was associated with the variables linked to early seedling growth and photochemical efficiency evaluated in this study.

A similar multivariate pattern has been reported in other crops. For example, in spinach plants inoculated with Bacillus circulans, principal component analysis (PCA) successfully differentiated plant groups based on root growth, biomass accumulation, and photosynthetic activity, with photosynthetic performance being closely associated with growth-related variables (Shahid et al., 2020).

Spearman correlation analysis revealed significant associations among variables related to early seedling growth, chlorophyll fluorescence, and gas exchange. Strong positive correlations were observed among emergence, root length, and root dry mass, indicating coordinated responses during early seedling establishment. In addition, shoot dry mass was positively correlated with the effective quantum yield of PSII (ΦII) and the maximum quantum yield of PSII (Fv/Fm), and negatively correlated with the non-photochemical quenching yield (YNPQ), highlighting the close relationship between biomass accumulation and photochemical performance under the conditions evaluated. These correlations represent patterns of association among the measured variables and should not be interpreted as evidence of causal relationships.

The combined use of Principal Component Analysis (PCA) and Spearman correlation is widely employed in studies involving plant growth-promoting bacteria, as these multivariate approaches facilitate the identification of association patterns between treatments and physiological and morphological traits. In maize, Zakavi et al. (2022) identified distinct groupings in PCA and significant correlations among root biomass, root length, and shoot growth in plants inoculated with different Bacillus strains under both normal and salt-stress conditions. Likewise, in the present study, PCA associated the 100 µL dose with variables related to early growth and photochemical efficiency, whereas the 150 and 200 µL doses exhibited distinct association patterns. These findings suggest differential physiological responses among treatments; however, the multivariate analyses identify patterns of association and do not, by themselves, establish biological superiority or causal relationships.

Finally, the results of this study suggest that the 100 µL dose was associated with variables related to early seedling growth and photochemical efficiency. However, these findings should be interpreted within the context of the experimental conditions adopted. Further studies under field conditions and at later stages of crop development are required to confirm the consistency of these responses and to improve our understanding of the underlying physiological mechanisms.

5. Conclusion

Bioinoculation with Bacillus spp. was associated with favorable responses in the initial establishment and morphophysiological performance of maize seedlings under the experimental conditions evaluated. Among the tested doses, the 100 µL treatment showed the most favorable performance, being associated with greater root growth and higher photosystem II photochemical efficiency. Since this study was conducted under controlled conditions and during the early stage of seedling development, the findings are limited to the experimental conditions evaluated. Therefore, further studies under field conditions and during later stages of crop development are required to validate these results and improve the understanding of the effects of Bacillus spp. bioinoculation on maize development.

Acknowledgements

The authors express their gratitude Seed Laboratory, Federal Institute of Education, Science and Technology of Goiano (IFGoiano), Rio Verde, South Goiania Highway, Km 01, Rural Area, Rio Verde-GO, 75901970, Brazil, the Research Support Foundation of the State of Goiás (FAPEG), and the Coordination for the Improvement of Higher Education Personnel (CAPES) for institutional support and for granting the master’s scholarship notice 38/2022.

Data Availability Statement

The entire data set supporting the results of this study was published in the article itself.

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*

e-mail: [e-mail]

Editor:

Takako Matsumura Tundisi

Publication Dates

• Publication in this collection
  12 Oct 2026
• Date of issue
  2026

History

• Received
  12 Dec 2025
• Accepted
  21 Aug 2026