Effect of Methylobacterium on the yield and quality of cauliflower (Brassica oleracea)

Efeito do Methylobacterium no rendimento e na qualidade da couve-flor (Brassica oleracea)

Abstract

The soil used is sandy in texture, low in nutrients, and low in organic matter, and therefore has low cation exchange capacity. Therefore, the objective was to evaluate the effectiveness of improving their physicochemical and biological properties by inoculating the roots with a solution of Methylobacterium and liquid compost in cauliflower cultivation, as well as integrating a treatment through an automated drip irrigation system. This biostimulant, biofertilizer, and biocontrol agent solution was used, which has applications in agriculture to improve soil health and crop productivity. The experiment was set up under a completely randomized block design with four treatments (T0, T1, T2, and T3), corresponding to doses of 0, 250, 333, and 400 mL per 200 L of water, respectively. The results indicated that treatment T3 significantly optimized the plant's physiological response to salt stress, increasing both its antioxidant capacity and chlorophyll α concentration. Likewise, physical characterization revealed significant differences in the morphological parameters of cauliflower, suggesting greater metabolic resistance and improved nutritional quality. Finally, ultrastructural analysis of the epidermis and stomata using microscopy showed that, while the control treatment (T0) had a collapsed surface, treatment T3 showed a functional and turgid ultrastructure. This demonstrates that the inoculation applied mitigates the phytotoxic impact of the substrate and optimizes the metabolic potential of the crop.

Keywords:
Brassica oleracea; Methylobacterium; biostimulation; antioxidant capacity

Resumo

O solo utilizado é de textura arenosa, pobre em nutrientes e matéria orgânica, apresentando, portanto, baixa capacidade de troca catiônica. Diante disso, o objetivo foi avaliar a eficácia da melhoria de suas propriedades físico-químicas e biológicas através da inoculação das raízes com uma solução de Methylobacterium e composto líquido no cultivo de couve-flor, bem como integrar um tratamento por meio de um sistema automatizado de irrigação por gotejamento. Foi utilizada esta solução de bioestimulante, biofertilizante e agente de controle biológico, que tem aplicações na agricultura para melhorar a saúde do solo e a produtividade das culturas. O experimento foi instalado sob delineamento de blocos casualizados com quatro tratamentos (T0, T1, T2 e T3), correspondentes a doses de 0, 250, 333 e 400 mL por 200 L de água, respectivamente. Os resultados indicaram que o tratamento T3 otimizou significativamente a resposta fisiológica da planta ao estresse salino, aumentando tanto sua capacidade antioxidante quanto a concentração de clorofila α. Da mesma forma, a caracterização física revelou diferenças significativas nos parâmetros morfológicos da couve-flor, sugerindo maior resistência metabólica e melhor qualidade nutricional. Finalmente, a análise ultraestrutural da epiderme e dos estômatos por microscopia mostrou que, enquanto o tratamento controle (T0) apresentava uma superfície colapsada, o tratamento T3 apresentava uma ultraestrutura funcional e túrgida. Isso demonstra que a inoculação aplicada atenua o impacto fitotóxico do substrato e otimiza o potencial metabólico da cultura.

Palavras-chave:
Brassica oleracea; Methylobacterium; bioestimulação; capacidade antioxidante

1. Introduction

Current trends in organic farming and environmental sustainability are constantly evolving, driving conceptual and technological innovations aimed at improving quality of life. Anthropogenic impacts increasingly drive ecological and evolutionary processes at many spatio-temporal scales, demanding greater capacity to predict and manage their consequences (Thrall et al., 2011). Salinity is the main factor limiting crop development and yield due to the physiological stress caused by salts (Cabezas Gutiérrez et al., 2022). Salinization is one of the most serious threats requiring comprehensive management of both soil and water (Sahab et al., 2021; Yu et al., 2025).

Salinity is one of the major abiotic stress factors affecting agricultural yield (Kumari et al., 2022). The main focus of current research on saline soil improvement includes physical, chemical, and biological strategies (Li and Li, 2025). Saline stress causes physiological and biochemical changes in plant metabolism, which determine their survival and productivity under these conditions. Plants have developed tolerance mechanisms to cope with this stress (Lamz and González, 2013).

The use of organic soil amendments presents a promising approach to mitigating yield losses and promoting sustainable agricultural production in saline-alkaline soils (Wang et al., 2023). The bioremediation of a saline-sodic soil in the municipality of Tlahuelilpan, Hidalgo, was evaluated through the use of organic and chemical amendments (Trejo, 2019). Plant growth-promoting bacteria are a key ally for sustainable agriculture (Boukhatem et al., 2022). The use of beneficial symbiotic and non-symbiotic free-living bacteria that promote plant growth as an external source of nitrogen is one of the main research concerns for sustainable crop production in the 21st century (Lee et al., 2009). Beneficial soil microorganisms, such as arbuscular mycorrhizal fungi and plant growth-promoting bacteria, especially when sourced from saline environments, can alleviate salt stress in plants through multiple mechanisms (Tedeschi et al., 2023).

The rhizosphere microbiome is crucial for agriculture because a wide variety of root exudates and plant cell debris attract unique and distinct patterns of microbial colonization (Khoso et al., 2024). One of the main bacterial genera in the plant microbiome that colonizes the entire plant system is the genus Methylobacterium is used as a biostimulant, biofertilizer, and biocontrol agent, and has potential applications in agriculture to improve soil health, crop productivity, and environmental sustainability (Ayyamuthu Rajarathinam Uma et al., 2025). Symbiotic strains of Methylobacterium constitute an important part of the plant microbiome. Methylobacteria can synthesize unusually high levels of phytohormones, called cytokinins, including their most active form, trans-zeatin (Palberg et al., 2022). Recently, the use of new products derived from compost, such as compost tea, is increasing due to their positive effects on crops. This perspective wants to give an updated shot at the effect of compost tea in horticulture (Pilla et al., 2023).

The effects of compost tea on crops can vary considerably depending on the waste material used, the quality of the compost, the production process and parameters, and the interaction between horticultural species and the application rate (Campana et al., 2025). The use of compost and compost tea offers promising solutions in organic farming practices (Hakimi et al., 2024). Compost teas are organic extracts obtained by mixing mature compost with tap water under controlled conditions. The application of these extracts could be a biostimulant and ecological alternative for sustainable agriculture (González-Hernández et al., 2022).

The objective of this research was to evaluate the agronomic performance of radish cultivation in sandy soil, characterized by its high and low nutritional levels. The effect of root inoculation using a solution of Methylobacterium S23 and liquid compost in four concentrations was analyzed, establishing a control group to determine the effectiveness of the treatments. The vegetative and soil responses were characterized using physicochemical analyses, spectrophotometry, and scanning electron microscopy.

2. Materials and Methods

2.1. Location and characterization the experimental area

The study was conducted at the experimental field of the Faculty of Agricultural, Food, and Environmental Engineering of the José Faustino Sánchez Carrión National University (UNJFSC), located in Huacho, Peru. The experimental area is situated at an altitude of 50 meters above sea level, at geographical coordinates -11.13 and -77.61.

2.2. Experimental design and cultivation process

The commercial hybrid cauliflower (Brassica oleracea var. botrytis L.) “Esmerald” was used as the biological material. The experiment was set up using a completely randomized block design (CRBD).

The cultivation process was divided into three main phenological stages: a) Sowing: Carried out in trays on April 23, 2025, b) Transplanting: Carried out in the final field on May 21, 2025 (28 days after sowing), c) Harvesting: This was carried out in stages; the first cut took place on September 12, 2025 (40% of production) and the second on September 29, 2025 (the remaining 60%). The synergistic effect of liquid compost and the nitrogen-fixing bacterium Methylobacterium symbioticum (strain SB23) was evaluated. The total dose of leachate was 2 L·ha⁻¹. The treatments were applied in two critical stages:

1. First application: At the time of transplanting (May 21, 2025), applying 50% of the total dose of both products.

2. Second application: 30 days after transplanting (June 20, 2025), corresponding to 58 days after sowing, applying the remaining 50%.

2.3. Methodology for the spectrophotometric quantification of bioactive compounds and plant pigments

2.3.1. Extraction procedure

The leaves were washed, rinsed and frozen at −40 °C for at least 16 h and freeze-dried (Biobase, CN) for 74 h at a pressure < 0.4 mbar using a temperature ramp starting from −40 °C up to 20 °C. After which they are pulverized into fine powder (IKA, AM20, Germany). The samples were extracted with acidic (HCl)-methanol/water (50:50, v/v, pH 2) in a gyratory orbital shaker (Benchmark scientific, R5010. USA) for 90 minutes (at room temperature) in dark conditions, by covering the samples with aluminum foil during the extraction and quantification. A solvent/solid ratio of 20 was used for the extractions. Thereafter, the extracts were centrifuged at 10,000 g for 20 min (4 °C) (Hermle Labortechnik GmbH, Wehingen, Germany) and the supernatants was separated. A second extraction used acetone: water (70:30 v/v). In a second extraction, acetone: water (70:30 v/v) was used. The supernatant was obtained using the same steps as in the first extraction. The supernatants from each extraction were pooled and stored at −40 °C in the ULUF ultra-low freezer (Arctiko, Lammefjordsvej, Denmark) until analysis.

2.3.2. Determination of antioxidant capacities

• DPPH assay: The free radical scavenging activity was measured using DPPH (1,1-diphenyl-2-picrylhydrazyl) as the source of the free radicals, according to Brand-Williams et al. (1995) with some modification was used to assess sample leaf. Briefly, diluted extracts (10 μL) were placed in each of the 96 wells of the microplate, or the wells were left blank, and mixed with 200 μL of DPPH radical (60 μmol/L dissolved in 1:1 methanol/10 mmol/L Tris-HCl buffer, pH 7.5). After 10 min of incubation at room temperature, the absorbance at 520 nm was measured in the Synergy HTX Multi-Mode microplate reader (Biotek, Rochester, VT, USA). These determinations were made using a Trolox (an analog of vitamin E) standard curve, serial dilutions (0-100 µM). The results were expressed as µmol TE per gram of dry weight (d.w.).

• ABTS assay: The free radical scavenging activity of the extracts was determined according to the method of Re et al. (1999). The ABTS⁺ radical (2,2′-azo-bis (3-ethylbenzothiazoline-6-sulfonic acid)) was used to determine the Trolox equivalent antioxidant capacity (TEAC) of the sample leaves. The ABTS radical cation was produced by reacting the ABTS stock solution (7 mM) with 7 mM potassium persulfate in a 2:1 ratio, and the mixture was allowed to stand in the dark at room temperature for 16 h before use. Before use, the stock solution was diluted with 70% ethanol to an absorbance of 0.75 ± 0.05 to obtain the ABTS working solution. Next, 10 μL of each extract sample (or standard) was mixed with 100 μL of the ABTS solution in each of the 96 wells of the microplate, and the absorbance was measured after 10 minutes at 734 nm in the Synergy HTX multimode microplate reader (Biotek, Rochester, VT, USA). Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was used as the standard antioxidant, and the results were expressed in µM of Trolox equivalents per gram of dry weight (w.w.).

• PCL assay: A photochemiluminescence (PCL) assay was performed to measure the antioxidant capacity of samples leaves extracts with a Photochem® apparatus (Analytik Jena, Leipzig, Germany). The PCL is based on the photo-induced autoxidation inhibition of luminol (a photosensitiser) by antioxidants when exposed to UV light (Popov and Lewin, 1999) mediated from the radical anion superoxide and is suitable to measure the radical scavenging properties of single antioxidants as well as more complex systems in the nanomolare range. Antioxidant activity was analysed with ACW (hydrophilic condition) kit according to the manufacturer’s protocol. Data acquisition and analysis were carried out through PCLsoft® control and analysis software (Analytik Jena, Leipzig, Germany). The antioxidant capacity was determined by triplicate using the Inhibition and was expressed as μmol Trolox equivalents (TE)/g of dry weight, based on the Trolox standard curve ranging from 0.5 to 3.0 nmol/L (R2 = 0.992).

2.3.3. Determination of total phenolic content (TPC)

The total amount of phenols was determined using the Folin-Ciocalteau reagent, according to the method modified by Magalhães et al. (2010), with slight modifications. In summary, 50 μL of sample extracts, standard or ultrapure water (as a blank) were mixed with 50 μL of Folin-Ciocalteu reagent (diluted in deionized water (1:5, v/v)) in a 96-well plate and allowed to stand for 2 minutes at room temperature. Next, 100 μL of 0.3 M NaOH solution was added, and the plate was immediately transferred to a Synergy HTX multimode microplate reader (Biotek Instruments Inc., USA), which had been preprogrammed to shake, stand for 5 minutes, and read at 760 nm. A calibration curve was plotted using different concentrations (0.0–35.0 mg/L) of standard gallic acid solutions. The results were expressed in milligrams of gallic acid equivalent per gram of dry weight of the sample leaf (mg GAE/g DW).

2.3.4. Determination of total flavonoid content (TFC)

The total flavonoid content (TFC) of the sample leaf was determined using a colorimetric assay with aluminum chloride, according to the method described by Duan et al. (2021), with some slight modifications. In summary, 50 μL of extracts (or standard) were mixed with 100 μL of ddH₂O and 10 μL of 5% NaNO₂ in a 96-well microplate. The mixture was incubated at room temperature for 6 minutes, 10 μL of 10% AlCl₃ was added, and it was incubated at room temperature for another 5 minutes, after which 50 μL of 1 M NaOH was added. The absorbance at a wavelength of 510 nm was read on a Synergy HTX multimode microplate reader (Biotek Instruments Inc., USA). The total flavonoid content was calculated from a standard calibration curve of quercetin (0–100 mM) and expressed as mg quercetin equivalent (QE) per dry weight (mean ± SD, n = 3).

2.3.5. Determination of chlorophyll α, β, and carotenoids

The total carotenoid and chlorophyll content of the sample leaf was determined using the method described by Lichtenthaler and Buschmann (2001). In summary, 1 mg of fine powder was dissolved in 1 ml of methanol and stirred for 30 minutes at room temperature in the dark. After centrifuging the sample, the supernatant was transferred to a cuvette and the absorption spectra were obtained at 400-700 nm every 1 nm in a SPECORD Plus 50 spectrophotometer (Analytik Jena, Germany). The spectra were deconvoluted by applying a linear baseline and the Gauss Amp model of PeakFit v.4.12 software (Systat Software, Inc., USA). The absorbance (A) of the peaks at 470, 652.4, and 665.2 nm was used for the quantification of total carotenoids (x+c), chlorophyll α (Cα), and chlorophyll β (Cβ), respectively, and was calculated using Equations 1, 2, 3 and expressed in mg/g dry weight (d.w.).

$c_a\left(ug/ml\right)=16.72A_{665.2}-9.16A_{652.4}$ (1)

$c_{\beta }\left(ug/ml\right)=34.09A_{652.4}-15.28A_{665.2}$ (2)

$c_{\left(x+c\right)}\left(ug/ml\right)=\left(1000A_{470}-1.63c_a-104.96c_b\right)/221$ (3)

2.4. Ultrastructural methodology using (SEM)

A FEI Inspect S50 scanning electron microscope (SEM) was used to analyze the surface morphology of the leaves. The analysis was carried out at the Specialized Equipment Laboratory of the Faculty of Biological Sciences at the National University of San Marcos. The cauliflower (Brassica oleracea var. botrytis) samples were processed according to the following procedure:

1. Preparation and Chemical Fixation: The samples were pre-washed with distilled water. b) Subsequently, in order to achieve optimal preservation of the epidermal and stomatal morphology, standard fixation and post-fixation protocols were implemented. Glutaraldehyde was used as a protein cross-linking agent to stabilize the cellular architecture, followed by the application of osmium tetroxide. The latter acted as a fixative for membrane lipids and as a contrast agent for biological structures (Kim, 2020), c) Dehydration and Critical Point Drying: After fixation, the tissues were gradually dehydrated using an ascending series of absolute ethanol concentrations. This procedure is essential for removing intracellular water prior to drying. To prevent structural collapse and deformation of epidermal cells and stomata, critical point drying (CPD) was performed using EMS 850 equipment. This method uses liquid carbon dioxide as a transition fluid, allowing the solvent to be removed without the adverse effects of surface tension, thus preserving the original topography of the epidermis (Murtey and Ramasamy, 2016). Metallization and Image Optimization: Finally, the dry samples were coated with an ultrathin layer of gold using the sputtering technique, employing equipment from SPI Supplies. Metal deposition increases surface electrical conductivity, facilitating electron charge dissipation. This condition of electroneutrality is imperative for maximizing secondary electron emission, allowing the acquisition of high-resolution images necessary to characterize microtopography and stomatal morphology under water stress conditions.

2.5. Statistical analysis

The processing and analysis of the biometric measurements of cauliflower grown in four treatments, with a control treatment T0 and test treatments T1, T2, and T3, was based on a design (CRBD). To determine the existence of statistically significant differences between treatments, an analysis of variance (ANOVA) was applied, establishing a level of significance. In cases where significant effects were detected, the means were compared using Duncan's multiple range test (p < 0.05). All statistical support was performed using the specialized software InfoStat. The measurements obtained by spectrophotometric analysis were performed in triplicate, and the results were expressed as mean ± SD. One-way analysis of variance (ANOVA) and Tukey's test were used for comparison, using the open statistical software Jamovi version 2.6.26 solid (Jamovi Project, 2024; R Core Team, 2024).

3. Results and Discussion

3.1. Soil analysis

The results derived from the physicochemical characterization of the soil used in the experimental phase of cauliflower cultivation are summarized in Tables 1 and 2.

Table 1
Physical and chemical analysis of the soil.

Sand %	Clay %	Silt %	EC 1:2:5 mS cm-1	pH 1:2:5	OM %	N %	P available ppm	K available ppm	CaCO3%	Exchangeable cations (mEq/100 g soil)
										Ca	Mg	Na	K
88	5.60	6.40	27.00	8.80	0.40	198.05	27.92	176.55	4.50	5.26	0.66	0.13	0.25

• Source: ANOBA (Anoba Soluciones Analíticas & Consultoría, 2025). CEC: Cation exchange capacity, OM: organic matter, EC: electrical conductivity.

Table 2
Micronutrients analysis.

Cu Available %	Zn Available %	Mn Available %	Fe Available %	B Available%	Relation C/N	Relation Mg/K available	Relation Ca/Mg available	Relation (Ca+Mg)/K available
<1.00	<2.00	5.24	3.90	0.59	11.72	0.55	28.25	16.14

• Source: ANOBA (Anoba Soluciones Analíticas & Consultoría, 2025).

Due to its sandy texture and lack of organic matter (0.40%), the soil has a low cation exchange capacity (CEC). In order to optimize its physicochemical properties, root inoculation with a solution of Methylobacterium and liquid compost was carried out on the cauliflowers, integrating the treatment through an automated drip irrigation system. According to Fageria (2012), these practices are vital for agricultural sustainability as they enhance the physical structure, nutrient bioavailability, and biological activity (N fixation and mycorrhizae) of the substrate.

The proposal to use Methylobacterium is highly technical and appropriate, as these plant growth-promoting bacteria can mitigate abiotic stress in alkaline soils. According to Dourado et al. (2015), these bacteria not only fix nitrogen; but also secrete cytokinins and auxins that stimulate root development in poor media.

3.2. Evaluation of the physical characteristics of cauliflower

Root inoculation of a phytoremediation mixture based on Methylobacterium and liquid compost was performed on cauliflower crops, following the concentrations described in Table 3.

Table 3
Dosage of Methylobacterium solution and liquid compost.

Treatment	Dosi mL/200 L
T0 (control)	0
T1	250
T2	333
T3	400

In order to evaluate the effect of root inoculation with Methylobacterium and liquid compost on the morphological parameters of cauliflower, the data were subjected to analysis of variance (ANOVA) and Duncan's multiple range test (p < 0.05). The analyses, performed using InfoStat statistical software, showed significant differences between the treatments evaluated, as detailed in Table 4.

Table 4
Evaluation of the physical characteristics of cauliflowers by treatment groups according to the Table 1.

Treatments	Total, plant length (cm)	Number of sheets	Diameter Equatorial (cm)	Weight of Sheets (g)	Weight of Pellas (g)	Weight of stems (g)
T3	25.60 A	27.20 A	67.25 A	1216.00 A	1760.50 A	385.13 A
T2	25.20 A	25.75 A	67.25 A	1207.25 A	1284.63 B	370.38 A
T1	25.05 A	25.33 A	60.25 A	942.50 B	1059.88 C	320.00 B
T0	22.00 B	22.50 B	49.25 B	869.88 B	928.38 C	192.13 C

• Note: Means with the same letter in columns do not differ statistically (p>0.05).

3.3. Determination of antioxidant capacities in leaves, total phenol content (TPC), total flavonoid content (TFC), Carotenoids and chlorophyll

3.3.1. Determination of antioxidant capacities in leaves

According to the data presented in Table 5, treatment T3 showed statistically significant superiority (p < 0.05) in the induction of antioxidant defense systems compared to the control group and treatments T1 and T2. In the ABTS+, DPPH, and PLC-ACW assays, T3 recorded the highest levels of activity. The increase in hydrophilic antioxidant capacity (PLC-ACW) is noteworthy, where the value of 211.68 µM Trolox Equiv./g represented an increase of more than 100% compared to the control (85.45 µM Trolox Equiv./g) and treatment T2. The results suggest that the application of Methylobacterium in combination with liquid compost acts as an agent that mitigates oxidative stress, an intrinsic condition of crops in sandy soils with limited nutrient availability and low water retention capacity.

Table 5
Antioxidant capacities (uM Equiv. Trolox/g of dry weight) in leaves.

Antioxidant assay	Sample Leave				p-Value
	Control	T1	T2	T3
ABTS+	187.10±11.27a	187.70±5.78a	187.76±4.83a	204.84±4.70a	0.042
DPPH	20.24± 2.04b	23.03± 0.28b	24.19± 0.99b	30.67± 2.28a	< .001
PLC-ACW	85.45± 6.11b, c	63.23± 4.06c	102.06±4.46b	211.68±20.99a	< .001

• Source: Food Process Research and Engineering Laboratory (SL01LA21), Faculty of Sciences, José Faustino Sánchez Carrión National University. Values are expressed as means ± standard deviation (SD) from three separate measurements. Means sharing the same letter in a row do not differ significantly.

3.3.2. Determination of total phenol content (TPC), total flavonoid content (TFC), Carotenoids and chlorophyll

Table 6 shows that treatment T3 stands out as superior in terms of nutritional and photosynthetic quality. TFC (flavonoids) reached the highest value (79.86 mg QE/g DW), significantly exceeding the control and T2. This suggests that this treatment better stimulates secondary metabolic defense and antioxidant pathways. With regard to Chlorophyll α, it has the highest concentration (4.05 mg/g DW), indicating greater photosynthetic capacity and, potentially, a more vigorous plant status compared to the other groups. Plant biostimulants contain substance(s) and/or microorganisms whose function when applied to plants or the rhizosphere is to stimulate natural processes to enhance plant nutrient uptake, nutrient use efficiency, tolerance to abiotic stress, biocontrol, and crop quality (Hamid et al., 2021).

Table 6
Bioactive compound in sample leaves.

Bioactive compound	Sample Leave				p-Value
	Control	T1	T2	T3
TPC (mg GAE/g DW)	32.33±3.30a	32.32±2.29a	25.42±0.23b	30.68±1.93a, b	0.016
TFC (mg QE/g DW)	62.19± 1.58b	72.43± 5.34a, b	62.82± 5.46b	79.86± 3.31a	0.003
Chlorophyll α (mg/g DW)	3.66±0.14a	3.36±0.05b	3.25±0.12b	4.05±0.03a	< .001
Chlorophyll β (mg/g DW)	2.46±0.21a	2.51±0.06a	1.35±0.04c	2.08±0.08b	< .001
Total Carotenoids (mg/g DW)	0.28±0.02d	0.39±0.01c	0.77±0.02a	0.63±0.01b	< .001

• Source: Food Process Research and Engineering Laboratory (SL01LA21), Faculty of Sciences, José Faustino Sánchez Carrión National University. Values are expressed as means ± standard deviation (SD) from three separate measurements. Means sharing the same letter in a row do not differ significantly. GAE: gallic acid equivalent. QE: Quercetin Equivalent. DW: dry weight.

3.4. Ultrastructural analysis of the epidermis and stomata

An ultrastructural analysis of the epidermis and stomata of cauliflower leaves was conducted. The main objective was to determine the morphological response of the plant to the application of an assisted phytoremediation treatment under abiotic stress conditions (saline soil and nutrient deficiency). The treatment consisted of inoculation with Methylobacterium bacteria and the addition of liquid compost. The experimental plants were established and grown in a substrate characterized by high salinity and low nutrient content. The experimental design included four treatment groups (samples), designated as T0, T1, T2, and T3. The formulation and dosage of the inoculum applied to each treatment is described in detail in Table 3.

Heterogeneity in stomatal characteristics is found at many levels, from the size, frequency, and behavior of individual guard cells to the gas exchange of entire plants or plant stands (Weyers and Lawson, 1997). Guard cells are specialized pairs of cells in the leaf epidermis that form stomata for gas exchange between the interior of the plant and the surrounding atmosphere. They are morphologically distinct from other leaf cells and adjust stomatal opening by modifying turgor pressure in response to internal or environmental signals (Rasouli et al., 2020).

The micrographs corresponding to treatment T0, obtained at 600x and 1200x magnification, are shown in Figure 1 and Figure 2, respectively, revealing a regular distribution of stomata on the surface of the leaves, but with no signs of significant opening. Most stomata are closed, with only a small number of stomata slightly open, indicating low stomatal activity. The epidermal surface is smooth and homogeneous, with no visible alterations in the surrounding cells, suggesting that, in the absence of Methylobacterium application, the stomata remain closed, probably in response to suboptimal growing conditions.

Figure 1
Micrograph of T0 at 600x magnification.

Figure 2
Micrograph of T0 at 1200x magnification.

The micrographs corresponding to treatment T1, obtained at 600x and 1200x magnification, are shown in Figure 3 and Figure 4, respectively. When examining Figure 4, from the 1200x sample, the stomata show minimal opening, with a barely visible aperture, indicating that the stomata are in an early stage of development and not yet fully functional. The surrounding cells maintain a regular structure, with no signs of visible damage, suggesting that there is no severe stress. However, the lack of significant stomatal opening limits gas exchange and transpiration, which can negatively affect the efficiency of the plant's metabolic processes.

Figure 3
Micrograph of T1 at 600x magnification.

Figure 4
Micrograph of T1 at 1200x magnification.

Microstructural analysis of the leaf epidermis of cauliflower reveals a significant morphological transformation induced by the application of Methylobacterium (T1), in contrast to the control group (T0). In treatment T1, a significant increase in stomatal density and epidermal cells is observed, which have more pronounced reliefs and a more compact cellular organization than in T0. According to Abanda-Nkpwatt et al., (2006), methylotrophic bacteria of the genus Methylobacterium have the ability to synthesize phytohormones, specifically cytokinins and auxins (indoleacetic acid), which stimulate cell division and differentiation, thus explaining the greater microstructural vigor observed in T1. Likewise, the greater opening of the stomata in the inoculated treatment suggests an optimization of gas exchange; this coincides with the findings of Omer et al. (2004), who point out that the symbiotic interaction between these bacteria and the plant improves photosynthetic efficiency and water regulation. On the other hand, the more defined cuticular relief in T1 could be associated with greater deposition of epicuticular waxes, an adaptive response mediated by the biostimulant that reinforces the barrier against abiotic stress (Dourado et al., 2015).

In the micrograph obtained at 600x magnification of treatment T1, no significant differences in stomatal opening were observed compared to control treatment T0. Most stomata remain partially open, similar to treatment T0. Microstructural comparison of the leaf epidermis reveals that, under conditions of high salinity and nutritional deficit, the control treatment (T0) exhibits signs of cellular stress characterized by an irregular epidermal surface, loose cuticular folds, and a notable presence of closed or collapsed stomatal complexes, which is a typical response to prevent water loss through transpiration in saline environments (Munns and Tester, 2008).

When observing the sample at 1200x magnification, the stomata continue to show partial opening similar to that of treatment T0, with no marked difference in the number of open stomata. The surrounding cells continue to show a regular structure and no damage is observed, suggesting that the plant is not under stress, but the ability of the stomata to regulate gas exchange remains limited. In treatment T1, no significant differences in stomatal opening are observed compared to control T0. Although there is a slight tendency toward greater opening, this improvement is not pronounced enough to indicate a substantial change in stomatal activity. The stomata continue to show partial opening, suggesting that the application of Methylobacterium together with compost has a slight but not significant effect on stomatal regulation at this dose.

In Figure 3 and Figure 4, the cauliflower leaf shows a rough and wavy surface pattern, typical of Brassica leaves. This morphology is due to a dense layer of epicuticular wax that is deposited in complex crystalline structures, giving the leaf its hydrophobic properties and acting as a vital protective barrier against desiccation, pathogens, and environmental stress (Shepherd and Wynne Griffiths, 2006). The waxy pattern, with numerous stomata and structures key to gas exchange, ensures that the leaf maintains its natural defense and ability to efficiently regulate transpiration, which is crucial for plant health and productivity (Fernández et al., 2015).

In the image obtained at 600x magnification, shown in Figure 5 of treatment T2, a higher density of open stomata is observed compared to the control. Although most stomata are partially open, there is a more evident tendency toward greater opening compared to previous treatments. This slight improvement suggests that the application of Methylobacterium and compost is having a positive effect. The epidermis remains uniform and shows no visible alterations in the surrounding cells, indicating that there is no evident stress on the plant. When examining the sample at 1200x magnification, as shown in Figure 6, the opening of the stomatal aperture is more visible in several stomata, showing a moderate opening compared to the previous treatments. Although no completely open stomata are observed, there is better stomatal regulation than in the previous treatments.

Figure 5
Micrograph of T2 at 600x magnification.

Figure 6
Micrograph of T2 at 1200x magnification.

In contrast, the T2 treatment shows a significantly more organized and functional leaf architecture, with epidermal cells of greater turgidity and a more homogeneous stomatal distribution where open ostia predominate; this change suggests that inoculation with Methylobacterium and the addition of liquid compost act synergistically to mitigate osmotic stress. Probably through the production of osmolytes and phytohormones such as cytokinins, they maintain membrane stability and cell vigor (Dourado et al., 2015). Thus, while T0 reflects severe metabolic limitation due to the degraded substrate, T2 demonstrates morphological reprogramming that favors gas exchange and crop resilience, validating the biostimulant effect of methylotrophic bacteria under adverse soil conditions (Omer et al., 2004).

Treatment T2 shows an improvement in stomatal opening compared to treatments T0 and T1, although the stomata are not completely open. This treatment presents a more active stomatal response, suggesting that the application of eco-efficient bacteria has had a positive effect on the regulation of gas exchange and transpiration, promoting greater metabolic efficiency in the plant.

In the Figure 7, obtained at 600x magnification of treatment T3, there is clearly a greater presence of open stomata compared to control treatments T0 and T1. A greater number of open stomata can be observed compared to the previous treatments, indicating a more active stomatal response. The epidermis remains uniform and shows no visible alterations in the surrounding cells. When examining Figure 8, the sample at 1200x magnification, the stomatal opening becomes more evident, with several stomata showing a more pronounced opening compared to the other treatments. Although the density of open stomata in T3 is higher compared to the control and T1, the difference with treatment T2 is not significant enough to ensure that treatment T3 is clearly superior in terms of stomatal opening.

Figure 7
Micrograph of T3 at 600x magnification.

Figure 8
Micrograph of T3 at 1200x magnification.

The comparison between the epidermis of treatment T0 and the 1200x detail of treatment T3 reveals a profound structural recovery in response to conditions of high salinity and low fertility. In the T0 control, the leaf architecture appears compromised, with a flattened surface and stomata that tend to close as a mechanism to avoid salt-induced water stress (Munns and Tester, 2008). In contrast, the high-resolution micrograph of T3 (Figure 6) shows a robust leaf morphology, where the epidermal cells have sharply defined contours and superior turgidity; most notable is the detailed visualization of the stomatal apparatus, which has clearly open ostia and vigorous guard cells. This level of detail confirms that the maximum dose of Methylobacterium, in synergy with liquid compost, stimulates greater synthesis of phytohormones (cytokinins) and osmolytes that protect the integrity of the plasma membrane, facilitating efficient gas exchange even in adverse edaphic environments (Dourado et al., 2015; Omer et al., 2004).

In conclusion, the transition from a collapsed surface at T0 to a functional and turgid ultrastructure at T3 demonstrates that inoculation mitigates the phytotoxic impact of the substrate, optimizing the metabolic potential of the cauliflower crop. The presence of eco-efficient bacteria appears to be directly related to improved stomatal activity.

4. Conclusion

The combined treatment of Methylobacterium and liquid compost promotes stomatal opening by optimizing nutrient absorption and mitigating water stress. This regulation improves gas exchange, transpiration, and photosynthesis, creating a more efficient metabolic environment. Specifically, the T3 treatment significantly improved the physiological response and morphological parameters of cauliflower under salinity conditions. In addition to increasing the antioxidant capacity and vigor of the plant, this treatment had the highest concentration of Chlorophyll α, suggesting a more resistant crop with superior nutritional quality compared to the control group and other treatments.

Acknowledgements

The authors would like to thank José Faustino Sánchez Carrión National University for its collaboration and for providing the facilities necessary to carry out some research activities.

Data Availability Statement

The tabulated data on the physical characteristics of cauliflower, collected in two phases in the field at the Faculty of Agricultural, Food, and Environmental Engineering of the José Faustino Sánchez Carrión National University (UNJFSC).

References

• ABANDA-NKPWATT, D., MÜSCH, M., TSCHIERSCH, J., BOETTNER, M. and SCHWAB, W., 2006. Molecular interaction between Methylobacterium extorquens and seedlings: growth promotion, methanol consumption, and localization of the methanol emission site. Journal of Experimental Botany, vol. 57, no. 15, pp. 4025-4032. https://doi.org/10.1093/jxb/erl173 PMid:17043084.
  » https://doi.org/10.1093/jxb/erl173
• ANOBA SOLUCIONES ANALÍTICAS & CONSULTORÍA, 2025. Informe de ensayo IESA7072: Análisis de caracterización completa Peru: Anoba Soluciones Analíticas & Consultoría. (Suelo de Cultivo Franco-Arenoso; ID SA257072).
• AYYAMUTHU RAJARATHINAM UMA, P., RATHINASAMY, P., THANAKKAN, R., DHASHNAMURTHI, V. and MURUGAIYAN, S., 2025. Pink powerhouses: insights into the multifaceted role of Methylobacterium in climate-resilient farming. Folia Microbiologica, vol. 70, no. 6, pp. 1241-1266. https://doi.org/10.1007/s12223-025-01350-9
  » https://doi.org/10.1007/s12223-025-01350-9
• BOUKHATEM, Z., MERABET, C. and TSAKI, H., 2022. Plant growth promoting actinobacteria, the most promising candidates as bioinoculants? Frontiers in Agronomy, vol. 4, pp. 849911. https://doi.org/10.3389/fagro.2022.849911
  » https://doi.org/10.3389/fagro.2022.849911
• BRAND-WILLIAMS, W., CUVELIER, M. and BERSET, C., 1995. Use of a free radical method to evaluate antioxidant activity. Lebensmittel-Wissenschaft + Technologie, vol. 28, no. 1, pp. 25-30. https://doi.org/10.1016/S0023-6438(95)80008-5
  » https://doi.org/10.1016/S0023-6438(95)80008-5
• CABEZAS GUTIÉRREZ, A., CAMUS ARAYA, F., ESTEBAN CONDORI , W., GONZÁLEZ VALLEJOS, F.A. and MAZUELA ÁGUILA, P., 2022. El silicio (Si) y su efecto mitigador del estrés salino en cultivos hortícolas. Idesia, vol. 40, no. 1, pp. 129-133. https://doi.org/10.4067/S0718-34292022000100129
  » https://doi.org/10.4067/S0718-34292022000100129
• CAMPANA, E., CIRIELLO, M., LENTINI, M., ROUPHAEL, Y. and DE PASCALE, S., 2025. Sustainable agriculture through compost tea: Production, application, and impact on horticultural crops. Horticulturae, vol. 11, no. 4, pp. 433. https://doi.org/10.3390/horticulturae11040433
  » https://doi.org/10.3390/horticulturae11040433
• DOURADO, M.N., NEVES, A.A., SANTOS, D.S. and ARAÚJO, W.L., 2015. Biotechnological and agronomic potential of endophytic pink-pigmented methylotrophic Methylobacterium spp. BioMed Research International, vol. 2015, pp. 909016. https://doi.org/10.1155/2015/909016 PMid:25861650.
  » https://doi.org/10.1155/2015/909016
• DUAN, Y., EDUARDO MELO SANTIAGO, F., RODRIGUES DOS REIS, A., DE FIGUEIREDO, M.A., ZHOU, S., THANNHAUSER, T.W. and LI, L., 2021. Genotypic variation of flavonols and antioxidant capacity in broccoli. Food Chemistry, vol. 338, pp. 127997. https://doi.org/10.1016/j.foodchem.2020.127997 PMid:33091988.
  » https://doi.org/10.1016/j.foodchem.2020.127997
• FAGERIA, K., 2012. Role of soil organic matter in maintaining sustainability of cropping systems. Communications in Soil Science and Plant Analysis, vol. 43, no. 16, pp. 2063-2113. https://doi.org/10.1080/00103624.2012.697234
  » https://doi.org/10.1080/00103624.2012.697234
• FERNÁNDEZ, V., EICHERT, T. and DOMBRO, M., 2015. Impact of leaf surface properties on foliar spray retention and absorption. Journal of Plant Nutrition and Soil Science, vol. 178, no. 1, pp. 3-19. http://doi.org/10.1002/jpln.201400539
  » http://doi.org/10.1002/jpln.201400539
• GONZÁLEZ-HERNÁNDEZ, A.I., PÉREZ-SÁNCHEZ, R., PLAZA, J. and MORALES-CORTS, M.R., 2022. Compost tea as a sustainable alternative to promote plant growth and resistance against Rhizoctonia solani in potato plants. Scientia Horticulturae, vol. 300, pp. 111090. https://doi.org/10.1016/j.scienta.2022.111090
  » https://doi.org/10.1016/j.scienta.2022.111090
• HAKIMI, F., SEBBAR, A., BOUAMRI, R., SIDIKOU, A., EL JANATI, M. and BOUAZIZ, A., 2024. Effects of compost and compost tea on soil properties and nutrient uptake of the Moroccan date palm cultivar “Mejhoul” under organic cultivation. Journal of Ecological Engineering, vol. 25, no. 7, pp. 224-240. https://doi.org/10.12911/22998993/188334
  » https://doi.org/10.12911/22998993/188334
• HAMID, B., ZAMAN, M., FAROOQ, S., FATIMA, S., SAYYED, R.Z., BABA, Z.A., SHEIKH, T.A., REDDY, M.S., EL ENSHASY, H., GAFUR, A. and SURIANI, N.L., 2021. Bacterial plant biostimulants: A sustainable way towards improving growth, productivity, and health of crops. Sustainability, 13(5). Sustainability (Basel), vol. 2856, no. 5, pp. 2856. https://doi.org/10.3390/su13052856
  » https://doi.org/10.3390/su13052856
• JAMOVI PROJECT, 2024 [viewed 18 January 2026]. jamovi (Versión 2.6) [software]. Available from: https://www.jamovi.org
  » https://www.jamovi.org
• KHOSO, M., WAGAN, S., ALAM, I., HUSSAIN, A., ALI, Q., SAHA, S., POUDEL, T., MANGHWAR, H. and LIU, F., 2024. Impacto de las rizobacterias promotoras del crecimiento vegetal (PGPR) en la nutrición de las plantas y las características de las raíces: perspectiva actual. Estrés Vegetal, vol. 11, pp. 100341.
• KIM, K., 2020. Methanol fixation for scanning electron microscopy of plants. Applied Microscopy, vol. 50, no. 1, pp. 10. https://doi.org/10.1186/s42649-020-00028-5 PMid:33580311.
  » https://doi.org/10.1186/s42649-020-00028-5
• KUMARI, R., BHATNAGAR, S., DEEPALI, MEHLA, N. and VASHISTHA, A., 2022. Potential of Organic Amendments (AM fungi, PGPR, Vermicompost and Seaweeds) in Combating Salt Stress ... A Review. Plant Stress, vol. 6, pp. 100111. https://doi.org/10.1016/j.stress.2022.100111
  » https://doi.org/10.1016/j.stress.2022.100111
• LAMZ, A. and GONZÁLEZ, M., 2013. La salinidad como problema en la agricultura: la mejora vegetal una solución inmediata. Cultivos Tropicales, vol. 34, no. 4, pp. 31-42.
• LEE, M., LEE, G., YIM, W., HONG, I., PALANIAPPAN, P., SIDDIKEE, M., BORUAH, H., MADHAIYAN, M., AHN, K. and SA, T., 2009. Inoculation effect of Methylobacterium suomiense on growth of red pepper under different levels of organic and chemical fertilizers. Korean Journal of Soil Science and Fertilizer, vol. 42, no. 4, pp. 266-273.
• LI, Z. and LI, X., 2025. Global trends and research hotspots on the improvement of saline soil: insights from bibliometric analysis. Agricultural Water Management, vol. 321, pp. 109923. https://doi.org/10.1016/j.agwat.2025.109923
  » https://doi.org/10.1016/j.agwat.2025.109923
• LICHTENTHALER, H. and BUSCHMANN, C., 2001. Chlorophylls and carotenoids: measurement and characterization by UV‐VIS spectroscopy. Current Protocols in Food Analytical Chemistry, vol. 1, no. 1. https://doi.org/10.1002/0471142913.faf0403s01
  » https://doi.org/10.1002/0471142913.faf0403s01
• MAGALHÃES, L.M., SANTOS, F., SEGUNDO, M.A., REIS, S. and LIMA, J.L., 2010. Rapid microplate high-throughput methodology for assessment of Folin-Ciocalteu reducing capacity. Talanta, vol. 83, no. 2, pp. 441-447. https://doi.org/10.1016/j.talanta.2010.09.042 PMid:21111158.
  » https://doi.org/10.1016/j.talanta.2010.09.042
• MUNNS, R. and TESTER, M., 2008. Mechanisms of salinity tolerance. Annual Review of Plant Biology, vol. 59, no. 1, pp. 651-681. https://doi.org/10.1146/annurev.arplant.59.032607.092911 PMid:18444910.
  » https://doi.org/10.1146/annurev.arplant.59.032607.092911
• MURTEY, M. and RAMASAMY, P., 2016. Sample preparations for scanning electron microscopy – life sciences. In: M. JANECEK and R. KRAL, editors. Modern electron microscopy in physical and life sciences. Rijeka: IntechOpen, pp. 296-299. https://doi.org/10.5772/61720
  » https://doi.org/10.5772/61720
• OMER, Z.S., TOMBOLINI, R. and GERHARDSON, B., 2004. Plant colonization by pink-pigmented facultative methylotrophic bacteria (PPFMs). FEMS Microbiology Ecology, vol. 47, no. 3, pp. 319-326. https://doi.org/10.1016/S0168-6496(04)00003-0 PMid:19712320.
  » https://doi.org/10.1016/S0168-6496(04)00003-0
• PALBERG, D., KISIAŁA, A., JORGE, G.L. and EMERY, R.J., 2022. Un estudio de especies y cepas de Methylobacterium revela una producción generalizada y perfiles variables de fitohormonas citoquininas. BMC Microbiology, vol. 22, no. 1, no. 49. https://doi.org/10.1186/s12866-022-02454-9 PMid:35135483.
  » https://doi.org/10.1186/s12866-022-02454-9
• PILLA, N., TRANCHIDA, V., GABRIELLI, P., AGUZZI, A., CAPUTO, M., LUCARINI, M., DURAZZO, A. and ZACCARDELLI, M., 2023. Efecto del té de compost en la horticultura. Horticulturae, vol. 9, no. 9, pp. 984. https://doi.org/10.3390/horticulturae9090984
  » https://doi.org/10.3390/horticulturae9090984
• POPOV, I. and LEWIN, G., 1999. Antioxidative homeostasis: characterization by means of chemiluminescent technique. In: Methods in enzymology Academic Press, vol. 300, pp. 437-456. https://doi.org/10.1016/S0076-6879(99)00149-4
  » https://doi.org/10.1016/S0076-6879(99)00149-4
• R CORE TEAM, 2024 [viewed 18 January 2026]. R: a language and environment for statistical computing (Versión 4.4) [software]. Available from: https://cran.r-project.org
  » https://cran.r-project.org
• RASOULI, F., KIANI-POUYA, A., LI, L., ZHANG, H., CHEN, Z., HEDRICH, R., WILSON, R. and SHABALA, S., 2020. Sugar Beet (Beta vulgaris) guard cells responses to salinity stress: a proteomic analysis. International Journal of Molecular Sciences, vol. 21, no. 7, pp. 2331. https://doi.org/10.3390/ijms21072331 PMid:32230932.
  » https://doi.org/10.3390/ijms21072331
• RE, R., PELLEGRINI, N., PROTEGGENTE, A., PANNALA, A., YANG, M. and RICE-EVANS, C., 1999. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biology & Medicine, vol. 26, no. 9-10, pp. 1231-1237. https://doi.org/10.1016/S0891-5849(98)00315-3 PMid:10381194.
  » https://doi.org/10.1016/S0891-5849(98)00315-3
• SAHAB, S., SUHANI, I., SRIVASTAVA, V., CHAUHAN, P., SINGH, R. and PRASAD, V., 2021. Potential risk assessment of soil salinity to agroecosystem sustainability: current status and management strategies. The Science of the Total Environment, vol. 764, pp. 144164. https://doi.org/10.1016/j.scitotenv.2020.144164 PMid:33385648.
  » https://doi.org/10.1016/j.scitotenv.2020.144164
• SHEPHERD, T. and WYNNE GRIFFITHS, D., 2006. The effects of stress on plant cuticular waxes. The New Phytologist, vol. 171, no. 3, pp. 469-499. https://doi.org/10.1111/j.1469-8137.2006.01826.x PMid:16866954.
  » https://doi.org/10.1111/j.1469-8137.2006.01826.x
• TEDESCHI, A., SCHILLACI, M. and BALESTRINI, R., 2023. Mitigating the impact of soil salinity: recent developments and future strategies. Italian Journal of Agronomy, vol. 3, no. 18, pp. 2173. https://doi.org/10.4081/ija.2023.2173
  » https://doi.org/10.4081/ija.2023.2173
• THRALL, P., OAKESHOTT, J., FITT, G., SOUTHERTON, S., BURDON, J., SHEPPARD, A., RUSSELL, R., ZALUCKI M., HEINO, M. and FORD, R., 2011. Evolution in agriculture: the application. Evolutionary Applications, vol. 4, no. 2, pp. 200-215. https://doi.org/10.1111/j.1752-4571.2010.00179.x
  » https://doi.org/10.1111/j.1752-4571.2010.00179.x
• TREJO, N., 2019. Biorrecuperación mediante enmiendas orgánicas y químicas de un suelo salino sódico en el municipio de Tlahuelilpan, Hidalgo Hidalgo: Universidad Autónoma del Estado de Hidalgo. Tesis de doctorado. Biblioteca Digital UAEH.
• WANG, Y., GAO, M., CHEN, H., CHEN, Y., WANG, L. and WANG, R., 2023. Organic Amendments promote saline-alkali soil desalinization and enhance maize. Frontiers in Plant Science, vol. 14, pp. 1177209. https://doi.org/10.3389/fpls.2023.1177209 PMid:37692414.
  » https://doi.org/10.3389/fpls.2023.1177209
• WEYERS, J. and LAWSON, T., 1997. Heterogeneidad en las características estomáticas. Avances en la Investigación Botánica, vol. 26, pp. 317-352.
• YU, Y., BEN-GAL, A., SCUDIERO, E., KISEKKA, I., RENGASAMY, P. and YAO, R., 2025. Soil and water management to prevent salinization under changing climate conditions. Agricultural Water Management, vol. 322, pp. 110002. https://doi.org/10.1016/j.agwat.2025.110002
  » https://doi.org/10.1016/j.agwat.2025.110002

Edited by

• Editor:
  Takako Matsumura Tundisi

Publication Dates

• Publication in this collection
  20 July 2026
• Date of issue
  2026

History

• Received
  24 Jan 2026
• Accepted
  28 May 2026