Bacillus thuringiensis and Trichoderma asperellum as Biostimulants in Hydroponic Tendril Pea (Pisum sativum) Microgreens

Abstract

The study evaluated the effect of Bacillus thuringiensis (Bt) and Trichoderma asperellum (Ta) as biostimulants in hydroponically grown tendril pea (Pisum sativum) microgreens. A completely randomized experimental design was conducted under controlled conditions; the study included the root pea-spraying treatment with Bt, Ta, and their combination, alongside a non-inoculated control. The results showed that treatments with Ta significantly enhanced growth parameters, such as stem length and diameter, stipular leaf area, and fresh weight of the microgreens. Specifically, fresh biomass increased by 140% and dry biomass by 80% with Ta compared to the control, while combined treatment with Bt and Ta reduced nitrates by 39.6%. Bt and Bt + Ta increased chlorophyll b and carotenoids contents, suggesting improved photosynthetic activity. No significant differences in visual appearance were observed. In conclusion, the application of Ta and Bt can enhance tendril pea microgreens’ yield and certain biochemical (chlorophyll b and carotenoids) qualities without affecting their visual quality, supporting the application of these microorganisms as biostimulants. These findings underscore the potential to scale these treatments in commercial hydroponic systems, offering a sustainable approach to optimizing the production of this emerging crop type.

1. Introduction

In recent years, the influence of diet on human health has garnered considerable attention [1]. Within this framework, microgreens have gained recognition as a novel category of food products rich in nutrients and bioactive compounds [2,3]. Microgreens are the young shoots of vegetables, aromatic herbs, or edible wild plants harvested shortly after germination, typically between 10 and 14 days after sowing, when they develop cotyledonary leaves [4]. These innovative food items are increasingly popular globally due to their strong flavors, crunchy textures, and vivid colors [5,6].

Some of the most widely cultivated species’ production of microgreens include broccoli, cabbage, cauliflower, cilantro, kale, radish, sunflower, and pea [7,8]. Their widespread appeal is attributed to their abundant phytochemicals content and functional properties [9]. Microgreens are rich in antioxidant, such as ascorbic acid, carotenoids, and phenolic compounds, as well as bioactive compounds like isothiocyanates, and indoles, which play a crucial role in disease prevention [10,11,12].

For this reason, there is growing interest in studying seed treatments for microgreen production to enhance germination, yield, and quality [13,14]. Some studies have evaluated physical and chemical agents as seed treatments for microgreens, yielding positive results in seedling germination and development. For instance, Lee et al. [15] achieved significant improvements in beet and Swiss chard germination by treating the seeds with temperature and substances like hydrogen chlorite, hydrogen peroxide, and sodium hypochlorite. Saengha et al. [16] demonstrated that cold plasma increases mustard microgreens’ germination and growth.

Another less explored option is using microorganism as biostimulants for microgreens. The microbial biostimulants can positively influence microgreen production, as various studies in other production system have demonstrated their ability to promote plant growth through the production of beneficial compounds, such as auxins, cytokinins, and volatile organic compounds [17,18,19]. These substances stimulate key physiological processes, including cell division, cell expansion, and photosynthesis [20,21]. Additionally, microbial biostimulants can enhance nutrient uptake and water use efficiency while reducing nitrate accumulation in plant tissues, which is essential for the quality of the crops [22,23,24]

Various bacterial and fungal species, such as Trichoderma and Bacillus species, have shown the ability to enhance germination and development in several crops [25]. However, few studies are focused on seed treatments for microgreens with microbial biostimulants to improve their yield and quality. In this sense, Briatia et al. [26] were the unique authors reporting the inoculation of buckwheat (Fagopyrum esculentum) with Herbaspirillum sp. ST-B2, showing a significant improvement of seedling growth. Eissa et al. [27] found that using Bacillus megaterium in combination with Azotobacter chroococcum and Pseudomonas flourescens enhances sprout length and nutritional value in pea sprouts. Recently, Wang et al. [28] report that B. velezensis applied in chilli peper (Capsicum annuum) microgreens production resulted in a germination increase of 23.8% and rise of 55.7% in fresh biomass stimulated by the bacteria. Similarly, Maleva et al. [29] reported that inoculating Arthobacter sp. CTF1 on pea (Pisum sativum) seeds for microgreens production improved biomass, vigor index, and significantly increased photosynthetic pigment content.

On the other hand, the production of microgreens in hydroponic systems, combined with microbial biostimulants, represents a scarcely explored field with high potential to offer significant environmental advantages. Among these benefits, he efficiency of hydroponic systems in water and nutrient use stands out, allowing for waste reduction and the minimization of pollutant emissions [30,31,32]. Moreover, by promoting plant health and development, microbial biostimulants reduce dependence on chemical inputs such as fertilizers and pesticides, thus contributing to mitigating environmental impact [33,34]. Additionally, hydroponics provides a sustainable solution for food production in urban areas and regions with limited water resources, reinforcing food security [35]. Therefore, the present study aimed to evaluate the effect of B. thuringiensis and Trichoderma asperellum as biostimulants on the vegetative, biochemical, and quality parameters in hydroponic tendril pea (Pisum sativum) microgreens.

2. Materials and Methods

2.1. Location of the Experiment

The trial was conducted at the Applied Microbiology, Plant Pathology, and Postharvest Physiology Laboratory (MAFFP) of the Autonomous University of Chihuahua (UACH) Chihuahua, MX (28°39′24″ N 106°05′12″ W) during September and October 2024.

2.2. Plant Material

Commercial organic seeds of tendril pea microgreens (Pisum sativum L.), obtained from Johnny’s Selected Seeds (Winslow, ME, USA), were used for the test. These seeds were open-pollinated.

2.3. Microorganims

The microorganisms evaluated as biostimulants were strains of Bacillus thuringiensis Bt24 (Bt) and Trichoderma asperellum TaMFP1 (Ta), obtained from the MAFFP Laboratory at the Faculty of Agrotechnological Sciences, UACH.

2.4. Preparation of Bacterial Inoculum

For the preparation of the bacterial inoculum, Bacillus thuringiensis (Bt) was cultured in nutrient broth (BD Difco Laboratories, Sparks, Maryland, MD, USA; NB) for 72 h at 28 °C under agitation at 120 rpm (MaxQ 4450, Termo Fisher Scientific, Waltham, MA, USA). After incubation, the culture was subjected to a thermal treatment in a thermostatic bath at 80 °C for 10 min (ISO Temp 228, Fisher Scientific, Fairlawn, NJ, USA). Then, it was centrifuged at 7000 rpm for 10 min at 4 °C (Centrifuge 5430R, Eppendorf, Hamburg, Germany). After centrifugation, the supernatant was removed, and the pellet was resuspended in 20 mL of sterile distilled water. This centrifugation and resuspension process was repeated twice. Finally, the bacterial suspension was adjusted to 1 × 10⁸ CFU/mL, corresponding to an optical density of 0.4 at 600 nm, measured with a UV-visible spectrophotometer (Model 60S Evolution, Thermo Scientific, Waltham, MA, USA; Chandrasekaran et al. [36].

2.5. Preparation of Fungal Inoculum

T. asperellum was cultured on potato dextrose agar (BD Difco Laboratories, Sparks, Maryland, MD, USA; PDA) in petri dishes at 28 °C for seven days (Lab-Line Imperial III incubator, Fisher Scientific, Dallas, TX, USA). After incubation, fungal conidia were collected by scraping the mycelial surface with a sterile spatula. The collected conidia were then suspended in 20 mL of sterile saline solution at 0.85% (w/v). The suspension was shaken for two min using a vortex at speed 3 (Daigger Vortex-Genie 2; Scientific Industries Inc., Bohemia, NY, USA) and filtered through a syringe equipped with glass fiber filters to isolate the conidia. Conidial concentration was determined using a Neubauer counting chamber (Weber Scientific International Ltd., Teddington, UK) and adjusted to 1 × 10⁶ conidia/mL.

2.6. Establisment of the Experiment

Tendril pea seeds were washed twice with tap water to remove impurities and then soaked for 6 h in a 0.12% H₂O₂ solution. After soaking, the seeds were drained and directly distributed into germination trays with drainage holes (31 cm × 25 cm × 4.5 cm: Eassty^®, B0BQ3KWRZ1, USA) without substrate, at a density of 1 seed per cm² [31] and placed in darkness at 24 °C. Once germinated, two-days growth tendril pea seeds were root inoculated with 2 mL of the microorganism’s suspensions by spraying (atomizer-JR-24/410, MultiPlastic ^®, Tlajomulco de Zúniga, JAL, México).

The planted trays were placed in a plant growth room with a photoperiod of 16 h of light at 28 °C and 8 h of darkness at 18 °C, under a light intensity of 3500 lux provided by white LED lights with a full-spectrum range (Goodwill az-energy^®, model 20460, 30W, Tlaxcalancingo, PUE, México), and 70 ± 2% relative humidity. The microgreens were watered every two days with a Steiner nutrient solution (Soluponic^®, Inverfarms México, San Pablo, QRO, México), a formulation commonly used in commercial hydroponic systems due to its balanced nutrient composition and adaptability for various crops, including microgreens. The formulation was composed of (ppm): 126 NO₃⁻, 42 NH₄⁺, 31 PO₄³⁻, 274 K⁺, 181 Ca²⁺, 48.6 Mg²⁺, 112 SO₄²⁻, 1.3 Fe-EDTA, 0.8 Mn-EDTA, 0.3 Zn-EDTA, 0.06 Cu-EDTA, 0.4 B, and 0.06 Mo. The pH was adjusted to 6.0–6.5 using HNO₃ (20% v/v) with electrical conductivity at 2.3 mS/cm [37].

2.7. Morphological Parameters

The morphological characteristics of the microgreen seedlings were evaluated on day 12 after inoculation. Ten seedlings were randomly selected from the middle section of each repetition and cut at the base of the stem. Stem length and diameter were measuring using a digital caliper (Starret ^®, EC799A-6/150, Athol, MA, USA). Scanning the seedling (1200 dpi; Epson EcoTank^® L5590 CIICK57301, Seiko Epson Corp., Lapa, BTG, Philippines) and analyzing the images with ImageJ 1.46r software determined the stipular leaf area. The number of tendrils was also recorded, and fresh and dry biomass of the seedlings (stem and leaves) was measured using an analytical balance (XT-220A, Precisa Instruments^®, Zurich, Switzerland). Dry biomass was obtained by drying the seedlings in a forced-air convection oven (SMO3, Shel Lab^®, Cornelius, OR, USA) at 60 °C for 48 h. The water content of the seedlings was calculated using the following Formula (1):

$$\mathrm{Water} \mathrm{content} \left(\%\right)={\displaystyle \frac{\mathrm{Fresh} \mathrm{shoot} \mathrm{weight}-\mathrm{Dry} \mathrm{shoot} \mathrm{weight} }{\mathrm{Fresh} \mathrm{shoot} \mathrm{weight}}}\ast 100$$

(1)

2.8. Biochemical Parameters

2.8.1. pH Determination

Five grams of each sample were macerated in a vacuum-sealed bag using a spatula. The bag was then squeezed to extract the microgreen juice. The juice was transferred to a test tube, and the pH was measured using a pH meter (Checher^® pH Tester HI98103, Hanna Instruments, Woosocket, RI, USA).

2.8.2. Determination of Total Soluble Solids (TSS)

A drop of microgreen juice from each sample was placed on a digital refractometer (Automatic Refractometer Smart-1, Atago, Tokio, Japan), which was calibrated with distilled water beforehand. The results were expressed in degrees Brix (°Brix).

2.8.3. Color Index

The microgreen color was measured using the CIE Lab system, where L represents lightness, ranging from black (0) to white (100), while a and b correspond to the green-red and blue-yellow components. Measurements were taken with a digital colorimeter (Minolta Chroma Meter CR-310; Konica Minolta Optics, Osaka, Japan). The color index (CI) was determined based on the following Formula (2) [38]:

$$CI= {\displaystyle \frac{(1000\times a)}{(L\times b)}}$$

(2)

2.8.4. Photosynthetic Pigment Content

The content of photosynthetic pigments was determined according to the methodology proposed by Lichtenthaler and Wellburn [39]. A 0.1 g of fresh leaves were macerated with 4 mL of 80% acetone (v/v). The mixture was then centrifuged at 3000 rpm for 5 min. The supernatant was collected, and its absorbance was measured at 663, 470, and 645 nm using a UV spectrophotometer. The pigment concentrations were calculated using the following Formulas (3)–(5):

$${\mathrm{Chlorophyll} \mathrm{a} \left(\mathrm{mg}.{\mathrm{g}}^{-1}\mathrm{FW}\right)=(12.21\times \mathrm{A}}_{663}-2.81\times {\mathrm{A}}_{645})\times \mathrm{V}/(1000\times \mathrm{W})$$

(3)

$${\mathrm{Chlorophyll} \mathrm{b} \left(\mathrm{mg}.{\mathrm{g}}^{-1}\mathrm{FW}\right)=(20.13\times \mathrm{A}}_{645}-5.03\times {\mathrm{A}}_{663})\times \mathrm{V}/(1000\times \mathrm{W})$$

(4)

$$\mathrm{Carotenoids} \left(\mathrm{mg}.{\mathrm{g}}^{-1}\mathrm{FW}\right)=⌊{\displaystyle \frac{\left(1000 \times {\mathrm{A}}_{470}-3.27 \times {\mathrm{Chl}}_{\mathrm{a}}-104 \times {\mathrm{Chl}}_{\mathrm{b}}\right)}{229}}⌋\times \mathrm{V}/(1000\times \mathrm{W})$$

(5)

where: V = volume (mL) of 80% acetone, FW = fresh weight of cotyledonary leaves (g).

2.8.5. Antioxidant Activity

Ten grams of fresh samples were homogenized with 20 mL of 80% ethanol and then brought to a final volume of 100 mL with distilled water. The mixture was stirred using a magnetic stirrer (Isotemp Model 11-100-49SH, Fisher Scientific, Ottawa, ON, Canada) for 10 min and then filtered using filter paper (Whatman No. 1). Next, 0.1 mL of the extract was mixed with 3.9 mL of ethanolic solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH; 0.025 g/L). The mixture was shaken and kept in dark for 60 min at 25 °C. After this period, the absorbance was measured at 515 nm using a UV-vis spectrophotometer (Model 60S Evolution, Thermo Scientific, Waltham, MA, USA). The results were expressed as the percentage of DPPH radical inhibition, calculated using the following Formula (6) [40]:

$$\% \mathrm{DPPH} \mathrm{inhibition}=\left({\displaystyle \frac{{\mathrm{C}}_{\mathrm{abs}} -{\mathrm{SM}}_{\mathrm{abs}}}{{\mathrm{C}}_{\mathrm{abs}}}}\right)\times 100$$

(6)

where: C_abs = absorbance of the control (ethanolic solution of DPPH radical without extract), SM_abs = absorbance of samples extract.

2.8.6. Nitrate Content

Nitrate concentration was determined from fresh matter following the methodology of Toscano et al. [41]. A 1 g sample of fresh matter was homogenized in 3 mL of distilled water, and the mixture was then centrifuged at 4000 rpm for 15 min. Subsequently, 20 μL of supernatant was collected and mixed with 80 μL of 5% sulfuric acid-salicylic acid and 3 mL of 1.5 N NaOH. After 10 min, the samples were measured at 410 nm using a UV-visible spectrophotometer, and nitrate concentration was calculated based on a KNO₃ standard curve (0, 1, 2.5, 5, 7.5, 10 mM; R² = 0.9952).

2.9. Foliar Nutrient Content

Microgreens were dried in a forced-air convection oven (SMO3, Shel Lab^®, Cornelius, OR, USA) at 60 °C for 96 h. After drying, the samples were ground with a 1 mm mesh (Thomas Scientific 800-345-2100, Swedesboro, NJ, USA). The total nitrogen content (%) was quantified using the Kjeldahl method (Novatech^® Digester, Nashville, TN, USA and Micro Kjeldahl Labconco ^®, Rapid Distillation Unit, Kansas, MO, USA). The contents of Cu, Fe, Mn, and Zn were determined through tri-acid digestion (NHO₃, HClO₄, and H₂SO₄ in a ratio of 10:1:0.25) using 0.1 g of each sample [42]. Concentrations were measured with an atomic absorption spectrometer (Perkin Elmer Analyst 100, Waltham, MA, USA) and expressed in mg kg⁻¹. The contents of Ca, Mg, and K were determined using the previous digestion samples, diluted to 1% in deionized water. Quantifications were performed with an atomic spectrometer and expressed as percentages. The total phosphorus content (%) was determined by vanadate-molybdate method. The determination was carried out by UV-visible spectrophotometer at 410 nm [43].

2.10. Microgreens Quality

The quality of the microgreens was evaluated according to the scale proposed by Rennie et al. [44] (

Table 1. Microgreens quality evaluation scale based on visual analysis of the product.

Score	Description	Visual Quality
9	Essentially defect-free, freshly harvested. No deep visual defects	Excellent
7	Minor, non-objectionable defects. Some physical damage (<10%) The product is turgid (not wilted)	Good
5	Moderately objectionable defects, threshold of commerciality. Slight chlorosis (yellowing) Dry and wilted microgreens areas (<25%)	Fair
3	Excessive, unsellable defects. Discolored hypocotyls (blue, black) Cotyledon chlorosis (>25%) Dry and wilted plants (>50%)	Poor
1	Degraded and unusable product 100% chlorotic Mold present, bad odor Extensive rooting Apparent physical degradation (fluid present)	Very poor

):

2.11. Yield

The yield of microgreens was calculated using the following Formula (7):

$$\mathrm{Yield} \left(\mathrm{k}\mathrm{g}/{\mathrm{m}}^2\right)=\mathrm{Fresh} \mathrm{weight} \mathrm{of} \mathrm{seedlings} \left(\mathrm{kg}\right){ \times \mathrm{seedlings} \mathrm{per} \mathrm{m}}^2$$

(7)

A sowing density of 1 seed per cm² was considered for the calculation of seedlings per square meter.

2.12. Statistical Analysis

The experiment was set up under a completely randomized design. The treatments were Bt and Ta as biostimulants in pea microgreens, applied individually and in combination. A control group corresponded to uninoculated microgreens. All treatment were replicated five times. Data collected from morphological, biochemical, nutritional content parameters, quality, and yield were subjected to Shapiro-Wilk and Levene’s tests to assess normality and homogeneity of variances. Based on the results, the data were analyzed using analysis of variance (ANOVA) followed by Tukey test, Welch ANOVA followed by the Games-Howell test, or non-parametric Kruskal-Walli’s test followed by Dunn test (p < 0.05). Data processing was performed using JAMOVI software (The Jamovi Project 2024; jamovi Version 2.5.2.0 [computer software]; retrieved from https://www.jamovi.org (accessed on 12 May 2024)).

3. Results

The results of this study demonstrated that tendril pea microgreens development in a hydroponic system was significantly improved by root inoculation with T. asperellum and B. thuringiensis.

3.1. Morphological Parameters

The morphological development of the tendril pea seedling treated with the microorganisms was similar to that of the control plants but with significant differences in the vegetative parameters (p < 0.05) (Figure 1 and

Table 2. Morphological parameters of tendril pea microgreens treated with Trichoderma asperellum (Ta) and Bacillus thuringiensis (Bt) at 12 days post-inoculation.

Parameters	Treatments
	Control	Ta	Bt	Bt + Ta
Stem length (cm) 1	5.30 ± 0.86 c	8.97 ± 1.09 a	8.39 ± 1.57 b	7.86 ± 1.04 b
Stem diameter (mm) 1	1.75 ± 0.14 c	2.86 ± 0.18 a	2.45 ± 0.18 b	2.55 ± 0.22 b
Tendrills 2	3.70 ± 0.46 b	4.10 ± 0.30 a	3.84 ± 0.37 b	3.86 ± 0.35 b
Leaf area stipulate (cm2) 2	3.86 ± 1.11 b	7.09 ± 1.42 a	7.21 ± 2.01 a	6.65 ± 1.94 a
Fresh shoot weight (g) 2	0.30 ± 0.04 c	0.72 ± 0.07 a	0.64 ± 0.08 b	0.60 ± 0.09 b
Dry shoot weight (g)	0.038 ± 0.007 c	0.070 ± 0.008 a	0.067 ± 0.011 a	0.061 ± 0.009 b
Water content (%) 2	87.32 ± 1.90 c	90.26 ± 0.96 a	89.48 ± 1.75 b	89.76 ± 1.39 ab

Results are means ± S.D. of ten seedlings by replicate (five replicate). Different lowercase letters in the same row indicate significant differences according to Tukey test, Games-Howell test ¹ or Dunn test ² at the 0.05 level. Control = non inoculate plants, Ta = Trichoderma asperellum, Bt = Bacillus thuringiensis.

).

Stem length significantly increased with the use of microorganisms. Particularly, the Ta treatment improved this parameter by 69.25% compared to the control, while the Bt and Bt + Ta treatments increased it by 53.30%. Similarly, stem diameter rise by 63.43% with Ta and by 42.86% with Bt and Bt + Ta, regarding the control. On the other hand, the number of tendrils increased only with the Ta treatment, showing a 10.8% increase compared to the control. The stipular leaf area improved by 80.92% with the use of microorganism compared to the control, with no statistically significant differences among microorganism treatments.

Regarding plant biomass, the results of fresh weight, dry biomass and water content also increased, being the Ta treatment that with the highest percentage. The fresh weigh augmented by 140% with Ta and by 106.67% with Bt and Bt + Ta relative to the control. Dry biomass increased by 80.26% with Ta and Bt, followed by the Bt + Ta treatment, which showed a 60.53% increase. Lastly, water content in seedlings treated with Ta and Bt + Ta increased by 3.08% compared to the control.

3.2. Biochemical Parameters

Regarding the biochemical parameters, the microbial treatments improved some of them, showing statistically significant differences (p < 0.05;

Table 3. Biochemical parameters of tendril pea microgreens treated with Trichoderma and Bacillus at 12 days post-inoculation.

Parameters	Treatments
	Control	Ta	Bt	Bt + Ta
pH	6.6 ± 0.08 a	6.5 ± 0.06 b	6.5 ± 0.07 ab	6.5 ± 0.08 ab
TSS 1	9.4 ± 0.36 a	7.9 ± 0.22 b	8.2 ± 0.11 b	8.3 ± 0.13 b
Color Index	−21.3 ± 3.66 a	−23.0 ± 3.89 a	−23.7 ± 2.09 a	−21.80 ± 2.16 a
Chlorophyll a	1.05 ± 0.022 a	1.05 ± 0.007 a	1.06 ± 0.008 a	1.06 ± 0.008 a
Chlorophyll b	0.568 ± 0.052 b	0.693 ± 0.106 ab	0.829 ± 0.125 a	0.831 ± 0.105 a
Carotenoids 1	0.571 ± 0.015 b	0.597 ± 0.031 ab	0.622 ± 0.015 a	0.615 ± 0.017 a
Antioxidant Capacity	46.75 ± 0.46 a	46.72 ± 0.96 a	46.70 ± 0.46 a	44.48 ± 0.44 b
Nitrates	1193 ± 270 a	734 ± 171 b	706 ± 238 b	1025 ± 126 ab

Results are means ± S.D. of five replicate. Different lowercase letters in the same row indicate significant differences according to Tukey test, Games-Howell test ¹ at the 0.05 level. Control = non inoculate plants, Ta = Trichoderma asperellum, Bt = Bacillus thuringiensis. TSS = total soluble solid.

) as explained below.

The pH of the microgreens did not show significant differences between the Bt, Bt + Ta treatments and the control; however, the Ta treatment reduced the pH by 2.41% compared to the control. In the case of total soluble solids, the microbial treatments reduced their content by 13.48% compared the control. On the other hand, the color index showed no significant differences between the microbial treatments and the control, with an average value of −22.45, corresponding to a light green color.

Regarding the photosynthetic pigments, the chlorophyll a content did not differ significantly between the microbial treatments and the control. However, the chlorophyll b and the carotenoid contents increased by 46.13% and 8.31%, respectively, with the use of microorganisms in Bt and Bt + Ta, compared to the control. No statistical difference was found in chlorophyll b and carotenoid contents when treatments were compared each other.

The antioxidant capacity showed significant differences only with the Bt + Ta treatment, which resulted in a reduction of 4.86% on this parameter compared to the control. Finally, nitrate content varied among the microbial treatments, as the Ta and Bt treatments reduced nitrate levels by 39.65% compared to the control, but when treatments were combined (Bt + Ta) the nitrates were statistically similar than the control, Ta and Bt.

3.3. Foliar Nutrient Content

Overall, the nutrient content showed significant differences between the microbial treatment and the control (p < 0.05;

Table 4. Nutrient content of tendril pea microgreens treated with Trichoderma and Bacillus at 12 days post-inoculation.

Parameters	Treatments
	Control	Ta	Bt	Bt + Ta
N 1 (%)	6.9 ± 0.10 a	6.6 ± 0.05 ab	6.4 ± 0.17 b	6.2 ± 0.44 b
P (%)	0.7 ± 0.05 a	0.6 ± 0.05 a	0.7 ± 0.05 a	0.6 ± 0.05 a
K (%)	2.4 ± 0.05 a	2.6 ± 0.15 a	2.4 ± 0.10 ab	2.3 ± 0.09 b
Ca 1 (%)	1.1 ± 0.02 b	1.4 ± 0.07 a	1.3 ± 0.11 a	1.1 ± 0.05 b
Mg 1 (%)	0.9 ± 0.05 a	0.8 ± 0.02 a	0.8 ± 0.05 a	0.8 ± 0.01 a
Cu 1 (ppm)	15.7 ± 1.44 a	13.7 ± 0.27 ab	15.8 ± 0.76 a	14.9 ± 1.08 a
Fe 1 (ppm)	154 ± 3.67 a	147 ± 1.78 b	162 ± 8.28 a	165 ± 4.08 ab
Mn (ppm)	80.7 ± 1.15 ab	80.5 ± 1.19 b	83.5 ± 1.97 a	82.8 ± 1.74 ab
Zn (ppm)	47.6 ± 1.71 a	45.4 ± 1.47 a	48.2 ± 2.51 a	46.5 ± 2.18 a

Results are means ± S.D. of five replicate. Different lowercase letters in the same row indicate significant differences according to Tukey test, Games-Howell test ¹ at the 0.05 level. Control = non inoculate plants, Ta = Trichoderma asperellum, Bt = Bacillus thuringiensis.

). Using Bt and Bt + Ta reduced the N content by 8.69%, while the Ta treatment did not show significant differences compared to the control. In the case of P and Mg, no statistically significant differences were observed between the microbial treatments and the control, with an average of 0.65% and 0.83%, respectively. Regarding K, the Bt + Ta treatment reduced the nutrient content by 4.16%, but the Ta treatment and the control did not show significant differences. For Ca, the Ta and Bt treatments significantly differed from the control, enhancing the nutrient content by 22.7%.

In the case of Cu and Zn, no statistically significant differences were observed in their content between the microbial treatments and the control, with an average of 15.02 ppm and 46.42 ppm, respectively. On the other hand, only the Ta treatment showed significant differences compared to the control in Fe content, with a reduction of 4.54%.

3.4. Quality

The quality of pea microgreens at harvest did not show statistically significant differences between the microbial treatments and the control. Only minor non-objectionable defects were observed, mainly attributable to physical damage during harvest (<10%) placing them in the good to excellent category as previously reported in Table 1. The average quality score for all microgreens across treatments was 8.2. The individual scores by treatment were as follows; the control scored 8.2, the Ta treatment achieved 8.6, the Bt treatment recorded 7.8, and the combined Bt + Ta treatment scored 8.2.

3.5. Yield

Microbial treatments significantly improved the yield of tendril pea microgreens, being statistically superior than the control (p < 0.05; Figure 2). The Ta treatment showed the highest yield, with 7.20 kg/m², representing a 140.0% increase compared to the control. Similarly, the Bt and Bt + Ta treatments were superior to the control, with 6.43 and 6.01 kg/m² yields, respectively, reflecting a 107.33% increase. In contrast, the control recorded the lowest yield, with 3.0 kg/m².

4. Discussion

The results obtained in this study showed that microbial treatments significantly improved the morphological parameters of the microgreens. Regarding the morphological variables of the microgreen seedlings, the Ta treatment was the most effective in increasing the stem length of the seedlings, followed by Bt and the combination of both treatments. This fact could be related to their ability to produce plant growth hormones, such as auxins or cytokinins, or to induce the hormone production in the plants that promote cell division and expansion [45,46,47,48]. These results are consistent with previous studies reporting increases in the height of Brassica seedlings when using B. subtillis QST 713 in canola (Brassica napus) seeds. Similarly, Ouledsir-Mohandkaci et al. [49] obtained a 48.75% of increase in stem length in canola seedling after seed inoculation with B. clausii B8. On the other hand, stem diameter also increased with microbial treatments, an effect previously by Turan et al. [50] and Oulebsir-Mohandkaci et al. [49].

Stipular leaf area increased significantly with microbial treatments, suggesting that these treatments may improve photosynthesis and early seedling development [51]. This effect may be due to volatile compounds produced by B. subtilis, such as 3-hydroxy-2-butanone and 2,3-butanediol, which can alter cytokinin and ethylene homeostasis in leaves, helping to increase the leaf size [52,53]. Similarly, volatile compounds produced by Trichoderma spp. can produce substances like 6-pentyl-2H-pyran-2-one and 2-methyl-1-butanol that can promote plant growth [54]. Increased leaf area has been observed in other studies, as reported by Turan et al. [50], who found a 16.86% improvement in leaf area in cabbage seedling inoculated with B. subtilis TV-17C. Similarly, Arif et al. [55] reported that using B. cereus MZ-12 in Chinese cabbage seedling (Brassica campestris sp. chinensis) improved the leaf area by 23.5%. In the case of Trichoderma, an increase in leaf area has been reported in tomato seedling by 44.94% with seed inoculation planting [56].

Furthermore, in this study, the fresh and dry shoot biomass increased significantly with microbial treatments, ranging from 106.6% to 140.0% in fresh weight and 60.52% to 80.26% in dry shoot weight, values exceeding those reported in other studies. For example, Kang et al. [57] reported that inoculating Chinese cabbage seedlings (Brassica rapa) with B. subtilis JW1 improved fresh and dry shoot biomass by 12.14% and 14.18%, respectively.

Akhtar et al. [58] found an increase in fresh and dry biomass by 32.81% and 16.01% in cabbage seedling inoculated with B. subtilis TV-17C. This could be attributed to regulating ethylene in seedlings by ACC deaminase production by Bacillus, which improves biomass [59]. ACC deaminase degrades the ethylene precursor, favoring leaf growth [60].

On the other hand, the microbial treatments applied increased the water content in the microgreens. This is a relevant results as previous research has shown a positive correlation between water content, total chlorophyl, and fiber content [61]. Furthermore, the increase in water content is crucial for photosynthesis, which depends on the availability of carbon dioxide and water to produce carbohydrates, thereby enhancing growth and cellular activity [62]. High chlorophyll levels better capture the light for photosynthesis and facilitate fiber formation, contributing to the structural development of the plant and, ultimately, promoting its optimal growth [63].

Moreover, the microbial treatments did not significantly affect chlorophyll a content but increased chlorophyll b and carotenoids, which may be related to Bacillus ability to modulate the synthesis of photosynthetic pigments by regulating the expression of CsNRT1 genes and the activities of carbon photosynthetic enzymes and nitrogen metabolism [64].

This result is supported by studies showing improvements in chlorophyll content in plants treated with Bacillus genus. Kang et al. [57] reported an increase in chlorophyll a content by 25.94%, chlorophyll b by 10.65%, and carotenoids by 44.30% in Chinese cabbage seedlings (Brassica rapa) inoculated with B. subtilis JW1. Likewise, Turan et al. [50] found a chlorophyll increase of 5.73% in cabbage seedling inoculated with B. subtilis TV-17C.

The results of this study indicate that the color of the microgreens was not affected by the applied microbial treatments, which is relevant considering that visual appearance, while valued by consumers, is not a decisive factor in the microgreens preference [65]. This finding suggest that microbial treatments can improve other attributes without compromising product appearance. Previous studies highlight that consumers prioritize characteristics such as flavor, texture, and lower astringency in microgreens, and these organoleptic factors contribute more significantly to their acceptance [66,67].

In the case of pH, the values were reduced only by using Ta; however, the percentage was small, which does not significantly affect the microgreens’ tributes. Since pH has been reported to correlate with sensory attributes of microgreens, such as the overall quality, taste, bitterness intensity, and astringency [66].

Total soluble solids concentration is key to the flavor and aroma of microgreens, as it reflects sugar and acid content, attributes that influence consumer preference [68,69]. However, in this study, the use of microorganisms reduced TSS content by 13.15% a moderate decrease that could slightly affect expected sensory perception but also indicate a metabolic shift towards other potentially health-beneficial compounds.

Previous studies have shown that several microgreen species contain high levels of antioxidants, including phenolics, flavonoids, and carotenoids [70,71]. However, the antioxidant capacity of microgreens can vary due to factors such as species and growing conditions [72], which could be related to the decreased antioxidant capacity of the Bt + Ta treatment. In the other hand, the interaction between Trichoderma and Bacillus in the rhizosphere is complex and can influence the antioxidant capacity of plants. While both microorganisms are known for their beneficial effects, their combination does not always result in positive synergy [73]. The production of secondary metabolites by both microorganisms could interfere with each other, reducing their effectiveness in antioxidant capacity. Nonetheless, the decrease in antioxidant capacity in this study was just of 4.86% with respect to the control that may not affect the health benefits of microgreens.

Another important aspect of nutritional quality in microgreens production is nitrate content. Studies have reported that the daily consumption of vegetables with high nitrate concentrations poses a threat to human health, potentially causing gastric cancer and methemoglobinemia in infants and children [74,75,76]. The treatments applied in this study significantly reduced the nitrate content in microgreens when Bacillus and Trichoderma were used individually. However, although nitrate content in the microgreens is not regulated [77], the nitrate content in microgreens treated with microorganisms did not reach the maximum levels permitted by European Union regulation 1258/2011 for the leafy vegetables like brassicas, whose maximum levels are between 6000 and 7000 mg NO₃/Kg. Generally, microgreens tend to have lower nitrate content, depending on the harvest time, that baby or mature leafy greens of the same species [78]. This finding suggests than incorporating these microbial treatments could enhance the safety profile of microgreens, aligning with regulatory standards and potentially providing a competitive advantage for growers targeting health-conscious markets.

Overall, the macro and micronutrient content were not affected in this study, and where reductions occurred, they were less that 10%. This contrast with previous studies reporting that microorganisms can significantly influence plant nutrient uptake [79]. However, effects may vary depending on the composition of the nutrient solution and plant species. Microorganism’s influence on nutrient accumulation may also depend on growth medium, exposure time, and plant cultivar [80].

In this study, Bacillus and Trichoderma do not significantly affect the microgreens ‘quality index, suggesting that the microgreens maintain the physical characteristic of the microgreens, their essential characteristics that influence consumer acceptance [65]. This is consistent with report by Lastochina et al. [81], who indicate that Bacillus bacteria used as growth promoters or biocontrol agents in pre and post-harvest do not negatively affect cop quality.

The yield of the microgreens increased significantly with microbial treatments, suggesting that using Trichoderma and Bacillus can improve the productivity of this type of emerging crops. However, despite the ample information on growth stimulation in various crops by both microorganisms, more evidence of their use as biostimulants in microgreens is still needed [82].

Despite the benefits obtained in this study using B. thuringiensis and T. asperellum in the production of tendril pea microgreens, their effects can vary significantly among other microgreen species due to differences in physiology, nutrient uptake, and microbial interaction mechanisms. Additionally, different strains of these microorganisms might exhibit varying levels of biostimulants activity or synergy depending on the species of microgreens. The combination treatment (Bt + Ta) demonstrated mixed outcomes, suggesting potential antagonistic interactions that merit further investigation into microbial synergy and compatibility. It is essential to explore how these microorganisms affect other horticultural species and examine their synergistic effects with additional strains.

Future research should evaluate the impact of B. thuringiensis and T. asperellum across a broader range of microgreen species (e.g., kale, radish, sunflower) to enhance the generalizability of the findings. It would also be beneficial to explore the effects of different strain within the same microbial species and other beneficial microorganisms. Investigating combinations of microbial biostimulants with other agents, such as humic substances, silicon, or plant extracts, could help identify potential synergistic affects.

5. Conclusions

The use of Bacillus thuringiensis and Trichoderma asperellum as biostimulants in tendril pea microgreens significantly improved several growth parameters, such as stem length and diameter, stipular leaf area, and biomass, especially with the Trichoderma treatment. These results highlight the potential of these microorganisms to enhance the yield and morphological quality of microgreens in hydroponic systems, providing a sustainable alternative for their production.

Furthermore, the Trichoderma treatment significantly reduce nitrate levels, a key health consideration. The Bacillus and Trichoderma combination had a slight impact on antioxidant capacity without affecting visual quality, an important factor for commercial acceptance. The variability in the effectiveness of individual and combined treatments underscores the need for further research on microbial interaction and its influence on different microgreen species and cultivation conditions, pointing to the potential for future studies in this area.

In a broader context, incorporating these microorganisms in indoor microgreen production could optimize the productivity and quality of this emerging crop type and address global challenges in sustainable urban agriculture and food security. Biostimulants can reduce reliance on chemical inputs, aligning with the growing demand for healthy and environmentally responsible food in urban areas.