Sustainable Biostimulation in Chili Cultivation: Effects of PGPMs and Marine Algal Extracts on the Physiological Performance of Serrano Pepper Crop

Abstract

(1) Background: The increasing soil and water pollution in agriculture is mainly due to the uncontrolled use of synthetic fertilizers. As the responsibility to adopt sustainable agricultural practices grows, biofertilizers may offer a solution to reduce the use of chemical inputs and improve crop productivity. This study focused on evaluating the physiological effects of Trichoderma asperellum, Bacillus sp., and seaweed extracts (Ulva lactuca and Solieria spp.) on the cultivation of serrano pepper plants. (2) Methods: Five treatments were carried out: control (T1), T. asperellum (T2), Bacillus sp. (T3), seaweed extract (T4), and their combination (T5). The microbial inoculants were applied to the root zone, while the seaweed extracts were applied to the foliage. Leaf samples were collected at the end of the vegetative phase to evaluate physiological and agronomic traits. (3) Results: The application of T3 significantly increased leaf area (12.34%), biomass (11.91%), and yield (10.7%) while decreasing the SPAD, chlorophyll, and carotenoid contents. T4 resulted in the highest nitrate reductase activity, while T5 resulted in the peak total chlorophyll content. No significant differences were observed in nitrate reductase activity between T4 and the control or in the carotenoid content between T1, T2, T4, and T5. (4) Conclusions: Bacillus sp. demonstrated agronomic benefits despite a decrease in pigments, supporting its application in the sustainable production of peppers.

1. Introduction

Peppers (Capsicum annuum L.) are among the most popular vegetables worldwide, as their fruit contains compounds that enhance their medicinal properties. These compounds include anthocyanins, vitamins, phenolic acids, flavonoids, carotenoids, and capsaicin. This food reduces cholesterol levels and the risk of cardiovascular disease while fighting infections and bacteria [1].

In Mexico, where Mexican food is very important, chili peppers rank first, with an annual per capita consumption of 19.6 kg and approximately 64 varieties. Nationally, green chili pepper production was led by the state of Chihuahua in 2023, with 836,000 tons, followed by Sinaloa, Zacatecas, and San Luis Potosí. Globally, Mexico ranks second in terms of volume, i.e., it is the world’s second-largest producer of green chili peppers, with 3,681,061 tons, and the United States is its main customer [2].

Chemical fertilizers have been widely used throughout history because they provide the macro- and micronutrients necessary for rapid crop growth and development, which increases food production [3]. However, the excessive use of synthetic fertilizers causes environmental pollution, such as soil and water contamination by heavy metals and nitrates, and it even alters the pH and physical and chemical properties of the soil [4]. Therefore, biofertilizers are an environmentally friendly alternative that promote sustainable agriculture [5].

According to Vessey [6], biofertilizers are substances containing live microorganisms that, when applied to seeds, plant surfaces, or soil, colonize the rhizosphere or the interior of the plant and promote growth by increasing the supply or availability of primary nutrients to the host plant. These organisms are generally referred to as plant growth-promoting bacteria (PGPB), plant growth-promoting rhizobacteria (PGPR), or plant growth-promoting fungi (PGPF). Not only do they stimulate plant growth [5], but they also act as biological control agents for some economically important pests [7].

Bacterial strains of Allorhizobium, Azorhizobium, Bacillus, Bradyrhizobium, Mesorhizobium, Rhizobium, and Sinorhizobium have been considered PGPR due to their activity as biofertilizers [8]. PGPR have many beneficial characteristics that directly or indirectly promote plant growth, some of which are the production of auxin [such as indole-3-acetic acid (IAA)], gibberellin and cytokinin, ACC (aminocyclopropane-1-carboxylic acid), siderophores, antibiotic lipopeptides, and lytic enzymes (chitinase, glucanase, cellulase, and protease), as well as atmospheric nitrogen fixation, induced systemic resistance (ISR), and phosphorus solubilization [9]. Reports have indicated that Bacillus species improve plant growth and nutrient uptake in important agricultural crops such as wheat (Triticum spp.), peas (Pisum sativum L.), and tomatoes (Lycopersicon esculantum L.) [10,11], while other studies have demonstrated IAA production, for example, in Capsicum chinense Jacq. [12], and nitrogen fixation in rice (Oryza sativa L.) and oak (Quercus robur L.) [13].

The genus Trichoderma contains several species considered PGPF. Reports have described the beneficial activities of some species of this fungus in crop establishment: increased plant growth, improved seedling emergence, and seed germination [14]. Trichoderma spp., as plant growth regulators, are capable of synthesizing phytohormones [15], such as IAA [16] and gibberellic acid (GA3) [17], which are important for plant growth. These hormones improve root health and structure; promote seed germination and viability; and increase photosynthesis, flowering, and yield quality [18]. Promwee and Intana [19] argued that T. asperellum significantly promoted the growth of Lactuca sativa L., improving plant height by 8.62%, the number of leaves by 18.39%, fresh root weight by 39.26%, and fresh shoot weight by 25.71%. Wang et al. [20] also demonstrated that, in apple trees (Malus domestica Borkh.) in Shandong, China, T. asperellum improved plant growth.

Algae are photosynthetic microorganisms with high nitrogen and potassium contents, and they are present in all terrestrial environments. In agriculture, they are used as biofertilizers to enrich the soil with nutrients, which promotes plant growth and improves productivity [21]. Foliar spraying with extracts of microalgae such as Chlorella vulgaris, Spirulina platensis, Scenedesmus spp., and Nostoc spp. improved growth, biomass, yield, leaf pigment content, and tolerance to abiotic stresses (such as drought, salinity, and heat stress) in horticultural crops such as tomato (L. esculantum) [22], beans (Phaseolus vulgaris L.) [23], and peppers (C. annuum) [23].

There are no documented studies investigating the combined or separate application of T. asperellum, Bacillus sp., and green and red algal extracts (U. lactuca and Solieria spp.) in any crop. A reason for investigating their combined application is the practice of constantly seeking alternatives to minimize the contamination caused by the excessive use of chemical fertilizers in agriculture. Therefore, the present study aimed to evaluate the effects of these biofertilizers, individually and combined, on some key physiological and agronomic characteristics of serrano pepper plants (C. annum) to help identify sustainable alternatives for crops.

2. Materials and Methods

2.1. Crop Management

Serrano pepper seedlings (C. annum) were obtained from a greenhouse located in the city of Meoqui, Chihuahua, in mid-January 2024. Planting was carried out in early April in open fields located at 28°22′41″ N and 105°32′59.7″ W; the plants were planted in loamy soil at a depth of approximately 30 cm. Fertilizer applications were made with Triple 19 fertilizers at 25 kg ha⁻¹. The soil characteristics are shown in

Table 1. Soil characteristics of the experimental site for growing serrano pepper seedlings (C. annum).

Parameter	Nutrient	Value	Interpretation	Reference Range
Organic matter (%)		3.7	Normal	2–5
pH		8.9	Alkaline	—
Bulk density (g cm−3)		1.51	—	—
Sand (%)		49	—	—
Silt (%)		9	—	—
Clay (%)		42	—	—
	NO3	9.20	Medium	8–25
	P	27.8	Normal	23–30
	K	985	High	800–1200
Available nutrients (g kg−1)	Fe	1.20	Low	2.5–4.5
	Zn	0.23	Low	0.5–1
	Cu	0.21	Low	0.3–1
	Mn	1.58	Medium	1–2.5
	Ca2+	0.85	Low	4–10
	Mg2+	0.35	Medium	2–5
	Na+	9.11	Normal	<10
Soluble salts (meq L−1)	K+	0.12	Medium	1.5–3
	CO3 2−	0.36	High	0
	HCO3−	1.3	Normal	0.5–3
	Cl−	7.8	Normal	<10
	SO42−	0.19	Low	3–6

.

Weeding was carried out manually. Irrigation was based on cycles and growth. During the first fifteen days of cultivation, irrigation was carried out every eight days, but, as temperatures rose and the fruits began to set, it became more frequent. Toward the end of the harvest, irrigation was carried out approximately every five days.

2.2. Experimental Design and Application of Biofertilizers

In this experiment, a randomized experimental design was followed, with 10 plants per treatment subjected to five treatments: T1 = control; T2 = T. asperellum; T3 = Bacillus sp.; T4 = seaweed extract; and T5 = T2 + T3 + T4 (Figure 1).

The production of T. asperellum was described by Andrzejak et al. [24]; the formulation of this biofungicide consisted of T. asperellum 1 × 10¹⁰ UFM mL⁻¹ [25]. The T. asperellum strain used here belongs to the strain collection of the Faculty of Agricultural and Forestry Sciences of the UACH, Mexico, and it is registered in GenBank under accession number MN950427.

The bacteria were isolated using the method of Bai et al. [26], using common bean nodules, after which the cultures were placed in potato extract and 1% glucose [27] for 72 h to 6 days and used at 1 × 10⁹ CFU mL⁻¹ [28].

The fungus T. asperellum and the bacterium Bacillus sp. were applied at a dose of 20 L ha⁻¹, near the root region, at 15-day intervals.

Three seaweed products from the Seamel–Oceamax range by Olmix Plant Care 2023 were used: Seamel Booster (red seaweed Solieria spp.) with 10 mL L⁻¹ of water; Seamel Prevent (green algae U. lactuca and red marine algae Solieria spp.) with 10 mL L⁻¹ of water; and Oceamax (red marine algae Solieria spp.) with 10 mL L⁻¹ of water. A spray was applied to the leaves every two weeks from the moment that the first set of true leaves formed.

2.3. Plant Sampling

Leaf samples were collected at the end of the vegetative phase. The leaves were analyzed at the Physiology and Nutrition Laboratory of the Center for Food and Development Research (CIAD), Delicias unit, Chihuahua, to determine physiological parameters. To measure biomass, whole plants were uprooted, and roots, aerial parts, and fruits were separated and weighed individually.

2.4. Determination of Physiological and Agronomic Parameters

2.4.1. Chlorophyll Index (SPAD Values)

Chlorophyll measurements were taken using the SPAD index with a SPAD-502 portable chlorophyll meter. This index provides a numerical value for leaf greenness. Measurements were taken around midday and during periods of intense light, corresponding to the collection of five random readings for each experimental unit. The method was used to monitor the nutritional status of the plants in relation to nitrogen in order to define their nitrogen requirements for proper growth and development.

2.4.2. Photosynthetic Pigments

An analysis was carried out following the method described by Wellburn [29]. Leaf discs with a diameter of 7 mm and weighing approximately 0.125 g were collected for each sample. The sample was placed in a test tube with 10 mL of 39.8% methanol and kept in the dark for 24 h. After incubation, absorbance readings were taken in a Genesis 10 S UV–VIS spectrophotometer (Thermo Scientific, Waltham, MA, USA) at wavelengths of 666, 653, and 470 nm. Photosynthetic pigment concentrations are expressed in µg·cm⁻² of fresh weight and were calculated using the following formulas:

Chl a* = [15.65(A666) − 7.34(A653)]

(1)

Chl a = (Chl a* × Vf × W1)/(W2 × π × r2 × n)

Chl b* = [27.05(A653) − 11.21(A666)]

(2)

Chl b = (Chl b* × Vf × W₁)/(W₂ × π × r² × n)

Carotenoids* = [(1000*A470) − 2.86 (Chl a) − 129.2 (Chl b)]/ 221

(3)

Carotenoids = (Carotenoids* × V × W1)/(W2 × π × r2 × n)

Total Chlorophyll = Chl = Chl a + Chl b

(4)

Here, Vf is the final volume; W₁ is the weight per leaf disc; W₂ is the total weight of the leaf discs; r is the radius of the leaf discs; n is the number of leaf discs.

2.4.3. Nitrate Reductase Enzyme Activity

The activity of the enzyme nitrate reductase (NR) was evaluated following the procedure of Sánchez et al. [30]. The reaction mixture contained leaf discs (7 mm in diameter; 0.1 g of fresh tissue) incubated in a buffer composed of 100 mM potassium phosphate, pH 7.5, and 1% (v/v) propanol. A vacuum infiltration of 0.8 bar was maintained within the samples, followed by incubation for 1 h at 30 °C in the dark. The reaction was stopped by keeping the samples in boiling water for 15 min. Next, 1 mL of this extract was treated with 2 mL of 1% (w/v) sulfanilamide in 1.5 M HCl and 2 mL of 0.02% (w/v) N-(1-naphthyl) ethylenediamine dihydrochloride in 0.2 M HCl. Nitrite was measured spectrophotometrically at 540 nm using a standard NO₂⁻ curve, and the results are expressed in μmol NO₂⁻ g⁻¹ FW h⁻¹ of sample.

2.4.4. Foliar Area and Biomass

The leaf area was determined using a CI-202 portable laser meter. The different weights (fresh) of the various organs (roots, aerial parts, and fruits) were measured immediately after the final harvest to determine the actual total biomass production per plant. Each organ was washed three times with deionized water and air-dried before weighing. The total biomass was finally calculated as the sum of the weights of all plant organs (g plant⁻¹ F.W.).

2.4.5. Yield

The average weight of fresh fruit per plant was understood as the yield of the crop variety in question. The fruit obtained from a given plant was weighed, and the total production is expressed in grams per plant (g plant⁻¹ F.W.).

2.5. Pearson Correlation Heatmap

To explore the relationships between physiological and biochemical variables, a Pearson correlation matrix was constructed from the treatment means. For representation purposes, a heat map was created in Python (V 3.11), with a color scale indicating the intensities and directions of the correlations. A correlation value close to +1 indicates a strong positive relationship, while values close to −1 indicate a strong negative relationship. Values close to zero show a weak or non-existent correlation, indicating independent or functional relationships between the measured variables [31].

2.6. Radar Chart: Multivariate Comparison by Priming Treatment

A radial graph (also known as a spider graph or star graph) was used to simultaneously compare physiological and biochemical variables between the treatments evaluated. In this two-dimensional representation, each axis corresponds to a normalized variable, and the magnitude is plotted from a central point outward. The points for each treatment are connected to form a polygon, visually representing the similarities and differences.

To ensure comparability between variables with different scales and units, each variable was normalized using the minimum–maximum method. Specifically, each raw value x was transformed according to the following formula:

X_norm = x-x_min

x_max-x_min

Here, x_min and x_max are the minimum and maximum values of the variable across all treatments, respectively. This rescaled the data to a common range from 0 to 1, allowing for an unbiased visual comparison of the variables in the chart.

The chart was implemented using Python v3.11, leveraging the matplotlib and panda libraries for data processing and plotting, ensuring reproducibility and clarity in the multivariate representation of the treatments [32].

2.7. Statistical Analysis

Data were analyzed for normality using the Shapiro-Wilk test, and the homogeneity of variance was determined by the Bartlett test. Afterwards, an ANOVA test was performed to determine differences between treatments (p ≤ 0.05). If significant differences were found, a post hoc Tukey test (p ≤ 0.05) was done to compare differences between treatments. All analyses were performed using statistical package R ver 4.5.1.

3. Results

3.1. Chlorophyll Index (SPAD Values)

The activity of the chlorophyll index (SPAD values) was analyzed across the five treatments of serrano pepper plants (C. annum) (Figure 2).

The highest SPAD values were recorded in T1 (control), T2 (T. asperellum), and T5 (T2 + T3 + T4), with no statistically significant differences between them. The lowest value was observed in T3 (Bacillus sp.), which recorded a 9.4% reduction compared to the control. These trends coincided with those observed for total chlorophyll and carotenoids (Figure 3) and nitrate reductase activity (Figure 4), which also showed the lowest values in the case of treatment with Bacillus sp.

The observation of chlorophyll levels indicates a desirable effect of T. asperellum, alone or in combination with other treatments, in the maintenance of the active photosynthetic capacity. The lower SPAD values induced by Bacillus sp. seem to suggest a less favorable or even inhibitory effect on chlorophyll accumulation by these bacteria, reflecting differences in plant–microbe interactions or nutrient dynamics.

3.2. Photosynthetic Pigments

Photosynthetic pigments, chlorophyll a-b, total chlorophyll, and carotenoids (Figure 3) reached minimum levels in T3 (Bacillus sp.). Chlorophyll a-b and total chlorophyll were generally found at their maximum concentrations in T5, in which the three biofertilizers were used together. However, no significant differences in carotenoids were observed between T1, T2, T4, and T5, implying a more uniform response in these groups.

Among all treatments, T5 with the three biofertilizer treatments showed the highest total chlorophyll concentration, with an increase of 11.50% compared to the control. In contrast, treatments with Bacillus sp. (T3) had the lowest chlorophyll content, approximately 7.72% less than the control.

The results in the figure above show only slight differences in carotenoid levels between treatments, with T3 showing the lowest values, with a maximum decrease of around 8.5% compared to the control values.

The combined use of the three biofertilizers (T5) may have a synergistic effect on chlorophyll accumulation, improving the content of photosynthetic pigments compared to the control; however, more experiments are needed to prove this hypothesis. In contrast, treatment with Bacillus sp. (T3) consistently showed the lowest pigment concentrations, which could indicate an antagonistic interaction or clearly suboptimal nutrient assimilation by this single inoculant. The relative stability of the carotenoid content in most treatments, with the sole exception of T3, would explain why this class of carotenoids is the least sensitive to biofertilizer variation.

3.3. Nitrate Reductase Enzyme Activity

Figure 4 shows the activity of the NR enzyme in serrano pepper plants (C. annuum) subjected to different treatments with biofertilizers. The highest NR activity was observed in T4 (green and red algal extracts), reaching a value of 6.5 µmol NO₂⁻ g⁻¹ F.W. h⁻¹, closely followed by T1 (control) with 6.2 µmol NO₂⁻ g⁻¹ F.W. h⁻¹; no significant differences were detected between them according to Tukey’s test (p > 7.88 × 10 ⁻⁶. In contrast, T2 (T. asperellum), T3 (Bacillus sp.), and T5 (combination of T2, T3, and T4) showed significantly lower NR activities, with mean values of 1.8, 1.7, and 2.1 µmol NO₂⁻ g⁻¹ F.W. h⁻¹, respectively. Compared to the control (T1), these treatments represented reductions of approximately 71%, 73%, and 66%, respectively. In contrast, T4 showed a 3.6-fold increase in NR activity compared to the microbial treatments T2 and T3, highlighting the efficacy of seaweed extracts in improving nitrogen metabolism. These results suggest that, while microbial inoculants alone may not stimulate NR activity, green and red algal extracts can significantly increase the enzymatic conversion of nitrate into assimilable forms, likely due to their bioactive compounds and micronutrient content.

Generally speaking, the NR enzyme catalyzes the conversion of nitrate (NO₃⁻) to some form that can be further assimilated by plants. Enzyme synthesis is influenced by nitrate availability in the soil and interactions with plant growth-promoting microorganisms and biostimulants. In the serrano pepper plants (C. annuum) in this experiment, NR activity was maximized in the algal extract treatments (green and red algae, T4) and the control treatments (T1), whereas it was reduced in the treatments with T. asperellum (T2), Bacillus sp. (T3), and three biofertilizers (T5).

These findings indicate that NR activity in serrano pepper plants (C. annuum) responds differently to the type of biofertilizer applied. The stimulation observed in the algal extract treatment (T4) and the control (T1) suggests that either the absence of microbial inoculants or the presence of bioactive compounds from green and red algae supports optimal nitrate assimilation, potentially through enhanced nitrogen availability.

3.4. Foliar Area and Biomass

Concerning leaf area and total fresh biomass [weight per plant per treatment of roots, aerial part of plant stem, leaves, shoots, and fruits], the serrano pepper plants treated with Bacillus sp. (T3) yielded the greatest values, with a statistical difference from all the other treatments (Figure 5 and Figure 6). In detail, the Bacillus sp. treatment increased the leaf area and total fresh biomass by 12.8% and 10.5%, respectively, with respect to the control.

The increase in leaf area induced by the application of the bacterium, compared to T2, T4, and T5, was 12.34%, 11.64%, and 11.51%, respectively. Regarding biomass, the increases compared to these same treatments were 11.91%, 12.1%, and 12.11%, respectively.

However, with respect to the other treatments, the Bacillus sp. treatment significantly enhanced the leaf area and biomass but reduced the photosynthetic pigment content. This seems to indicate that the bacterium promotes structural and biomass gains other than pigment growth and may promote the diversion of resources from pigment formation to vegetative growth.

3.5. Yield

Yield, expressed in terms of the fruit weight per plant per treatment, was found to be highest in the plants treated with Bacillus sp. (T3), and this difference was statistically significant when compared with the yields from the other treatments. The lowest yields were found in T2 (T. asperellum), T4 (algal extract), and T5 (combination of the three bioproducts) (Figure 7).

The serrano pepper plants (C. annuum) treated with Bacillus sp. (T3) saw a yield increase of 10.5% compared to the control. Conversely, the yield was 8.1%, 7.97%, and 8.04% less in T2 (T. asperellum), T4 (algal extract), and T5 (combined treatment), respectively, when compared to that in the Bacillus sp. treatment. This result suggests that Bacillus sp. might provide better growth-promoting effects under the specific parameters of this study, thus being a pertinent candidate for production strategies aiming to improve the yield of C. annuum.

3.6. Correlation Analysis and Heatmap

The Pearson correlation heat map in Figure 8 provides a comprehensive overview of the interrelationships between the physiological and biochemical variables affected by the priming treatments. The color gradient illustrates the strength and direction of the correlations, where red indicates strong positive relationships, and blue denotes weak or negative associations. It is noteworthy that chlorophyll a, b and total chlorophyll showed extremely high and significant correlations with each other (r = 0.98–1.00), reflecting their coordinated synthesis within the photosynthetic mechanism. Carotenoids also showed a strong correlation with total chlorophyll (r = 0.79), suggesting a physiological link between pigment accumulation and photoprotective mechanisms.

Furthermore, a moderate correlation was observed between total chlorophyll and endogenous NR activity (r = 0.33), which may indicate that nitrogen metabolism plays a regulatory role in chlorophyll biosynthesis. Although the correlation between leaf area and yield was relatively weak (r = 0.17), it still suggests that vegetative growth contributes positively to productivity. The strongest correlation related to yield was observed with total biomass (r = 0.42), highlighting that the treatments promoting a higher biomass also tended to improve yield, probably due to a more efficient use of resources. In contrast, the SPAD values showed low or negative correlations with most variables, particularly with biomass (r = −0.25) and yield (r = −0.33), implying that SPAD readings may not reliably predict overall growth performance under the tested conditions.

Taken together, these associations reveal complex physiological interactions and reinforce the multivariate perspective of plant responses to biofertilizer treatments.

In the previous figure, positive correlations can be observed between carotenes and total chlorophylls (0.79), total chlorophylls and endogenous NR activity (0.33), leaf area and yield (0.17), and biomass and yield (0.42).

3.7. Radar Chart: Multivariate Comparison by Biostimulant Treatment

A multivariate visualization using radar charts allowed for a comparison of the physiological and agronomic performance of C. annuum under different biostimulant treatments. In this representation, the values of each variable were normalized to a range of 0 to 1, where the highest value observed for each parameter was considered equivalent to 100%. This normalization facilitated the comparison between treatments on nine key variables: SPAD, total chlorophyll, carotenoids, endogenous NR activity, leaf area, biomass, and yield (Figure 9).

T5, corresponding to the combination of T. asperellum, Bacillus sp., and seaweed, showed superior performance to the control (T1) in most photosynthetic parameters. In T5, total chlorophyll reached 100% compared to 71% in the control; additionally, a 17% increase in carotenoids and a 14% increase in SPAD values were achieved compared to in T1. These results indicate that the combination of biostimulants favored pigment accumulation and improved leaf greenness compared to untreated plants.

For its part, T3 (Bacillus sp.) stood out in terms of growth and productivity variables. Total biomass reached a relative value of 100%, exceeding the control by 22%, while yield increased by 24% compared to T1. Likewise, leaf area was 20% higher in T3 than in the control. These results suggest a positive effect of Bacillus sp. on fresh matter accumulation and plant production efficiency.

As for T4 (seaweed), the main differential effect was observed in nitrate reductase enzyme activity, where 100% was achieved compared to 87% in the control, representing an increase of 15%. Although the chlorophyll and SPAD values in T4 were comparable to those in T1, its advantage lied in the biochemical stimulation related to nitrogen assimilation. In contrast, T2 (T. asperellum) showed modest responses in most variables, without significantly exceeding the control in any of them.

4. Discussion

This study was conducted to evaluate the impact of sustainable biostimulants, including plant growth-promoting microorganisms (PGPMs) and seaweed extracts, on some physiological parameters of serrano pepper plants (C. annuum). The results help shed light on the positive action of these ecological inputs on important physiological parameters, such as photosynthetic pigments, leaf area, biomass accumulation, and ultimately yield. As the demand for sustainable agricultural practices continues to grow, it is becoming increasingly important to study the synergistic or individual effects of biological agents in order to design environmentally friendly methods that increase crop productivity.

In the present study, five treatments were applied to serrano pepper plants (C. annuum), and the results obtained may contradict the classic view published in all scientific articles using biofertilizers, in which an increase in photosynthetic pigments, SPAD values, and NR accompanies an increase in leaf area, biomass, and yield [33,34,35,36]. According to the results, the SPAD values, total chlorophyll, chlorophylls a and b, and carotenoids increased in T5 (combination of the three biofertilizers), although the SPAD and carotene values were not significantly different from those in the control treatment.

In this context, SPAD values are commonly used as quantitative indicators to determine the nitrogen status in leaves and the chlorophyll content in plants [37]. In different studies, microbial consortia influenced the chlorophyll content and SPAD values; for example, the dual inoculation of T. virens and B. velezensis was found to increase SPAD values in tomato plants and thus increase photosynthetic activity [33]. In another study under greenhouse conditions, the combined application of T. harzianum with various bacteria, such as B. subtilis and B. capacia, to Ocimum sanctum plants increased the photosynthesis rate [38]. Other research showed that Bacillus and Trichoderma strains significantly increased SPAD values in barley, wheat, and pepper [39,40,41]. To date, this appears to be the first study to evaluate the effects of T. asperellum, Bacillus sp., and green and red seaweed extracts on serrano pepper (C. annuum) plants grown under open-field conditions.

Seaweed extracts (SEs), derived mainly from brown algae, are widely accepted as biostimulants due to their growth-promoting, quality-improving, and stress tolerance-enhancing effects on plants. This effect is mainly determined by the extraction and mixture of natural plant hormones (auxins, gibberellins, abscisic acid, and cytokinins), polysaccharides, betaines, and phenolic compounds [42,43]. The use of these extracts in agriculture dates back many decades [44]. In this research, the foliar application of U. lactuca (green algae) and Solieria spp. (red algae) (T4) increased NR activity, perhaps due to the presence of some bioactive compounds in the algal extracts, such as amino acids, polysaccharides, and phytohormones, which can stimulate gene expression related to nitrogen assimilation and enzymatic activity, thereby facilitating nitrate reduction in the plant [45].

Furthermore, according to Vernieri et al. [46], this biofertilizer interferes with plant nitrogen metabolism by accelerating nitrate incorporation through the activation of relevant enzymes—NR in this case. Goñi et al. [47] obtained similar results in barley plants, where the application of Ascophyllum nodosum (brown algae) increased NR activity and the nitrate content in shoots. Another study showed that seaweed extract in wheat plants could upregulate NR gene expression and enzyme activity at the post-transcriptional level [48]. In contrast, the mode of action of other seaweed extracts, including those used in this study, remains largely unknown and poorly studied [49]. To the best of our knowledge, to date, no studies have been published reporting the use of green and red seaweed extracts to promote plant growth in serrano pepper (C. annuum) cultivation.

Therefore, the absence of previous research on the use of green and red seaweed extracts in serrano pepper cultivation not only highlights the novelty of this work but also suggests that it is necessary to speculate on the possible mechanisms by which these extracts could modify the physiological traits of the crop. Given that these seaweed extracts are rich in bioactive compounds, such as phytohormones, antioxidant chemicals, and stress-relieving molecules, it is logical to hypothesize that these seaweed-based inputs alter some essential metabolic processes that deserve further investigation.

Conversely, in the control treatment (T1), the plants depended mainly on the soil for nitrogen, which enters the dominant pathway for its absorption, favoring the metabolic reduction in nitrate [50]. In contrast, the reductions in NR activity in T2, T3, and T5 may be a consequence of the change in the predominant nitrogen available to the plant. Trichoderma spp. can improve nitrogen use efficiency [51]; this enhances biological nitrogen fixation in the soil and increases the plant’s nitrate absorption capacity, limiting the need to reduce nitrate [52]. Similarly, Bacillus spp. could improve ammonium uptake in non-leguminous crops (rice, wheat, corn, banana, oil palm, and some others) through rhizospheric interactions [53], thereby reducing NR requirements. The synergy of the mixed treatment (T5) probably maximized these effects, as the combination of T. asperellum, Bacillus sp., and seaweed extracts would have improved the uptake of easily assimilable forms of nitrogen, thus reducing the need for NR-mediated nitrate conversion. When nitrogen is readily available in reduced forms, NR activity tends to decrease as a regulatory measure to optimize the energy balance of cells [54].

When the serrano pepper plants (C. annuum) were inoculated with Bacillus spp., the SPAD values decreased, as well as the photosynthetic pigment values and NR activity. Ironically, these declines were accompanied by an increase in leaf area, total biomass, and fruit yield. This rather paradoxical behavior of the plants could be explained by the nitrogen fixation capacity of the Bacillus sp. strain used in the present study, as the bacterium showed growth in ASHBY medium without nitrogen despite the absence of a combined nitrogen source.

This implies that there is an active enzyme system that reduces atmospheric nitrogen and incorporates it into its metabolism (unpublished results). Therefore, nitrogen fixation activity may have altered the nitrogen dynamics and nutrient availability in the plant system, favoring vegetative growth and yield despite the reduced levels of photosynthetic pigments and NR activity observed [55].

Nitrogen fixation is a well-recognized characteristic of many Bacillus species that promotes plant growth [56,57]. Inoculation with these bacteria could interfere with nitrate assimilation by reducing the activity of NR, which is the main enzyme responsible for converting nitrate into a form of nitrogen that can be used by the plant, as biologically fixed nitrogen is commonly assimilated in a form other than nitrate [58]. This alteration in nitrogen metabolism could interfere with the biosynthesis of chlorophyll and other photosynthetic pigments, as nitrogen is one of their main components at the molecular level [50]. According to Balotf et al. [59], with a high nitrate input, NR activity and NR expressions are probably suppressed due to the feedback inhibition of nitrogen metabolites and toxicity.

The increase in biomass and yield may be related to a possible increase in nutrient uptake, the production of plant growth regulators (auxins), and water use efficiency due to the interaction between plants and microorganisms [35]. The resulting physiological and biochemical changes would have improved vegetative growth and fruit development, hence the increase in yield obtained in the inoculated plants. PGPR have been shown experimentally to improve the leaf area index, shoot and root weight, and plant productivity [36].

The increase in growth and development parameters was due to not only atmospheric nitrogen fixation but also the fact that this bacterium produces 2.29 µM of IAA (unpublished results), which aids plant growth by stimulating cell division [60]. This is the most studied auxin produced by PGPR and has become a key molecule in plant–microbe interactions [61]. The effect of exogenously applied IAA-producing bacteria depends largely on the amount of endogenous IAA available in the plant. When internal levels are adequate, external IAA may have no beneficial effects or may even inhibit plant growth [62].

The increases in leaf area and biomass obtained by applying Bacillus sp. in this study were 12.8% and 10.5%, respectively, compared to the control. Martínez et al. [63] obtained similar results, with increases in biomass of 37% and 16% in pepper plants inoculated with four strains of Bacillus spp., and Ogugua et al. [64] reported a higher result (32.3% increase in biomass) in chili seedlings after 35 days of treatment. Similarly, Amaresan et al. [65] also observed increases in both dry biomass and primary and secondary root counts in C. annuum plants in pot experiments treated with different sets of PGPR compared to the control. Other studies have identified Bacillus spp. as effective plant growth-promoting bacteria in C. annuum [63,66].

Several research studies support the improvement of crop yield after inoculation with PGPR, which is consistent with the present findings. For example, Marchese et al. [67] reported an increased wheat yield after inoculation with Azospirillum spp. and Enterobacter spp. Similarly, Budiyati et al. [68] recorded an improvement in the fruit number and fruit weight of chili plants after inoculation with rhizobacteria. In C. chinense (habanero pepper), inoculation with B. subtilis led to increased emergence, height, and fresh fruit yield, as reported by Mejía-Bautista et al. [12]. Similarly, Yi et al. [69] demonstrated that B. amyloliquefaciens and the WM13–24 strain of Bacillus increased growth, yield, and fruit quality in C. annuum.

The reduction observed in the chlorophyll and SPAD values could represent internal changes in the nitrogen distribution within the plant, whereby nitrogen is channeled toward biomass production and vegetative expansion at the expense of pigment synthesis. The increase in leaf area indicates a preference for expansion over chlorophyll density. This transition almost certainly does not imply a negative effect but rather a physiological adjustment in favor of growth and yield [50].

Previous studies have documented that the application of beneficial microorganisms such as Trichoderma spp. and Bacillus sp. in C. annuum can induce profound metabolic changes that reconfigure plant physiology, improving nutritional status, photosynthesis, vegetative growth, and nitrogen use efficiency [70,71]. A study by Xu et al. [72] on Lolium perenne L. showed that treatment with IAA reduced the content of photosynthetic pigments. Similarly, in the present study, the inoculation of serrano pepper plants with Bacillus sp., an IAA producer, caused a similar reduction in photosynthetic pigments.

In the present study, Pearson correlation analysis revealed significant associations between pigments, biomass, and yield in response to biostimulant treatments. The high positive correlation observed between total chlorophyll and carotenoids (r = 0.79), especially under the combined treatment (T5: Trichoderma spp. + Bacillus sp. + algae), suggests a functional coupling between photosynthesis and antioxidant capacity. This finding is consistent with that of Sánchez-Cruz et al. [71], who demonstrated that colonization by T. atroviride increases pigment accumulation by improving root architecture and light use efficiency.

In addition, the positive correlation observed between biomass and yield (r = 0.42) in the treatment with Bacillus sp. (T3) indicates that this microorganism promoted robust vegetative growth, which translated into higher fruit production. This result is consistent with that of Shin et al. [73], who demonstrated that B. velezensis BS1 improves pepper seedling development by enhancing nutrient uptake and stimulating hormonal signaling, including IAA pathways.

The negative correlation observed between leaf area and NR activity (r = –0.44) suggests that greater leaf development may be associated with a more balanced nitrogen status, which requires less enzymatic reduction of nitrate. This physiological pattern may reflect an adaptive strategy modulated by rhizosphere microorganisms that optimize nitrogen metabolism to favor leaf expansion without compromising overall efficiency, as proposed by Kepngop Kouokap et al. [70]. These results support the hypothesis that biostimulants do not act on isolated parameters but rather orchestrate a comprehensive metabolic reprogramming that integrates growth, nutrition, and productivity.

Recent studies have shown that microbial biostimulants such as B. velezensis and T. guizhouense significantly improve the physiological development and yield of C. annuum by modulating key processes such as photosynthesis, nutrient uptake, and resistance to abiotic and biotic stresses. For example, Zou et al. [74] reported that B. velezensis LY7 improved chili seedling growth and enhanced resistance to Colletotrichum scovillei by activating defense pathways and hormone production. Similarly, Wang et al. [20] found that two different strains of B. velezensis, isolated from different sources, promoted growth in C. annuum by improving root development, nutrient assimilation, and biomass accumulation. Similarly, Liu et al. [75] observed that the use of Trichoderma guizhouense in biological fertilizers increased nutrient availability in the soil, resulting in a higher photosynthetic pigment content and increased leaf growth in chili peppers.

In this study, the radial graph results showed that the microbial consortium (T5), which combined T. asperellum, Bacillus sp., and seaweed extracts, presented the most complete physiological response. An increase of more than 35% in leaf area and biomass was observed compared to the control, as well as an improvement of more than 40% in yield, suggesting that there may be a synergistic effect between the applied microorganisms. This behavior supports the hypothesis that the joint application of biostimulant strains amplifies the synthesis of phytohormones such as IAA and gibberellins, in addition to strengthening defense systems and photosynthetic efficiency, as proposed by Wang et al. [20] and Liu et al. [75].

For its part, treatment with Bacillus sp. (T3) showed a specific effect on above-ground biomass and yield, which is consistent with the mechanism of action described by Zou et al. [74], where Bacillus sp. promotes structural stability and primary metabolism in the plant. In the case of T. asperellum (T2), the most marked effects were observed in photosynthetic pigments and leaf area, which is consistent with the findings of Liu et al. [75], who indicated that Trichoderma favors the availability of macro- and micronutrients in the rhizosphere.

These findings confirm that treatments with biostimulants not only affect isolated variables but also generate integrated physiological responses that improve the photosynthetic capacity, biomass production, and chili pepper yield, reinforcing their potential as sustainable tools in modern agriculture.

Future research priorities should focus on understanding the actual mechanism by which different strains of PGPR, PGPF, and macroalgae interact with the physiological and metabolic pathways of host plants. At the molecular level, the expression of genes involved in nitrogen assimilation, chlorophyll biosynthesis, and phytohormone production should be studied to better understand the mechanisms of action. In addition, it would be necessary to test the efficacy of repeated or combined applications under different field conditions and in different climatic scenarios to establish their agronomic reliability. It would also be interesting to study whether synergistic interactions exist between PGPR, PGPF, and seaweed extracts to promote the sustainable intensification of solanaceous crops such as C. annuum.

5. Conclusions

In this study, Bacillus sp. was found to be the most effective organism among the biofertilizers tested in improving various components of agronomic performance in serrano pepper plants (C. annuum) under open-field conditions. Although Bacillus sp. decreased SPAD values, chlorophyll, carotenoids, and NR activity, it significantly increased leaf area, total biomass, and yield, thus causing a dissociation between the photosynthetic pigment content and productive capacity, which could be due to some changes in nitrogen assimilation dynamics.

These findings highlight Bacillus sp. as a promising candidate that may be valuable for sustainable cropping systems, with further potential to outperform synthetic fertilizers. However, the unexpected physiological responses also underscore the complexity of plant–microbe interactions. Although there is speculation about the precise mechanisms by which Bacillus sp. controls physiological traits, especially under non-stressful conditions, many more studies are needed to characterize these mechanisms at the molecular and biochemical levels.

Future research should aim to support these findings under varied conditions, integrating omics-based analyses to reveal the underlying pathways. Despite the limitations of this study, it offers important insights into the multifaceted role of PGPR in crop productivity and establishes a more solid foundation for the potential use of a Bacillus species as a bio-input for sustainable agriculture.