Plant material. We placed 166 Ancho chili pepper plants (C. annuum) of the San Luis variety (Pacific Seed Company^©) and 55 tomato plants (S. lycopersicum) of the Rio Grande variety (CIIASA Seeds^©) in 2 L pots with a mixture of substrate (55% sand, 40% soil and 5% agrolite) previously sterilized in an autoclave (120 °C

/ 103421.4 Pa / 6 h) and kept under greenhouse conditions.

Microbiological material. The phytopathogenic bacteria BV801 and BV865, isolated simultaneously from the diseased tissue of chili pepper plants affected by bacterial spot (López-Vielma et al., 2016) and determined as the cause of the disease (Rico-Aguilar et al., 2019) were obtained from the collection of microorganisms of the Phytopathology Laboratory of the Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco, A.C. (CIATEJ). The bacteria were reactivated from cryoconservation at -80 °C in liquid culture media. For X. euvesicatoria, strains BV801 and BV865, medium NYG (0.5% peptone, 0.3% yeast extract, 2 % glycerol, 7.0 pH, agar 1.6% when required) was used. On the other hand, P. syringae pv. tomato strain DC3000, was placed in a King (KB) liquid medium (2% peptone, 0.8% K2HPO4 [1 M], 0.6% MgSO4•7H2O [1 M], 1.2% glycerol, 7.2 pH, agar 1.6% as needed). Subsequently, the media were incubated at 28 °C, for 24 h, in the dark and shaken at 250 rpm. The bacteria were cultivated in liquid media all night (18-20 h), then planted by centrifugation at 10000 rpm for 5 min. Later, the supernatant culture medium was discarded and sterile deionized water was used to wash, repeating the process of centrifugation and washing three times. Next, the concentration of bacterial cells was adjusted to an optic density (OD) of 600 nanometers (nm), OD600 of 1.0 (OD600=1.0≈1x10⁸ UFC mL^-1) with a spectrophotometer (Eppendorf^®, BioPhotometer).

Production of Hrp proteins in the pilot plant: The Hrp proteins were produced in a pilot plant based on the methodology by Valerio-Landa et al. (2021a), extrapolated for applying in a tray-style solid fermentation bioreactor (45x30x3.5 cm). The bacterium X. euvesicatoria strain BV801 previously grown in a rich NYG medium and adjusted to OD600 = 1.0 (≈1x10⁸ UFC mL^-1), was applied at a volume of 2.5 mL per tray. The bacteria were then spread onto the induction medium for subsequent cultivation (0.2% of pulverized C. annuum tissue, 1.5% food-grade agar, pH of 7.0). The tray was closed using Kraft paper and sealed with tape, the tray was sterilized with the induction medium; after bacterial inoculation, they were placed in the solid fermentation bioreactor, and incubated at 28 °C +/-3 °C in the dark. The harvest of the bacterial cells was carried out after 48 h by scraping and collecting the bacterial lawn with sterile deionized water. Finally, sterile deionized water was used to wash away the excess xanthan at 10000 rpm for 30 min at 4 °C. Bacterial lysis was carried out using sonication in an exposure time of 15 min with an ultrasonic liquid processor (Sonics VCX 130), using a frequency of 20 Khz, an amplitude of 40%, 30 s on / 30 s off cycles at 130 W, placing the samples in ice. After bacterial lysis, the protocol for protein extraction according to Valerio-Landa et al. (2021a) continued; the extracted proteins constituted the product named BioFensa (invention patent application MX/a/2020/013638; Valerio-Landa et al., 2023).

Experimental design. To evaluate the biological effectiveness of BioFensa (BF), it was tested against the strain DC3000 of P. syringae pv. tomato in tomato plants (Experiment 1) and against X. euvesicatoria (strains BV801 and BV865) in chili pepper plants (Experiments 2 and 3). Three experiments were performed in totally randomized experimental designs, each one with five treatments (11 repetitions per treatment): (1) diseased plant (PE, with bacterial inoculation); (2) healthy plant (PS, without bacterial inoculation); (3) one commercial chemical inducer (Actigard^®, 0.003 g mL⁻¹); (4) one commercial αβ Harpin protein inducer (Messenger Gold^®, 0.003 g mL⁻¹); (5) BioFensa (1 μg mL^-1). On the other hand, to determine the optimum dose for the application of BioFensa against X. euvesicatoria strain BV801 in chili pepper plants (Experiment 4), a bioassay was held in a completely randomized experimental design with seven treatments (eight repetitions): (1) diseased plant (PE); (2) healthy plant (PS); (3) one chemical inducer (Actigard^®);

(4) one commercial αβ Harpin protein inducer (Messenger Gold^®), BioFensa at 0.01 (5); 0.1 (6) and 1 (7) µg mL^-1 (Table 1). For all induction treatments (Actigard^®, Messenger Gold^®, BioFensa), they were applied 24 h before the infection with bacteria. The 3 mL treatments were applied by spraying on all of the plants’ leaves (adaxial and abaxial).

Table 1

Controls			Inducer
	Healthy plant	Diseased plant	Actigard®	Messenger Gold®	BioFensa
Ingredient	Without phytopathogen	With phytopathogen	Acibenzolar- S-metilo	Commercial harpin	Protein inducer of X. euvesicatoria
Dose (3 mL/plant)	Not applicable	108 UFC/mL	0.0003 g/mL	0.03 g/mL	0.01, 0.1, 1 μg/mL

Phytopathological response variables. To evaluate the severity of the disease, 30 days after the experiment began, response variables were recorded by quantifying the spots: number of chlorotic and necrotic spots, as well as the total number of spots observed on leaves with disease symptoms.

Statistical analysis of data. The data obtained from the response variables were analyzed statistically using a one-way analysis of variance and LSD tests: All statistical tests were evaluated at a significance level of 5% (p≤0.05). The information was processed using the package for statistical analysis Statgraphics Centurion ver. XV.II (http://www.statgraphics.com/).

The results indicate that the symptoms of the bacterial speck (P. syringae pv. tomato, strain DC3000) in tomato plants, evaluated by the number of leaves with necrotic and chlorotic spots, as well as the number of total spots per plant (Figure 1), significantly decreased with the application of BioFensa (1 μg mL⁻¹).

Figure 1

In comparison with the untreated diseased plants, the treatment with BioFensa prevented the appearance of spots by 53% (LSD, p≤0.05). Specifically, the average number of total spots per treated plant was 15±3, in comparison with 32 ± 4 in the diseased control (PE).

On the other hand, for the bacterial spot in ancho chili pepper plants treated with BioFensa (1 μg mL⁻¹) and challenged with the X. euvesicatoria strain BV865 (Figure 2), they showed a lower number of total spots, similar to those treated with commercial resistance inducers (Messenger Gold^® and Actigard^®). Specifically, the plants treated with BioFensa showed a significant reduction (LSD, p≤0.05) of 60% in the total number of spots, compared to the diseased control (PE).

The analyses of variance and the LSD tests LSD (p≤0.05) indicate that the treatment with BioFensa prevents disease severity by showing a lower occurrence of foliar tissue symptoms, ranging from chlorotic to the presence of necrosis, as observed through visualization, as stated in Rico-Aguilar et al. (2019). Therefore, the application of Hrp proteins (BioFensa) significantly prevents the incidence of P. syringae pv. tomato, DC3000 (bacterial speck) in tomato plants and X. euvesicatoria, strain BV865 (bacterial spot) in chili pepper plants.

Figure 2

The concentration of 1 µg mL^-1 of BioFensa was enough to reduce the level of the spots of the disease, which could be attributed to the activity of the molecular patterns associated to pathogens (PMAP) found in the Hrp proteins of BioFensa. These results are consistent with earlier studies by Valerio-Landa et al. (2021b), who suggest that Hrp proteins can be recognized by the immunity receptors of plants, unleashing a new robust defense response by the immunity receptors by the plants.

The evaluation of strain BV801 of X. euvesicatoria in chili plants treated with BioFensa (1 μg mL⁻¹) displayed a reduction of only 13% in the total number of spots, which was not statistically significant (LSD, p≤0.05) with the diseased treatment (PE) (Figure 3). This means that the concentration of 1 μg mL⁻¹ of BioFensa may not have been optimal for the case of strain BV801, in comparison with strain BV865 (Figure 2). The variability in the efficiency observed may be due to the intrinsic virulence of strain BV801 and the saturation of the PMAP receptors in the plants, which coincides with studies that indicate that high concentrations of inducers can lead to the desensitization of defense receptors (Boller and Felix, 2009; Abdul et al., 2020). When comparing the different treatments, a greater reduction in foliar spots was found in tomato plants with strain DC3000 and in chili plants with strain BV865, since strain BV801 displayed a greater degree of disease virulence. Likewise, when comparing the effect of the commercial inducers, a similar behavior was observed between Messenger Gold^® and BioFensa, in relation to the necrotic spots (Figure 3). In addition, the treatment with Actigard^® displayed a greater degree of the disease than the diseased control (PE) in relation to the total stains (Figure 3). The plants that were pretreated with BioFensa (1 μg mL⁻¹) displayed the same degree of disease than the diseased control plants (PE). This result may be due to the fact that the ability of chili plants to perceive PMAP inducers and activate their physiological state of resistance (PSR) seems to have been affected by the high concentrations of Hrp proteins during this investigation. In order to prove this hypothesis, an experiment was carried out to evaluate the effect of different doses of BioFensa in chili plants infected with the strain BV801 of X. euvesicatoria (Figure 4).

Figure 3

The plants treated with different concentrations of protein solutions (0.01, 0.1 and 1 μg mL⁻¹) for the control of X. euvesicatoria strain BV801 in ancho chili plants (Figure 4) displayed few symptoms of the disease when the concentration of 0.01 μg mL⁻¹ was used, in relation to the total spots on the leaves, achieving a 41% reduction of the disease. For necrotic lesions on leaves, a reduction of 51% was found, for the concentration of 0.1 μg mL⁻¹, 38% was found, and at 1 μg mL⁻¹ there were no significant differences were found in comparison with the diseased control (-15% the 1 μg mL⁻¹ treatment in comparison with PE), therefore no biological effectiveness was registered, causing a negative effect on the plant with this concentration (Figure 4, necrotic spots). In this regard, Zehra et al. (2021) mention that the effectiveness of the inducers can vary, depending on a series of factors, including the type of inducer, the plant species and environmental conditions. This coincides with the results of Figure 4, and could therefore be due to the inducers of effectors in plants working better at lower concentrations, since plants have a limited number of receptors, and at low concentrations, they allow for the activation of the adequate defense response. At higher concentrations, receptors saturate and can cause negative regulation mechanisms, cause toxicity disorganize signaling and divert energy resources from other vital processes such as growth, leading to energy depletion (Boller y Felix, 2009; Abdul et al., 2020).

Figure 4

The findings of this study show that a concentration of 0.01 or 0.1 µg mL^-1 of BioFensa was enough to induce PSR and significantly reduce the severity of the bacterial speck and spot in tomato and chili plants, respectively (LSD, p≤0.05). This response could be related to the activity of the activity of molecular patterns associated to pathogens (PMAP) found in Hrp proteins, which are recognized by the innate immunity receptors in plants, thus activating the defense responses. Previous studies such as those by Boller and Felix (2009) have proven that similar proteins, such as flagellin (flg22) and even peptides such as systemin (18 amino acids) can induce defense responses in sub-nanomolar concentrations. Diverse studies, including the revision by Zehra et al. (2021) have proven that microbial elicitors, such as proteins and peptides, can unleash an Induced Systemic Resistance (ISR, mediated by jasmonic acid and ethylene). The phenomenon, characterized by a generalized and long-lasting state of alert, prepares the plant to respond faster and more effectively to a wide spectrum of pathogens. The activation of ISR is unleashed by the perception of elicitors, which act as danger signals and set the plant’s defense machinery on motion (PMAP) when recognized by specific receptors in the surface of plant cells, starting a cascade of intracellular signaling that culminates in the activation of defense genes, as well as the establishment of local and systemic resistance, and the production of effector proteins. In this context, harpin protein Hpa1 has proven to have several beneficial effects on plants, including the promotion of growth and the induction of resistance to phytopathogens. Earlier investigations such as thos by Wang et al. (2020) revealed that Hpa1 can significantly mitigate the symptoms of the tobacco mosaic virus (TMV) in Pinellia ternata, suggesting an effective stimulation of the resistance to the disease, showing that Hpa1 has a significantly superior antiviral activity than the control and that is protective effect surpasses it curative effect. Additionally, Hpa1 induced a notorious increase in the activity of enzymes related to the defense (polyphenol oxidase, peroxidase, catalase and superoxide dismutase), as well as in the expression of genes associated to the resistance of diseases by the plant (PR1, PR3, PR5 and PDF1.2).

This suggests that the Hrp proteins in BioFensa may be acting through a similar mechanism, unleashing the production of antimicrobial defenses and strengthening cell barriers. Additionally, the variability in the response to different concentrations and types of pathogens underscores the importance of optimizing the dose of resistance inducers to maximize their effectiveness. In this sense, literature also suggests that the combination of resistance inducers with other biological or chemical treatments may offer a more robust strategy for the management of diseases (Walters et al., 2013).

The results from this study on the effectiveness of the biological inducer BioFensa (Hrp proteins) in the reduction of the bacterial speck in tomato and bacterial spot in ancho chili peppers coincide with the existing literature on the use of resistance inducers in plants. Boller and Felix (2009) pointed out that PMAPs such as flagellin are recognized by specific receptors and act as powerful elicitors of defense responses at sub-nanomolar concentrations, a mechanism similar to the one observed with BioFensa, which displayed a greater effectiveness at low concentrations (0.01 μg mL⁻¹). This phenomenon has also been documented by Abdul et al. (2020), who pointed out that the efficiency of resistance inducers may depend on the concentration used and the type of pathogen, reflecting the findings of this study, where higher concentrations did not always improve the defensive response. In addition, Walters et al. (2013) discussed the challenges and need to optimize the doses of inducers to maximize their effectiveness, a recommendation that resonates with the results regarding BioFensa being more effective at low concentrations (0.01 μg mL⁻¹). The capacity of resistance inducers to activate systemic defense responses in plants has been well documented (Ryals et al., 1996; Durrant and Dong, 2004) and the results provide additional evidence that BioFensa can induce a physiological state of resistance, significantly reducing the severity of diseases. These findings suggest that BioFensa may be a valuable tool in the biological control of pathogens that cause diseases, promoting a more sustainable agriculture and reducing the dependency on chemical pesticides (Pieterse et al., 2012; Van Loon et al., 2006).

Therefore, further evaluations are required to analyze the most effective

concentrations for the reduction of diseases in tomato plants caused by P. syringae pv. tomato DC3000, as well as in chili pepper for X. euvesicatoria strain BV865 based on the results of X. euvesicatoria strain BV801, where a higher concentration of application of Hrp protein-based inducers displayed a greater effectiveness in the reduction of the bacterial spot, making BioFensa an alternative as a possible tool in the control of important phytopathogenic bacteria for agricultural production.