A total of 100 seeds of Lactuca sativa cv Mimosa lettuce (Category S2, 2018 harvest) were used per treatment, with germination percentage of 89%. They were purchased from an agricultural trading house. The seeds were treated manually by soaking them in various concentrations of the effector protein rBASIDIN (50 µg mL^-1, 75 µg mL^-1 and 100 µg mL^-1) for 30 min. After soaking, the seeds were aseptically air-dried and used throughout the study. Seeds treated with distilled water and Tris-HCl 10 mmol L^-1 were used as controls. Once this seed treatment process was completed, the physiological and biochemical qualities were evaluated using the following laboratory tests:

To determine electrical conductivity, the method proposed by (Veronesi et al., 2014) was employed, with some modifications, using three replicates of 25 seeds per treatment. The seeds were previously weighed with a precision analytical balance (0.0001) and were subjected to imbibition with rBASIDIN at different concentrations (50 µg mL^-1, 75 µg mL^-1 and 100 µg mL^-1) for 30 min. As a control, the seeds were soaked in distilled water and 10 mmol L^-1 of Tris HCl buffer. Afterward, the seeds were dried with paper towels, transferred to a container containing 10 mL of autoclaved distilled water and kept in a BOD at a temperature of 25 °C. Electrical conductivity was determined after 24 hours using a conductivity meter (CD, Lutron Electronics model 4322), and the results were expressed as µS cm^-1 g^-1 of seeds. The conductivity of autoclaved distilled water was measured as a blank control (CO). The calculation of electrical conductivity was determined according to the following equation:

Where:

EC= total electrical conductivity, in µS cm^-1 g^-1;

Rce = electrical conductivity reading of soaked seeds;

CO= electrical conductivity of distilled water (blank control); and

Ms= mass of 25 seeds, in g^-1.

The activity of enzymes that are part of the plant defense system against the action of pests was evaluated to verify the effect of rBASIDIN as an inducer. To analyze the enzymatic activity of the enzymes chitinase, glucanase and catalase, the lettuce seeds were treated by immersion for 30 min, in each treatment, at the concentrations previously described. Sixty seedlings of each treatment were used (four replicates of 15 seedlings).

Crude enzymatic extracts were obtained by macerating the seedlings in liquid nitrogen, followed by the addition of 1 mL of 0.1 M potassium phosphate buffer, pH 6.8, and ethylenediaminetetraacetic acid (EDTA). The samples were processed in an ultrasonicator (Gex ultrasonic processor model 130, 130W) on ice until the tissue was completely disrupted, with a pulse of 8 s, at intervals of 10 s and an amplitude of 70%. Then the samples were centrifuged for 5 min at 17,530 g. Immediately afterwards, 200 µL aliquots of the aqueous phase were collected and diluted in 800 µL of the appropriate buffer for analysis of the activity of each enzyme. An aliquot of 30 µL of each sample was separated for quantification by the Bradford methods (Bradford, 1976) using BSA as standard. The specifics of the methods for each enzyme are described below.

In the germination test, there was a significant difference (p ≤ 0.05) between treatments, as can be seen in Figures 1A, B . After four days of germination, the lettuce seeds that received treatments with rBASIDIN at concentrations of 50 µg mL^-1 and 75 µg mL^-1 showed higher germination percentages than those of the controls, reaching average values of 79% and 85% of germinated seeds respectively, while the seeds treated with distilled water showed 58% germination and 39% for buffer exposure ( Figure 1A ). Subsequently, the seeds treated with the rBASIDIN protein at a concentration of 100 µg mL^-1, had a lower germination percentage, of 35%, compared to the controls ( Figure 1A ). Furthermore, the germination percentage of the lettuce seeds treated with rBASIDIN at concentrations of 50 µg mL^-1 and 75 µg mL^-1 at seven days did not differ significantly compared to the control seeds treated with distilled water ( Figure 1B ), but differed from the buffer control treatment and the treatment with rBASIDIN at a concentration of 100 µg mL^-1 ( Figure 1B ).

Treatment of lettuce seeds with rBASIDIN at concentrations of 50 µg mL^-1 and 75 µg mL^-1 did not hamper seedling development (uniformity and greater formation of normal seedlings) ( Figure 2 ).

There was a greater increase in the length of seedlings obtained from seeds that were treated with rBASIDIN at a concentration of 50 µg mL^-1 (0.72 cm) in comparison with the other treatments, which presented an average length of 0.58 cm in the treatment with 75 µg mL^-1, distilled water (0.58 cm), 100 µg mL^-1 (0.56 cm) and buffer (0.50 cm) ( Figure 3A ). Regarding the length of the primary root, statistical differences were observed between treatments. rBASIDIN at a concentration of 50 µg mL^-1 presented an average root length of 1.51 cm, which was statically equal to the average length of seedlings obtained from the treatment of seeds with rBASIDIN at a concentration of 75 µg mL^-1, but statistically different from the other treatments ( Figure 3B ). The largest seedlings were obtained in the treatments with rBASIDIN ( Figure 3C ).

The fresh mass of lettuce seedlings varied according to the treatment to which the seeds were submitted. The treatments with rBASIDIN at concentrations of 50 µg mL^-1 and 75 µg mL^-1 showed statistically greater fresh mass, with averages of 0.22 g seedling^-1 and 0.19 g seedling^-1, respectively ( Figure 4A ). In the other treatments, there was no significant difference, with averages of 0.07 g seedling^-1 to 0.12 g seedling^-1.

For dry mass ( Figure 4B ), the plants from seeds treated with rBASIDIN at a concentration of 50 µg mL^-1 (0.022 g seedling^-1) were statistically equal to those submitted to 75 µg mL^-1 (0.019 g seedling^-1), but differed from the others. Lettuce seedlings from seeds treated with rBASIDIN at a concentration of 75 µg mL^-1 did not differ from treatments with rBASIDIN at a concentration of 100 µg mL^-1 (0.012 g seedling^-1), or the controls, distilled water (0.012 g seedling^-1) and buffer (0.007 g seedling^-1).

The electrical conductivity (EC) of the seeds in all treatments with rBASIDIN had lower values compared to the control treatments. Although a linear increase in the amount of electrolytes released by the seeds was observed with increasing rBASIDIN concentration, there was no statistical difference ( Figure 5 ). At a concentration of 50 µg mL^-1, a lower CE occurred, of 851 µS cm^-1 g^-1, in relation to the control treatments, water (2,105 µS cm^-1 g^-1) and buffer (1,785 µg cm^-1 g^-1) ( Figure 5 ).

The treatment of lettuce seeds with different concentrations of rBASIDIN stimulated the enzymatic activity positively or negatively depending on the concentration used. When lettuce seeds were treated with rBASIDIN at a concentration of 75 µg mL^-1, the seedlings showed an increase in exochitinase activities ( Figure 6A ). For seeds that were treated with rBASIDIN at a concentration of 100 µg mL^-1, there was a sharp decrease in enzyme activity in seedlings, with activity still lower than that of control treatments ( Figure 6A ). For endochitinase activity, rBASIDIN interfered negatively, as can be seen in Figure 6B . The concentrations of 75 µg mL^-1 and 100 µg mL^-1 induced lower enzyme activity compared to the controls, and the protein at the concentration of 50 µg mL^-1 showed no difference in activity compared to the controls ( Figure 6B ).

Regarding the activity of β-1-3 glucanase, the maximum increase in activity was observed in lettuce seedlings from the seeds treated with rBASIDIN at a concentration of 75 µg mL^-1, which presented enzymatic activity of 62.39 µmol glucose h^-1 mg^-1 ( Figure 6C ), a result statically different from the other treatments. For the treatment of seeds with rBASIDIN at a concentration of 50 µg mL^-1, the enzyme activity in seedlings was 33.39 µmol glucose h^-1 mg^-1 which was not significantly different from the control treatments in comparison with the other treatments, except for the treatment with rBASIDIN at a concentration of 100 µg mL^-1. For the seeds treated with distilled water, the seedlings had activity of 32.44 µmol glucose h mg^-1, and the buffer was 32.82 µmol glucose h^-1 mg^-1. Lower activity of glucanase was observed in the treatment of seeds with rBASIDIN at a concentration of 100 µg mL^-1 (17.93 µmol glucose h^-1 mg^-1).

The activity of the enzyme catalase (CAT) in seedlings from seeds treated with different concentrations of rBASIDIN, was higher than in seedlings seeds treated with rBASIDIN at a concentration of 100 µg mL^-1, with an average value of 2.88 Abs 240 min^-1 mg^-1 protein. The seedlings from seeds treated with rBASIDIN at concentrations of 50 µg mL^-1, 75 µg mL^-1 and the control with distilled water had, respectively 1.40; 1.56 and 2.71 Abs²⁴⁰ min^-1 mg ^-1, while the value for the treatment with buffer was, 0.93 Abs²⁴⁰ min ^-1 mg ^-1 ( Figure 6D ). Static analysis showed a significant difference only between seed treatments with rBASIDIN at a concentration of 100 µg mL^-1 in relation to the control treatment with the buffer and treatments with rBASIDIN at concentrations of 50 and 75 µg mL^-1 ( Figure 6D ).

Seed germination is the first critical stage in the life of plants, since seeds are exposed to a wide range of environmental stresses that can affect their germination. To enhance or preserve the germination and productivity of different crops, some seed treatment methods are used, such as priming (Nile et al., 2022), pelletizing (Javed et al., 2022) and seed coating (Paravar et al., 2023).

Seed preparation with biotic and abiotic elicitors results in better germination, and can induce plants’ immunological memory, which is maintained stably throughout the development stages and can also be transmitted over generations (Kalaivani et al., 2016; Nile et al., 2022). In this sense, we tested the effect of the rBASIDIN effector regarding its ability to induce germination and physiological changes in lettuce seedlings through seed treatment.

The average germination percentage of lettuce seeds increased due to treatment with rBASIDIN at low concentrations. This increase was observed in treatments with rBASIDIN at 50 and 75 µg mL^-1 four days after sowing ( Figure 1A ). The beneficial effect on germination observed for these concentrations may have occurred as a result of the action of rBASIDIN on membrane permeability, since cell membrane homeostasis is a prerequisite for normal germination and good physiological functioning of seeds (Dhaliwal and Angeles-Shim, 2022).

The first germination count is an indication of the seeds’ vigor, since seeds that germinate more quickly and produce a higher percentage of normal seedlings are predisposed to be more vigorous (Brasil, 2009). Thus, the treatment of seeds with BASIDIN at the lowest concentrations produced seedlings with uniform development ( Figure 2 ). This is an important characteristic since uneven growth of seedlings results in yield losses (Ebone et al., 2020).

Still in this context, at the end of the germination test (seven days after sowing), the seeds treated with the rBASIDIN effector at the concentrations mentioned above presented a germination percentage in accordance with the percentage established by the batch manufacturer ( Figure 1A ). This fact, added to the early germination of seeds on the fourth day, showed that these concentrations were efficient in increasing seed germination. In line with our results, other studies have shown positive results from the use of elicitor/effector molecules derived from pathogens for seed treatment. The use of trehalose, a mycogenic elicitor isolated from the cell wall of Thrichoderma atroviride in broccoli seeds provided a remarkable increase of 94% in the seed germination rate (De Britto et al., 2021). The biostimulant PSP1 formulated based on an elicitor protein from the fungus Sarocladium strictum demonstrated promising results for the germination potential of wheat seeds in field tests (Martínez et al., 2024).

Increasing the concentration of rBASIDIN used to 100 µg mL^-1 caused a decrease in the germination rate, both in the first count (four days) and in the second (seven days). This suggests that this dose may have caused a toxic effect on the seed embryo. Other researchers have reported the need for caution when choosing treatment concentrations for seed conditioning (Mahesh et al., 2017; Vázquez-Hernández et al., 2019; De Britto et al., 2021).

Therefore, our data indicate that priming of lettuce seeds with the M. perniciosa effector rBASIDIN at appropriate concentrations can induce physiological and biochemical changes that stimulate seeds to germinate faster and increase the germination percentage of Lactuca sativa cv (mimosa lettuce).

Seedlings emerging from seeds treaed with rBASIDIN at low concentrations showed more promising growth than control seedlings originating from seeds treated with distilled water and buffer. For seedling growth, a significant effect of seed pretreatment with the effector at a concentration of 50 µg mL^-1 was observed, with average shoot length of 0.72 cm and root length of 1.51 cm ( Figures 3A, B ). It is possible to hypothesize that metabolic events caused by rBASIDIN, at this concentration in the seeds triggered better root and shoot growth, by positively influencing the physiological quality of the seedlings, assuming that the seedlings that grow longer are more vigorous (Kalaivani et al., 2016).

In the present study, an increase in the dry and fresh mass of seedlings that developed from seeds treated with rBASIDIN was also observed, depending on the concentration (50 and 75 µg mL^-1). This shows that seedlings from these treatments can become more vigorous and productive, since in lettuce crops, fresh matter production is directly related to the plant’s leaf area (Moura Neto et al., 2022). Likewise, a comparable increase in dry and fresh mass parameters was observed in pepper seedlings after seed treatment with abiotic elicitors (Abhayashree et al., 2016).

The use of effectors in other studies has demonstrated positive effects on the parameters analyzed here. For example, the use of the harpin effector HpaXpm in Arabidopsis thaliana seeds promoted better root development and higher fresh mass content of the plants I comparison with the control treatment (Liu et al., 2020). However, our results demonstrate great potential of using rBASIDIN to obtain more vigorous and anatomically developed seedlings. However, a field study is necessary to verify whether these qualities remain in an uncontrolled environment.

The electrical conductivity test allows assessing the integrity of cell membranes when seeds are soaked in water. The lower the conductivity value, the better the seed vigor will be, indicating that the cell membranes are intact. On the other hand, a high result indicates that the seeds are deteriorated and have low vigor (Veronesi et al., 2014). Hence, this test can also be used to verify whether the application of pretreatment to seeds before sowing causes any damage, since it allows detecting the deterioration process (Vashisth and Nagarajan, 2010; Adetunji et al., 2021).

Thus, in the electrical conductivity (EC) analysis of lettuce seeds treated with rBASIDIN, we observed that for all concentrations of the effector used, there was a decrease in solute leakage in relation to the control treatments, thus presenting a lower EC at the concentration of 50 µg mL^-1 ( Figure 6 ). This result suggests that rBASIDIN does not damage the membrane integrity of lettuce seeds. The reduction of electrolytes in seeds treated with rBASIDIN is an important result since the physiological quality of seeds is influenced by the integrity of the cell membrane system. For example, Chomontowski and Podlaski (2020) reported that the application of solid matrix priming and zeolites to sugar beet seeds reduced the electrical conductivity and enhanced the germination capacity of the seeds four days after sowing. In addition, they found that seeds with low vigor had 58% higher electrical conductivity than seeds with high vigor.

In the treatments of lettuce seeds with rBASIDIN, an association between lower electrical conductivity and a higher germination rate at concentrations of 50 and 75 µg mL^-1 of rBASIDIN occurred. On the other hand, although at a concentration of 100 µg mL^-1 the seeds suffered lower electrolyte leakage in relation to the controls, they also had lower germination percentage. One hypothesis to explain this finding is that rBASIDIN at this concentration may negatively affect water absorption by seed tissues, hindering or preventing the start of the germination process and compromising radicle emergence (Chomontowski and Podlaski, 2020; Adetunji et al., 2021).

However, the results of the electrical conductivity test indicated that the protein effector rBASIDIN at concentrations of 50 and 75 µg mL^-1 does not impair the physiological quality of lettuce seeds.

The plant defense system can be activated by elicitors, and effectors can generate an elicitor response (Martínez et al., 2024). In plant biology, elicitors are considered extrinsic or foreign molecules that act at very low concentrations and induce defense responses in plants (Vázquez-Hernández et al., 2019). They can bind to specific receptor proteins located in plant cell membranes and are highly specific in the production of secondary metabolites (Lavanya et al., 2022). In some cases, effectors can generate a priming effect in plants, whereby a previous treatment can improve the plant’s response to future challenges. This activation process constitutes a type of biological memory, where the plant remembers the initial exposure to stress and uses this to strengthen its defenses (Yang et al., 2022). In view of this, we investigated the protective effect of the effector rBASIDIN when applied to lettuce seeds, evaluating the activity of enzymes related to plant defense.

The chitinases evaluated exhibited different behaviors according to the data shown in Figures 6A and B . For exochitinase activity, a statistically significant difference was observed in seedlings treated with rBASIDIN at a concentration of 75µg mL^-1 in relation to the other treatments, with activity approximately twice as great as the two controls used ( Figure 6A ). However, the endochitinase activity in the seedlings was negatively influenced by the controls when using rBASIDIN at concentrations of 75 and 100 µg mL^-1, and the other treatments did not differ from each other ( Figure 6B ). According to Kasprzewska (2003), the type of chitinase produced during resistance induction is closely related to the stimulus to which the plant is subjected, which leads us to believe that the type of chitinase evaluated may have varied based on the inducer used.

Chitinases belong to the category of pathogen response proteins, since they directly attack the structural components of insects and fungi (Shoresh and Harman, 2008). The type of action of exokinase is through the hydrolysis of chitin into oligosaccharides. Endochitinases cleave the chitin chain randomly, releasing a mixture of soluble oligomers predominantly diacetylchitobioses (GlcNac)₂ (Shoresh and Harman, 2008; De Oliveira et al., 2024).

There are reports that chitinase induction in plants is generally nonspecific and increases under biotic and abiotic stresses. In addition, they can accumulate gradually or systematically in other tissues (Vaghela et al., 2022). In this context, we suggest that the higher level of exochitinase in seedlings treated with rBASIDIN, and the statically equal increase in relation to the controls of the endochitinase enzyme in seedlings emerged from seeds treated with rBASIDIN at concentrations of 50 and 75 µg mL^-1 may increase the protection of the lettuce plants in case they suffers any biotic or abiotic stress. Plants that express both enzymes simultaneously are more resistant than plants that express only one of the enzymes at the same level, since the action of the two enzymes together has a synergistic effect that improves the plant’s defense (Bolar et al., 2001).

The activity of the enzyme β-1-3 glucanase was positively influenced in seedlings from seeds treated with rBASIDIN at a concentration of 75 µg mL^-1, with the other treatments being the same as the controls ( Figure 6C ). Increased β-1-3 glucanase activity in seedlings may indicate activation of defense mechanisms, since this enzyme is part of the group of proteins related to defense processes during pathogenesis. This enzyme acts by degrading the pathogen’s cell wall, limiting its spread (Perrot et al., 2022). Furthermore, this type of enzyme plays important roles in cell division, trafficking of materials through plasmodesmata and resistance to abiotic stresses, and is also involved in the formation of flowers until seed maturation (Balasubramanian et al., 2012). In this sense, seedlings of Pennisetum glaucum 7042S (susceptible cultivar) originating from seeds treated with different elicitors presented significantly higher β-1-3 glucanase activity compared to the controls, when inoculated with the pathogen Sclerospora graminicola (Sacc.) (Lavanya et al., 2022).

Although the main interest in β-1-3 glucanases is related to their possible role during plant-pathogen interactions, these enzymes are also involved in several physiological and developmental processes of plants, such as cell division, seed germination, embryogenesis, fruit ripening and the control of callose degradation in plasmodesmata (Perrot et al., 2022; Lyalina et al., 2023).

Another enzyme analyzed by us was catalase, an antioxidant enzyme responsible for removing excess reactive oxygen species (ROS) during stress and for breaking down peroxide into oxygen and water (Billah et al., 2021). In lettuce seedlings, catalase activity had a marked increase when rBASIDIN was applied to the seeds at a concentration of 100 µg mL^-1. Interestingly, excess ROS can compromise germination and seedling establishment (Soares et al., 2020), so increased activity of catalase may help maintain a strictly regulated concentration of ROS, to improve the adaptive response of lettuce seedlings grown from seeds treated with rBASIDIN. It is worth noting that even with the marked increase in the activity of the antioxidant enzyme catalase at this concentration, this occurrence demonstrated the inefficiency of this enzyme in the detoxification process of the seedling tissues, as evidenced by the reduction in the number of normal seedlings, decreasing the germination rate ( Figure 1 ).

Thus, we hypothesize that the ROS accumulation exceeded the scavenging potential of antioxidants and regulation of only a few components of the antioxidant system may not be sufficient to detoxify superoxide radicals. In this sense, it was shown that despite the high activity of superoxide dismutase and peroxidase enzymes, ROS concentrations (O^2- and H₂O₂) were considerably higher in in Choysum (Brassica rapa var. parachinensis) seedlings coming from seed treatment with NaCl (Kamran et al., 2021). This was associated with a sharp decline in germination rate, seed germination index and inhibition of seedling growth.

Therefore, because the rBASIDIN effector from seed treatment promotes the activation of the metabolic pathways of enzymes involved in the plant defense mechanism, these results are satisfactory and can be explored in lettuce protection induction programs.