Bacillus thuringiensis and Silicon Modulate Antioxidant Metabolism and Improve the Physiological Traits to Confer Salt Tolerance in Lettuce

We investigated the impact of Bacillus thuringiensis as seed treatment and application with silicon on lettuce plants exposed to salinity levels (4 dS m−1 and 8 dS m−1). Results revealed that leaves number, head weight, total yield, relative water content (RWC), and chlorophyll a and b declined considerably due to two salinity levels. Oxidative stress markers, i.e., hydrogen peroxide (H2O2), superoxide (O2−), and lipid peroxidation (MDA) dramatically augmented in stressed plants. On the other hand, leaves number, total yield, RWC, and chlorophyll a, b in stressed lettuce plants were considerably enhanced because of the application of Si or B. thuringiensis. In contrast, EL%, MDA, and H2O2 were considerably reduced in treated lettuce plants with Si and B. thuringiensis. In addition, the treatment with Si and B. thuringiensis increased head weight (g) and total yield (ton hectare-1), and caused up-regulation of proline and catalase, superoxide dismutase, peroxidase, and polyphenol oxidase activity in lettuce leaves under salinity conditions.


Introduction
Lettuce plants (Lactuca sativa L.) belong to the Asteraceae family. It is an important and popular vegetable crop grown as an annual plant, which is used as a salad crop. It is a rich source of vitamins and antioxidants such as vitamins C, carotenoids, and fiber content [1]. Some studies suggest that lettuce may have originated in the Mediterranean region [2], it is a very important commercial crop in North and Central America, Asia, Europe, and Egypt [3]. Japan, China, the U.S.A, Spain, Italy, and India are among the largest producers [4,5]. Environmental stress factors such as salinity [6][7][8][9], drought [10][11][12][13][14][15][16], chilling, and heat stress [17,18] negatively affect crop productivity in many plants. Salinity is an abiotic harmful stress factor, affecting structure, physiochemical characters, and the ecology of the soil, as well as the growth and plant yield [19][20][21]. The total area of saline and sodic soils was determined (831 million ha) worldwide according to the FAO report [22]. The salt-affected soils were divided into two main groups according to their physical, biological, and chemical properties [23]. The first group is saline soil, which contains large quantities of neutral soluble salts, mainly sodium chloride and sodium It was observed that lettuce plants exposed to salinity stress showed a considerable increase in MDA (pFW9 and Figure 1A). Plants treated with low (S1) and high (S2) levels of salinity resulted in 44% and 70% increases in MDA content compared with the control in the first year, and in the second year the increases were 44% and 87%, respectively. Conversely, MDA significantly declined in the stressed lettuce plants treated with B. thuringiensis and Si treatment. Si application significantly decreased MDA content in lettuce at a low salinity level (14% and 15%) compared with untreated stressed plants at a low level of salinity, 4 dS m −1 . Also, MDA decreased significantly with Si treatment at a high level of salinity 8 dS m −1 (S2; 21% and 20%) compared with untreated stressed plants at high level. B. thuringiensis led to decreased MDA and gave the best results in MDA content (29% and 30%) at the low level of salinity and (21% and 23%) at the high level, compared to the untreated plants at low salinity levels in both seasons.
A remarkable increase in super oxide (O 2 − ) and hydrogen peroxide (H 2 O 2 ) formation was observed in the exposure lettuce plants to both salinity levels. H 2 O 2 was significantly augmented in stressed lettuce plants ( Figure 1B) Additionally, the obtained results in Figure 1C showed that O 2 − dramatically increased in the stressed lettuce plants at a low level of salinity (138.6% and 160.59%) and at a high level (208. 16%   It is evident from the recorded results in Figure 1D that EL% considerably augmented in the lettuce stressed plants; the low salinity level led to a significant increase (56.14 and 63.04%) compared to control plants in both seasons. Moreover, the high level of salinity was more deleteriously effective on EL% (225.75% and 261.85%) compared with control treatment. However, Si application significantly declined EL% in the stressed lettuce plants at the low level (33.73% and 42.11%) and the high level (29.5% and 27.67%). Additionally, B. thuringiensis had a helpful effect and reduced EL% (55.13% and 55.27%) in the stressed plants with low salinity level in both seasons. Moreover, B. thuringiensis significantly declined EL% in the stressed treated plants at high salinity level (33.82% and 28.92%) compared with lettuce stressed untreated plants at high salinity level in both seasons.

Relative Water Content (RWC%) and Proline Content
The data in Figure 3A exhibited that RWC in lettuce plants was significantly declined under both levels of salinity, the low level led to significant reductions (36.96% and 37.18%) compared with control plants in the two seasons. Meanwhile, the decrease was higher under a high level of salinity (48.32% and 48.65%) compared with control treatments in both seasons. Contrariwise, Si application considerably increased RWC in stressed lettuce plants at the low level (28.78% and 33.93%) compared with untreated stressed plants in the two seasons, respectively. Furthermore, RWC was significantly increased with B. thuringiensis application and recorded the best results with a low salinity level (61.4% and 67.4%) and under a high salinity level (37.16% and 33.79%) in comparison with stressed untreated plants and the control treatment.
It is evident from the recorded results in Figure 3B that, proline content significantly augmented in lettuce plants under two salinity levels, the high level gave the high content of proline in lettuce plants (123.66% and 100%) compared to the control treatment in both seasons, respectively. Conversely, the application of Si led to the regulation and diminishing of the accumulation of proline in stressed plants with a low concentration of salinity (40.48% and 41.17%) compared with stressed untreated plants in both seasons; the greatest effect on proline content was achieved with B. thuringiensis as seed treatment (72.41% and 76.14%) in comparison with stressed untreated plants in the two seasons.

Ascorbic Acid (AsA)
The results presented in Figure 4 indicated a significant reduction in ascorbic acid (AsA) in lettuce under two salinity levels (35.71%, 51.39%, and 22.03%, 63.63%) in comparison with the control in both seasons, respectively. Conversely, application of Si and B. thuringiensis displayed a significant increase in ascorbic acid in salt-stressed lettuce plants. Si led to a significant increase in ascorbic acid in the stressed plants with salinity levels (54.52%, 59.32% and 31.25%, 52.27%) in the two seasons. Likewise, B. thuringiensis gave the maximum results of AsA particularly, with the low salinity level (67.85% and 55.25%) in both seasons, respectively, in comparison with untreated stressed plants and control also.

Chlorophyll A and B Concentration
Lettuce plants grown at the different salinity levels had significantly reduced chlorophyll a and b concentrations ( Figure 5A,B). A low salinity level led to significant reduction in chlorophyll a (41.66% and 42.85%) compared with the control in both seasons, while the decrease in chlorophyll a was greater under a high salinity level (55.6% and 105.7%) ( Figure 5A). Application of Si and B. thuringiensis induced the increase in chlorophyll a concentration, B. thuringiensis gave the maximum concentration of chlorophyll a in the stressed plants with low salinity level (70% and 84.21%) followed by Si treatment (62.5% and 65.78%) compared with untreated stressed plants.
It is noticeable in Figure 5B that chlorophyll b considerably declined in lettuce stressed plants; chlorophyll b significantly declined at a low level of salinity (45.36% and 42.85%) compared with the control treatment in both seasons. Additionally, chlorophyll b was significantly reduced at the high salinity level (60.82% and 55.02%) in the two seasons. Nevertheless, Si and B. thuringiensis led to remarkable increases in chlorophyll b. The best result (91.5% and 81.48%) was achieved with B. thuringiensis treatment in the stressed plants at low salinity level in comparison with the untreated stressed plants.

Number of Leaves, Head Weight and Total Yield
Considerable differences were recorded in number of leaves, head weight, and total yield of lettuce plants ( Figure 6A-C). Our results revealed that both salinity levels (4 dS m −1 and 8 dS m −1 ) caused a considerable reduction in the number of lettuce leaves in the first year (24.50% and 32.92%) and in the second year (27.73% and 35.71%) ( Figure 6A). Similarly, head weight was also significantly decreased (30.02%, 52.94% and 31.87%, 56.23%) compared to the control in each of the two seasons, respectively ( Figure 6B). Obtained results in Figure 6C

Discussion
In the current study, we provided evidence of Si exogenous application and B. thuringiensis seed treatment in the mitigation of salt stress effects on lettuce plants. The exposure of lettuce plants to two different salinity levels (4 dS m −1 and 8 dS m −1 ) showed harmful effects on number of leaves, head weight, and total yield. This damaging effect of salinity on these characteristics might be due the impact of salinity on decreasing water and nutrients uptake from the soil, decreasing the elongation of roots, and cell division in shoots, consequently decreasing growth and physiological characteristics [6,7], resulting in the reduction in morphological characters and total yield in lettuce plants. Also, salinity may induce ion toxicities such as high Na + , Cl − , or sulfate (SO 4 2− ) which decrease the uptake of essential elements like nitrogen, phosphorus, and potassium [52]. The increase in salinity concentration in soil may result in a reduction in water potential and affect many biochemical and physiological processes [53]. Similar results have been reported by Alomran et al. [54] and Kim et al. [55]. Contrariwise, B. thuringiensi and Si application led to improvements in number of leaves, head weight, and total yield in stressed lettuce plants. This positive effect of B. thuringiensi and Si could be due to the essential role of B. thuringiensis in mitigating the adverse effect of salinity by reducing ethylene and production of indole acetic acid by B. thuringiensis that not only enhanced root elongation but also increased NPK concentration in stressed plants [56]. In addition, Si application increased N and P uptake and could regulate the absorption and mobility of N in the plant, maintaining the optimum level of N and increasing N use efficiency [57], consequently improving the yield of stressed plants.
Ascorbic acid, chlorophyll a, b, and RWC decreased significantly according to the adverse effects of salinity. This unfavorable effect of salinity, mainly the high salinity level, could be due to the harmful impact of salinity on AsA and the reduction of secondary metabolites [58], decrease in energy transport from PSII to PSI [59], and adverse effect on the chloroplast structure and formation i salt stressed plants [3]. The injurious impact of salinity on chlorophyll was also due to the decrease in stomatal movements and the damage of many biological processes. These results are in harmony with those recorded by Abdelaal et al. [20] and Islam et al. [60] in stressed sweet pepper plants. Additionally, salinity adversely affects RWC, which could be due to the detrimental effect of salinity on the cell wall, increasing the synthesis of stress ethylene, which plays vital role in the loss of membrane stability and chlorophyllase activation, decreasing osmotic potential and water status, consequently decreasing RWC % [61,62]. The damaging influence of salinity on AsA, chlorophyll a, b, and RWC can be mitigated by silicon and seed treatment with B. thuringiensis. These results, perhaps due to Si, can play a supportive role in increasing the uptake and concentration of K + , as well as reducing Na+ uptake [17], which enhances enzyme activity and improves water status, photosynthesis, and RWC [63]. Similarly, the positive role of B. thuringiensis in increasing AsA, chlorophyll a, b, and RWC in stressed lettuce plants is perhaps due to its valuable effect on root growth and ability to increase water availability. Also, plant growth promoting rhizobacteria (PGPR) can produce exopolysaccharides which enhances the soil structure and increases the availability of soil water [64], improving physiological characters, especially chlorophyll a, b, and RWC, under stress conditions [65,66].
Salinity stress negatively influenced and significantly increased EL%, proline content and MDA. This negative impact on EL% is probably due to its injuring effect on the cell membrane and permeability process. Similar results were recorded by Abdelaal et al. [20]. Treatment of lettuce plants with Si and B. thuringiensis resulted in a decrease in EL%, proline content, and MDA. This synergistic impact of Si and B. thuringiensis treatment was probably due to the helpful roles of Si and B. thuringiensis in improvement of the membrane integrity and the permeability of the plasma membrane. Salinity stress has a deleterious effect on lettuce plants and increases proline content; proline was significantly augmented as a response to salinity, and this harmful influence of salinity might be due to a decrease in the oxidation of proline to glutamate, consequently increasing proline content [67]. Moreover, the application of Si and seed treatment with B. thuringiensis led to the adjustment of the osmotic balance and regulation of proline content in stressed lettuce plants. MDA considerably increased in lettuce plants under low salinity levels, a high increase was recorded with the high level of salinity, this increase could be due to the fact that MDA is one of the reactive compounds and a signal for many stresses, particularly salinity, and causes harmful effects to proteins, lipids, and the electron transport chain [68]. Contrariwise, the stressed lettuce plants indicated a significant reduction in MDA because of the application of SI and seed treatment with B. thuringiensis. This helpful effect in decreasing MDA could be due to the role of Si and B. thuringiensis in improving phenol content and enzyme activity, which protects proteins and lipids from oxidative stress and reduces MDA formation in the stressed lettuce plants. These results are in accordance with those recorded by Sharma et al. [69]. Also, Si application can decrease the tocopherol radical and oxidative damage in the plant cells, and consequently increase α-tocopherol, which may neutralize MDA content by decreasing reactive oxidative anions and helped to stabilize membrane integrity [70].
It is well known that antioxidant enzymes play a crucial role in plant tolerance to stress. In the present study, we found that CAT, SOD, POX, and PPO were accumulated significantly in the stressed lettuce plants, and this accumulation is an important approach that helps plants to deal with numerous stresses and as the main scavenging enzymes involved in ROS scavenging in several plants [20,34,36]. Moreover, application of SI and B. thuringiensis led to adjusted CAT, SOD, POX, and PPO activity in stressed lettuce plants. The significant effect of B. thuringiensis, perhaps due to the production of growth substances such as auxin, cytokinin, and nutrient availability, as well as the upregulation of essential enzymes [65]. Interestingly enough, Si application can activate the plant defenses by boosting the activity of enzymes CAT, SOD, POX, and PPO, whic, scavenge ROS and protect the cells from oxidative damage [71].
A very important signal under salinity stress conditions is ROS generation, which causes membrane disturbance and increases EL% and MDA. Under the two salinity levels, the excessive accumulation of O 2 − and H 2 O 2 was recorded in lettuce plants; this accumulation of reactive compounds can cause oxidative stress to lipids, proteins, and nucleic acids [65]. Also, the increase of reactive oxygen species was recorded under drought stress in sugar beet plants [72]. Nonetheless, the harmful impact of salinity was overcome by Si application and B. thuringiensis, which reflect a significant reduction in O 2 − and H 2 O 2 levels. Therefore, it is possible that seed treatment with B. thuringiensis can obstruct ROS formation by adjusting enzymatic and non-enzymatic antioxidants. Also, B. thuringiensis was reported to counteract the oxidative stress of ROS under saline conditions in sweet pepper and keep the cell membrane from being damaged [6]. Additionally, Si application led to mitigation of the harmful influence of salinity in lettuce plants and significantly decreased the levels of O 2 − and H 2 O 2 , because Si plays a vital role as an anti-stress compound [71,73]. In summary, salinity stress caused a reduction in number of leaves, head weight, total yield of lettuce plants, and increase in EL%, MDA, and reactive oxygen species. Nonetheless, the application of Si and seed treatment with B. thuringiensis led to mitigation of the harmful influence of salinity by increasing the photosynthesis process and RWC, and regulating proline content and enzyme activity in stressed lettuce plants.

Experimental Designe and Plant Materials
The experiments were performed in pots at Kafrelsheikh Univ., Botany Dept. during 2019/2020 and 2020/2021 seasons, to examine the effect of Bacillus thuringiensis MH161336 106-8 CFU/cm 3 as seed treatment and Si (potassium silicate at 2.7 mmol L −1 ) as foliar spray on lettuce under the different salinity levels (4 dS m −1 and 8 dS m −1 ). The biochemical and physiological characteristics were performed at the EPECRS Excellence Center and PPBL Lab., Kafrelsheikh University. The seeds of lettuce (Lactuca sativa L.) cv. SUSANA were arranged in three groups; one of them was treated with B. thuringiensis and the other two went without treatments. The seeds were surface sterilized for 5 min by sodium hypochlorite 2.5%, 70% ethanol for 1 min, and then washed with distilled water four times. B. thuringiensis MH161336, the final concentration of Bacterial cultures was 10 6-8 CFU/cm 3 [42], seeds were kept at room temperature for 6 h and then sown in trays in the nursery on the 17 th and 19 th of September in both seasons, respectively. The transplanting was done in pots 40 cm in diameter fifty six days after the sowing, each pot having two plants. The plants were divided into three collections (control, B. thuringiensis treatment, potassium silicate at 2.7 mmol L −1 ). Saline water with two salinity levels (4 dS m −1 and 8 dS m −1 ) from NaCl was used to irrigate the plants and the group with Si was treated with potassium silicate at 2.7 mmol L −1 twice, 15 and 30 days after transplanting. Fertilizer containing nitrogen, phosphorus, and potassium (NPK) (48:72:48 kg ha −1 ) was used in one dose before transplanting. The treatments were arranged in a completely randomized design with three replications and the samples were selected for morphological, biochemical, and physiological studies at 60 days from transplanting, while head weight and plant and total yield were calculated at 80 days from transplanting.

Electrolyte Leakage Assay (EL%)
Ten fresh leaf discs (1 cm 2 ) of lettuce were placed into bottles in 25 cm 3 deionized water. Bottles were shaken for 20 h, electrical conductivity was recorded for each, and then flasks were heated (80 • C) for 1 h, and the samples were shaken again (at 21 • C) for 20 h. Final conductivity was calculated for each flask. Electrolyte leakage % was calculated with the following formula: initial/final conductivity × 100 [76].

Enzymes Assay
Frozen lettuce leaves were used for protein extraction; the frozen leaves were ground in liquid nitrogen using ice cold mortar and pestle. Protein was extracted according to Bradford [77]. Enzyme activity was assayed in supernatant; CAT activity was assayed using spectrophotometer at 240 nm based on the rate of H 2 O 2 consumption as µmol min −1 mg protein −1 [78]. A SOD activity was measured at 560 nm as µmol min −1 mg protein −1 according to Giannopolitis and Ries [79]. Peroxidase (POX) activity was assayed as described by Hammerschmidt et al. [80]. Polyphenol oxidase (PPO) activity was assayed according to the method described by Malik and Singh [81].

Determination of Proline
Proline in fresh lettuce leaves was determined using a spectrophotometer at 520 nm and calculated as µg g −1 FW [83].

Determination of Ascorbic Acid
Samples of leaves were taken at the harvesting date to determine ascorbic acid (AsA) (vitamin C mg 100 g −1 FW) according to the Association of Official Analysis Chemists (A.O.A.C) [84].

Chlorophyll A and B Determination
The extraction from fresh leaves was prepared using N-N Dimethyl formamide and the chlorophyll a and b was determined at 647 and 664 nm using a spectrophotometer [85].

Morphological and Head Yield Characters
Number of leaves, head weight, and total yield were recorded.

Statistical Analysis
The results were statistically analyzed using ANOVA procedures [86] using the MSTAT-C statistical software package. Duncan was used to compare the means between treatments [87] when the difference was significant (p ≤ 0.05).

Conclusions
Our results offer insights into the possible efficiency of B. thuringiensis as a seed treatment and Si (potassium silicate at 2.7 mmol L −1 ) as a foliar application in the alleviation of the deleterious impacts of salinity in lettuce plants and to increase yield production. These treatments cause increases in the number of leaves, chlorophyll a and b, and RWC, as well as total yield under the salinity levels. On the other side, the stress signals such as ROS (O 2 − and H 2 O 2 ), MDA, and EL% were reduced considerably in stressed lettuce plants as a positive effect of these treatments. Therefore, we recommend the application of Si as a foliar application and B. thuringiensis as a seed treatment to mitigate the adverse impact of salinity on lettuce plants by improving the antioxidant system and improving plant production in the agro-biological system.