**3. Leaf osmotic potential**

To determine the osmolality (c), 1 g of fresh weight from fully expanded leaves was homogenized in a mortar and mixed with distilled water to reach a final volume of 20 mL. After extraction using a millipore filter, the sap was utilized to determine

*Impact of Biofertilizers on Plant Growth, Physiological and Quality Traits of Lettuce… DOI: http://dx.doi.org/10.5772/intechopen.108710*

the osmolality using a freezing point osmometer (Gonotec Osmomat 030, Germany). The osmotic potential was determined using the following formula according to the Van't Hoff equation [18]:

$$\text{sys}\left(MPa\right) = -\text{c}\left(mos\,mol\,\text{kg}^{-1}\right) \times 2.58 \times 10^{-3} \,\text{J}$$

#### **3.1 Leaf relative water content**

Fresh, turgor, and dry weights of the leaf samples were determined. The relative water content of the leaves was calculated with the following formula:

*FW DW TW DWx* – / – 100

#### **3.2 Lettuce weight, leaf area, and its number at harvest**

The yield of lettuce is expressed as g plant−1. At the same time, the number of leaves per plant was recorded. Afterward, the leaf area was determined by leaf area meter (Li-3100, LICOR, Lincoln, NE, USA) and indicated as cm2 plant−1.

#### **3.3 Evaluation of dry matter and total soluble solids**

Dry weight (DW) was obtained in a forced-air oven at 70°C until constant weight. Dry matter (DM) was measured by weighting fresh (FW) and dried lettuce material and expressed in percentage (DM = 100 × DW/FW). Besides, total soluble solution (TSS) was measured with a digital refractometer and was expressed in percentages.

#### **3.4 Measurement of EC, pH of lettuce leaf**

The electrical conductivity (EC) and lettuce leaf pH were determined.

#### **3.5 Statistical analysis**

Data were exposed to ANOVA test using SAS-JUMP/7. In addition, Fisher's LSD test was used to compare the averages at a 5% significance level.

## **4. Results and discussion**

It was determined that lettuce weight was statistically affected by salt and biofertilizers. The lowest lettuce weight was obtained from salt (229 g) and then control (235 g) treatments (**Figures 2** and **3**). The heaviest lettuce was obtained from mycorrhiza (369 g) followed by microalgae (346 g). The biofertilizers decreased the salt effect and increased the lettuce weight. Compared to salty conditions, microalgae + salt, mycorrhiza + salt, and bacteria + salt applications increased lettuce weight by 19.2, 21.3, and 20.08%, respectively. Biofertilizers provided an increase in lettuce weight compared to control. Under without salt conditions, microalgae, bacteria, and mycorrhiza applications increased by 47.2, 30.6, and 57.2%, respectively (**Figure 3**). The fungal colonization, PGPR, and microalgae biofertilizers may stimulate the rate

#### **Figure 2.**

*Images of lettuce plants with the following applications: control (a), bacteria + salt (b), mycorrhiza + salt (c), microalgae + salt (d) 16 days before harvest (20 March 2020).*

of photosynthesis. Mycorrhiza may benefit plants by stimulating growth-regulating substances, increasing photosynthesis, improving osmotic adjustment under drought and salinity stresses, and increasing resistance to pests [19]. As cytokinin hormone

*Impact of Biofertilizers on Plant Growth, Physiological and Quality Traits of Lettuce… DOI: http://dx.doi.org/10.5772/intechopen.108710*

#### **Figure 3.**

*Effects of the biofertilizers on lettuce weight (g plant−1) under salt stress.*


*LSD; minimum significant difference, mean followed by the same letter in each column are not significantly different according to LSD test (probability level of 0.05).*

#### **Table 1.**

*The effect of biofertilizers on lettuce leaf number and dry matter under salt stress.*

production is a relatively common trait of PGPR and mycorrhizal fungi [20], cytokinin production may ameliorate salt stress. The cytokinins can enhance stomatal opening and photosynthesis. Stimulation of shoot biomass of lettuce plants grown in saline by the cytokinin-producing microorganisms implies considerable root-to-shoot cytokinin signaling [3].

Salt stress had a decreasing effect on the number of leaves in the lettuce plant. Compared to salt stress microalgae, bacteria and mycorrhiza applications increased the number of leaves by 9.3, 6.4, and 2.8%, respectively (**Table 1**). Under without salt conditions, the increasing effects of biofertilizers on leaf number were 26, 17, and 22% in microalgae, bacteria, and mycorrhiza, respectively.

The differences between the applications were found to be statistically significant for leaf dry matter. Compared to saline conditions, an increase in biofertilizer + salt applications has been achieved. Microalgae + salt, bacteria + salt, and mycorrhiza + salt increased the dry matter by 2.64, 8.05, and 18.4%. The higher plant dry matter accumulation with biofertilizers under salinity could be related to a higher source activity due to higher stomatal conductance and photosynthesis [21]. Beneficial microorganisms increase the production of cytokinins and they can enhance stomatal opening under salinity stress. Compared to control conditions, microalgae and bacteria applications increased dry matter production. Algal biofertilizer increased by 13.05% and bacterial biofertilizer increased by 9.8%. On the contrary, dry matter in lettuce leaves decreased in the mycorrhiza application may be due to faster growth.

The "L" represents brightness from the color parameters measured using a Hunter colorimeter (**Table 2**). The brightness values of lettuce plant increased in biofertilizer + salt applications compared to salty conditions. Increases of 23.01% were achieved in microalgae + salt application, 1.89% in bacteria + salt application, and 27.80% in mycorrhiza + salt application. When comparing control conditions and biofertilizer applications, increases in biofertilizer applications were determined. An increase of 58.53% was achieved in algae application, 49.43% in bacteria application, and 63.86% in the mycorrhizal application. Compared to saline conditions, an increase of 6.93% in microalgae + salt application, 7.40% in bacteria + salt application, and 9.82% in mycorrhiza + salt application was determined in "a" value. Kardüz et al. [22] found a significant effect of mycorrhiza application on the "a" value, which shows the green color of the lettuce leaves. Compared to the control, the "b" color value increased by 11.46, 1.83, and 8.85% in algae, bacteria, and mycorrhiza applications, respectively. According to saline conditions, an increase of 14.88, 23.75, and 11.34% was determined in microalgae + salt, bacteria + salt, and mycorrhiza + salt applications, respectively.

The highest EC value in lettuce leaves was 19.45 in bacteria + salt application and the lowest value was 8.32 in the microalgae application (**Figure 4**). Compared to saline conditions, 2.27 and 4.37% decreases in EC values were determined in microalgae + salt and mycorrhiza + salt applications, respectively. Compared to the control, decreases of 36.63, 33.96, and 12.03% were determined in the EC values of microalgae, bacteria, and mycorrhiza applications, respectively.

Lettuce leaves had the highest pH value of 5.95 in mycorrhiza and the lowest value of 5.81 in bacteria + salt application. Compared to the control, increases of 1.19 and 1.70% were recorded in the algae and mycorrhiza treatments, respectively. Compared to saline conditions, 1.35 and 0.68% decreases were recorded in microalgae + salt and mycorrhiza + salt applications, respectively (**Figure 4**). The salt stress decreased the TSS value. Lettuce leaves have the highest and lowest TSS of 5.60 and 2.73%, respectively, in control and mycorrhiza treatments.

TSS increases were recorded in biofertilizer + salt applications compared to a single biofertilizer application. Under salt stress, bacteria + salt was the biofertilizer application that showed the highest TSS value of 4.95% (**Figure 4**).

The highest acidity value in lettuce leaves was determined as 3.06% in mycorrhiza + salt application and 1.91% in microalgae application. Compared to saline conditions, 16.98, 40.56, and 44.33% acidity increases were recorded in microalgae + salt, bacteria + salt, and mycorrhiza + salt applications, respectively. Compared to the control, acidity decreases of 24.50, 47.82, and 2.37% were determined, respectively, in microalgae, bacteria, and mycorrhiza biofertilizers (**Figure 4**). TSS, titratable acidity, *Impact of Biofertilizers on Plant Growth, Physiological and Quality Traits of Lettuce… DOI: http://dx.doi.org/10.5772/intechopen.108710*

**Figure 4.** *The effect of biofertilizers on pH, EC, total soluble solids, and acidity of lettuce grown under salt stress.*


*LSD; minimum significant difference, mean followed by the same letter in each column are not significantly different according to LSD test (probability level of 0.05)*

#### **Table 2.**

*The effect of biofertilizers on lettuce leaf color "L," "a," and "b" values under salt stress.*

#### **Figure 5.**

*Effects of biofertilizers on osmotic potential of lettuce leaf under salt stress and control conditions.*

ascorbic acid, and dry matter were reported to be higher in the fruits of tomato plants inoculated with mycorrhiza than in those that were not inoculated [23].

The osmotic potential was found to be low in saline conditions (−0.52). Biofertilizer + salt combinations reduced this effect and increased the osmotic potential compared to saline conditions. The highest osmotic potential was obtained with −0.19 MPa in microalgae + salt application, followed by mycorrhiza with −0.22 Mpa and microalgae + salt applications with −0.23 Mpa. Biofertilizers had a stress-reducing effect under salinity and increased osmotic potential (**Figure 5**). Root colonization by AMFs can induce the production of the major groups of organic solutes and induce the accumulation of specific osmolytes, such as proline, soluble sugars, and amino acids [3].

Relative water content in the lettuce leaf was determined as the lowest in the salt application (68.5%) and the highest (85.6%) in mycorrhizal plants. Biofertilizers increased the relative water content and reduced stress in lettuce leaves under salt stress (**Figure 6**). Mycorrhizal plants generally show higher stomatal conductance and transpiration rates than non-mycorrhizal plants [24], even under salinity stress [25], this has been associated with improved leaf water status.

The lowest stomatal conductivity was determined in salt stress (115 mmolm−2s −1) and the highest (310 mmol mmolm−2s −1) in mycorrhizal biofertilizer alone. Biofertilizers increased the stomatal conductivity of lettuce leaves under salt stress and control conditions (**Figure 7**). Compared to saline conditions, salt + biofertilizer applications had a positive increasing effect on stomatal conductivity; microalgae + salt, bacteria + salt, and mycorrhiza + salt applications increased 63.8, 59.2, and 50.0%, respectively. Higher stomatal conductance and higher photosynthesis were reported under NaCl stress in pepper plants inoculated with PCPG [21]. Yao et al. [26] reported that PGPR prevented salinity-induced ABA accumulation in cotton seedlings. The ABA may mediate stomatal and photosynthetic responses to salinity stress [27], and the effects of plant-microorganism interactions on ABA status may enhance the growth of salinized plants.

*Impact of Biofertilizers on Plant Growth, Physiological and Quality Traits of Lettuce… DOI: http://dx.doi.org/10.5772/intechopen.108710*

**Figure 6.** *Effects of biofertilizers on relative water content of lettuce leaf under salt stress and control conditions.*

**Figure 7.** *Effects of biofertilizers on stomatal conductance of lettuce leaf under salt stress and control conditions.*
