Impact of Biofertilizers on Plant Growth, Physiological and Quality Traits of Lettuce (Lactuca sativa L. var. Longifolia) Grown under Salinity Stress

Abstract

This study aims to reveal the responses of biofertilizers to the detrimental effects of salt stress on lettuce cultivation. Presidential variety lettuce seeds belonging to Syngenta company were used as plant material. Microalgae Chlorella vulgaris, beneficial bacteria, and mycorrhizal fungi are used to reduce salt damage in lettuce plants grown under salt stress. The experiment was carried out on eight different applications; (1) control, (2) salt (50 to 75 mM NaCl), (3) micro microalgae, (4) microalgae + salt, (5) bacteria (6) bacteria + salt, (7) mycorrhiza, (8) mycorrhiza + salt. The biofertilizers decreased the salt’s detrimental effects 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 increased pH, EC, total soluble solids, titratable acid, and total dry matter in lettuce leaves under salt stress. Biofertilizers had a stress-reducing effect under salinity and increased leaf osmotic potential, leaf water relative content, and leaf stomatal conductance. Microalgae Chlorella vulgaris, mycorrhiza, and beneficial bacteria are recommended as stress relievers when growing lettuce in saline agricultural soils or with saline irrigation water.

1. Introduction

When plants are exposed to adverse environmental conditions, such as nutrient deficiency, lack of water, low or high temperature, ultraviolet radiation, salinity, insufficient oxygen, heavy metal toxicity, diseases, and pests, their growth is adversely affected. This condition is called stress. Stress can last for a long time or be temporary for a short time. Agricultural productivity is decreasing due to the detrimental impacts of climate change. Therefore, in order to extend sustainable agriculture and to increase crop products for food in the world, it seems necessary to use the appropriate solutions to decline the negative effects of stresses on agricultural plants [1]. Salinity is one of the most important abiotic stress factors that adversely affects growth and development in plants, limiting yield and quality. The salinity-affected area is expected to reach about 50% of total agricultural land by 2050. Salinity stress generates various detrimental effects on plants’ morphological, physiological, biochemical, molecular, and agronomic characteristics and decreases productivity. Reduced plant growth under salinity stress is due to decreased nutrients, hormonal imbalance, generation of reactive oxygen species (ROS), ionic toxicity, and osmotic stress [2].

In recent years, improvements in beneficial microorganisms have raised the tendency to use biofertilizers as valuable tools in sustainable agriculture. Biofertilizers have various benefits for plant growth. They regulate the soil texture and activate the soil biologically. It has been reported that many biofertilizers suppress plant pathogens and protect the plant against soil-borne diseases, so they are known as environmentally friendly. In terms of agricultural sustainability, biofertilizers do not harm the ecological system and do not contain harmful substances, they are proportionally cheaper when compared to commercial chemical fertilizers. Biofertilizers stimulate plant growth and produce phytohormones, thus increasing the yield and quality of the plant. In the fight against salinity, biofertilizer applications are widely preferred all over the world because they significantly increase salt tolerance [3].

One of the most effective alternatives among biofertilizer applications is mycorrhiza. Mycorrhizal fungi, which have the ability to establish a symbiotic relationship with plant roots, take carbohydrates that they cannot synthesize from the plant itself and contribute to the ability of plants to take in more water and nutrients by expanding their root domain thanks to their hyphae [4, 5]. It has been reported that the positive effect of mycorrhiza is not only to increase the intake of water and nutrients but also to increase the tolerance of plants to abiotic and biotic stress conditions [4, 6, 7]; mycorrhiza and beneficial bacteria have taken their place in the biofertilizer industry in recent years. The effectiveness of these fertilizers has positive effects on the nutrition of the plants by increasing the solubility of nutrients in the root area, with benefits, such as lowering the pH in the root zone, secretion of chelators, production of special ion carrier proteins [8, 9, 10, 11, 12]. While the solubility and availability of nutrients, such as phosphate, Fe, Zn, and Mn, increase by decreasing the pH in the root zone, some bacteria also fix the nitrogen to the soil from the air. It is reported that PGPR (plant growh promoting rhizobacter) bacteria that promote plant growth produce hormones, fix nitrogen in the air, and dissolve phosphate [13].

Chlorella vulgaris, one of the microalgae species with the highest biotechnological applicability, has been widely commercialized and is used as a food supplement for humans and as a feed additive for animals. These algae, a member of Chlorophyta, is seen as an alternative protein source due to their high protein content of 42–58% and has been cultivated for various purposes by many countries [14]. Instead of chemical fertilizers, which are generally used as a nitrogen source in agriculture, the use of C. vulgaris with high protein content will be a cheaper and environmentally friendly application. However, studies on the use of microalgae as biofertilizers, both in the world and in our country, are limited.

The origin of lettuce is accepted as Anatolia, Caucasus, and Turkestan regions. Some researchers stated that different forms of salad and lettuce are found in central Europe and southern Europe and the Canary Islands, some African countries, Mesopotamia, Kashmir, Nepal, and even Siberia [12]. Its Latin name “Lactuca sativa L. var. longifolia” was used in this study. It is also called Romain lettuce or Cos lettuce. This lettuce is a species whose leaves are longer than wide, the leaves overlapping each other and often forming a loose and oval core.

The aim of this study is to determine the effect of microalgae, bacteria, and mycorrhiza biofertilizers on plant growth, yield, and plant nutrient content of lettuce grown under salt stress. It also revealed the effects of using less chemical fertilizers in lettuce cultivation, thus saving fertilizer and protecting the environment, as well as the yield and quality of the plant.

2. Material and method

2.1 Plant material and growing conditions and experimental design

The present study was conducted in a glasshouse between the spring and the summer of 2019 at the University of Cukurova, Adana, Turkiye (36°59’N, 35°18’E, 20 m above sea level). Lettuce seeds (Lactuca sativa L. var. longifolia “Presidental”) were provided by Syngenta seed company. Seed sowing was done on 28 October 2019 and seedlings were planted on 13 December 2019 (Figure 1). The lettuce plant was grown in a two-liter capacity pot filled with cocopeat substrate and irrigated nutrient solution. Lettuce plants were grown with eight treatments and three replications. Randomized blocks experimental design, with 10 plants in each replication (30 plants per treatment) was used. The treatments are as follows:

1. Control

2. Salt

3. Microalgae

4. Microalgae + Salt

5. Bacteria

6. Bacteria + Salt

7. Mycorrhiza

8. Mycorrhiza + Salt

Figure 1.

Lettuce seedlings were transferred to pots containing cocopeat.

The pH and EC of the nutrient solution during the trial were measured daily. The pH was kept between 5.5 and 6.0, and the EC was fixed at 1.5 and 2.7 μS cm⁻¹. Two stock solutions for nutrition were used: stock A (Potassium nitrate, calcium nitrate, ammonium nitrate, and Fe-EDDHA) and stock B (potassium sulfate, mono-potassium sulfate, magnesium sulfate, microelements, zinc sulfate, boric acid, manganese sulfate, and ammonium molybdate). The nutrient solution consists of nitrogen (N) 212 ppm, phosphorus (P) 30 ppm, potassium (K) 305 ppm, calcium (Ca) 205 ppm, magnesium (Mg) 60 ppm, Iron (Fe) 3.0 ppm, manganese (Mn) 0.4, Boron (B) 0.40 ppm, Zinc (Zn) 0.50 ppm, copper (Cu) 0.05 ppm, and molybdenum (Mo) 0.07 ppm. The first salt application of 50 mM NaCl was made on February 28, 2020. The salt concentration was increased to 75 mM NaCl on 20 March 2020. Lettuce plants were harvested on April 6, 2020.

2.2 Biofertilizers

“Chlorella vulgaris” strain of microalgae was used in 2×10⁷ mL⁻¹ concentration. The inoculation was diluted 40 times before its use [16]. For 1 L nutrient solution, 25 mL of algae was added from the 2×10⁷ mL⁻¹ concentration. Three bacterial species (Bacillus subtilis, Bacillus megaterium, and Pseudomonas fluorescens) were obtained from NGB Company (Next Generation Biotechnology) with a trading name “Rhizofill.” A mixture of 50 mL of the colony (1×10⁹ mL⁻¹) was added every 10 days in a 50 L nutrient solution. The commercial mycorrhiza was obtained from ERS Company (Bioglobal). The mixture was composed of Glomus intraradices, Glomus aggregatum, Glomus mosseae, Glomus clarum, Glomus monosporus, Glomus deserticola, Glomus brasilianum, Glomus etunicatum, and Gigaspora margarita (1×10⁴ w: w). During seed planting, 1000 spores’ plants⁻¹ were used.

2.3 Leaf stomatal conductance

During the experiments, by using Delta T Devices brand AP4 model portable porometer, the gas passing from stomas in mature leaves was recorded [17].

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 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]:

$$\psi s\left(MPa\right)=-\text{c}\left(mosmolk{g}^{-1}\right)\times 2.58\times {10}^{-3}.$$

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:

$$FW–DW/TW–DWx100$$

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 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 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].

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).

Figure 3.

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

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.

Treatments	Leaf number per plant	Dry matter (%)
Control	37.00 de	6.97 c
Salt	36.00 e	7.95 b
Microalgae	45.33 a	7.88 c
Microalge + salt	39.33 cd	8.16 b
Bacteria	42.00 bc	7.66 bc
Bacteria + salt	38.33 de	8.59 ab
Mycorrhiza	44.00 ab	5.19 d
Mycorrhiza + salt	37.00 de	9.42 a
P	0.0001	0.0001
LSD0.05	2.768	0.926

Table 1.

The effect of biofertilizers on lettuce leaf number and dry matter 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).

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.

Treatments	L	A	B
Control	32.93 e	−11.19 ab	39.87 de
Salt	37.94 d	−12.83 c	37.63 e
Microalgae	52.20 ab	−10.59 a	44.44 ab
Microalgae + salt	46.67 c	−11.94 b	43.23bc
Bacteria	49.21 a-c	−11.16 ab	40.60 cd
Bacteria + salt	38.66 d	−11.88 b	46.57 a
Mycorrhiza	53.96 a	−11.91 b	43.40 bc
Mycorrhiza + salt	48.49 bc	−11.57 b	41.90 b-d
P	0.0001	0.0021	0.0004
LSD0.05	4.796	0.828	2.965

Table 2.

The effect of biofertilizers on lettuce leaf color “L,” “a,” and “b” values 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)

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.

Figure 4.

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

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, 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].

Figure 5.

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

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.

Figure 6.

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

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.

Figure 7.

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

5. Conclusion

The biofertilizers decreased the salt’s detrimental effects 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 increased pH, EC, total soluble solids, titratable acid, and total dry matter in lettuce leaves under salt stress. Biofertilizers had a stress-reducing effect under salinity and increased leaf osmotic potential, leaf water relative content, and leaf stomatal conductance. Microalgae Chlorella vulgaris, mycorrhiza, and beneficial bacteria are recommended as stress relievers when growing lettuce in saline agricultural soils or with saline irrigation water.

Conflicts of interest

The authors declare that they have no conflicts of interest.

Thanks

The authors thank us for using the greenhouse and laboratory facilities of the Department of Horticulture, Faculty of Agriculture, Çukurova University.