Possibility of overcoming salt stress of lettuce plants using humic acid and mycorrhiza

INTRODUCTION

Edible crisped lettuce (Lactuca sativa var. capitata L. 'Calmar') belongs to the family Asteraceae, or Compositae. Lettuce was cultivated by the ancient Egyptians, Greeks and Romans and were widely spread to every continent and is grown everywhere except in the hottest tropical lowlands (Shannon and Grieve, 1999). The harvested area all over the world is 472552 fed. and the world production is 45449152 tones (FAOSTAT, 2019(. In Egypt, production of lettuce is 203510 tones and the total area grown was 9592.8 fed. for lettuce and chicory (FAOSTAT, 2019).Lettuce is one of the most commonly consumed vegetables worldwide, but its nutritional value has been underestimated. Lettuce is low in sodium, fat and calories. It is a good source of iron, folate, vitamin C and fiber. Lettuce is also a good source of various other health-beneficial bioactive compounds (Kim et al., 2016). However, lettuce is a salt moderately-sensitive crop where salinity affects its quality, yield, and production (Grieve et al., 2012 (.

Soil salinity is one of the major abiotic stresses that hamper crop growth and productivity worldwide. It has been stated that approximately 20% of irrigated land worldwide is salt-affected, which represents one-third of food-producing land (Gregory et al., 2018). Moreover, the salt-affected areas are increasing at a rate of 10% yearly for various reasons, including low precipitation, high surface evaporation, poor cultural practices, and irrigation using saline water (Shrivastava and Kumar, 2015). This issue has been further aggravated by the continued trends in global warming and climatic changes. Therefore, living with salinity is the only way of supportive agricultural production in the salt affected soil. So that, it is must to finding the best management to alleviate salt hazard (Al-Rawahy et al. 2011).

In current years, exogenous protectants such as osmoprotectants, phytohormones, humic compounds, antioxidants and mycorrhiza have been found useful to alleviate the salt-induced damages (Khan et al., 2019). The development of methods and strategies to ameliorate the harmful effects of salt stress on plants has received considerable attention (Senaratna et al., 2000).

Humic acids are rich in mineral nutrients like potassium, calcium, magnesium, zinc, iron, copper and organic acid (Tahir et al., 2011; Canellas et al., 2015).  Photosynthetic activity of lettuce improved with all levels of humic acid due to enhancement of chlorophyll content and mesophyll conductance. Humic acid can stimulate N metabolism and photosynthesis activity of lettuce to improve yield (Haghighi et al., 2012). Aydin et al. (2012) indicated that adding Humic acid treatment to bean plants has great potential in alleviating salinity stress on plant growth in saline soils of arid and semi-arid areas and appeared to be highly effective for soil conditioners in vegetable growth, to improve crop tolerance and growth saline conditions and enhanced plant root and shoot dry weight by allowing nutrients and water to be released to the plant as needed.  Also, El-Hamdi et al. (2016) indicated that humic acid exhibited a protective effect against salinity stress that increased fresh and dry weight and improving physicochemical and biological properties of saline soils.

Arbuscular Mycorrhizal Fungi -inoculated plants develop better than non-inoculated plants under salt stress, according to several studies. (Al-Karaki, 2000; Cantrell and Linderman, 2001; Giri et al., 2003; Sannazzaro et al., 2007; Zuccarini and Okurowska, 2008). Arbuscular Mycorrhizal Fungi are soil-borne fungi that can increase resistance to several abiotic stress factors and significantly improve plant nutrient uptake (Sun et al., 2018).  Arbuscular Mycorrhizal Fungi have the capability to improvement the uptake of inorganic nutrients in almost all plants, specifically of phosphate (Smith et al., 2003; Nell et al., 2010).

 The present study therefore, was conducted to evaluate the potential of humic acid and mycorrhiza, to alleviate the salt-induced deleterious effects on growth and chemical characteristics of lettuce under the environmental conditions of El-Behera Governorate.

MATERIALS AND METHODS

Two pots experiments were conducted at Ahmed Rami village, El-Bostan, EL- Beheira Governorate, Egypt, during the successive winter seasons of 2018 and 2019 to investigate the effect of humic acid and inoculation with vesicular-arbuscular mycorrhizal (VAM) fungi under different salinity levels on the growth and chemical properties of Crisp head lettuce, iceberg, (Lactuca sativa var. capitata L. 'Calmar'). The physical and chemical analyses of the soil (Table 1) were carried out before transplanting according to Black (1965).

Table (1): Some Physical and chemical properties of the experimental soil.

The lettuce, iceberg seeds, cv. Calmar produced by Nelson Garden seed company based in Sweden, were purchased from a local seeds market, and sown in plastic pots (35 cm inner diameter, and 30 cm height), each pot was filled with 12 kg of soil, and placed in the open field. The seeds were planted on 10 and 13 of October 2019 and 2020, respectively. Each treatment was composed of five replicated pots with four plants in each pot. Each experiment includes 24 treatments which were the combinations between four salinity levels (Tap water, 2.0, 4.0 and 6.0 mS/cm) and six treatments; i.e., five protecting treatments: humic acid at 1.5 g / L, humic acid at 3.0 g / L, inoculation of mycorrhizal (VAM), (humic acid at 1.5 g / L + mycorrhiza), (humic acid at 3.0 g / L + mycorrhiza) in addition to the control treatment (distilled water). The source of humic acid is potassium humate.

Mycorrhizal (VAM) root colonization: the process of root infection takes place at the age of 20 days from planting. All VAM fungal structures (hyphae, arbuscules, and vesicles) found in the roots were counted. Stained root pieces were examined under a dissecting scope at 40X magnification and extent of colonization was assessed by the grid-line intercept method (Giovannetti and Mosse, 1980). The recommended concentrations of adding treatments were applied as a soil application. All precautions were followed during weighing, dissolving and adding. Each treatment was applied three times after planting. The first application was conducted in the three specific leaves phase (20 days) after sowing and the others were applied with one-week intervals (Smoleń and Sady 2012; Fouda, 2016). Harvesting was done after 50 days of planting during both seasons. All experimental pots received identical levels of nitrogen, phosphorus and potassium fertilizers. Ammonium nitrate (33.5% N) at the rate of 60 kg N/fed. was equally divided and side dressed after 21, 28 and 35 days after planting, Calcium super phosphate (15.5 % P₂O₅) at the rate of 150 kg P₂O₅ /fed. was base dressed before planting and potassium sulphate (48 % K₂O) at the rate of 50 kg K₂O /fed. was equally divided and side dressed after 21 and 28 days of planting. All other agricultural practices were adopted whenever they were necessary and as commonly recommended for the commercial production of lettuce, iceberg.

The layout experiment was split plots system in a Randomized Complete Blocks Design (RCBD) with three replications. The salinity levels were arranged in the main plots and the protecting treatments of humic acid, mycorrhiza and their combination, were considered as sub- plots.

Data Recorded

      Vegetative growth parameters and chlorophyll contents

Lettuce plants were harvested after 55 days from transplanting and the measurement of vegetative growth parameters were performed immediately. Three lettuce plants from each treatment were randomly taken to measure:

Number of leaves per plant; It was estimated as an average of the selected plants.

Plant fresh weight (g);The whole plant sample was weighted and the average weight plant^-1(g) was calculated.

Plant dry weight (g); The collected plant samples were oven dried at 70 Cº in a forced air oven till obtaining a constant weight to obtain shoots dry weigh (g plant^-1) and the dried tissues were ground for further analysis., then the percentage of dry matter was calculated.

Total leaf chlorophyll contents (SPAD index); were measured using spad-502 chlorophyll meter devise (Konica Minolta, Kearney, NE, USA).

Head weight(g); was determined as the weight of the three selected randomly edible heads.

Root characteristics

Root length (cm); it was measured for plant samples randomly taken, and the average root length (cm) was calculated.

Root fresh weight (g); The whole fresh root sample was weighted and the average root weight (gm) was calculated

Root dry weight (g); The collected fresh root samples were oven dried at 70 C^º in a forced air oven till obtaining a constant weight to obtain root dry weigh (g).

Chemical contents

After harvest, chemical contents were achieved immediately.

Total nitrogen was determined calorimetrically according to Evenhuis and De Waard (1980). Phosphorus was determined using ammonium molybdate stannous chloride method (A.O.A.C, 1992). Potassium and sodium were measured using a flame photometer as explained by Singh et al. (2005). Then the ratio of K/Na was calculated. Calcium was determined by using the versinate titration method, as described by  Johnson and Ulrich (1959).

Statistical analysis

      All the obtained data were statistically analyzed by CoStat program (Version 6.4, Co Hort, USA, 1998–2008). Least significant difference (LSD) test was applied at 0.05 level of probability to compare means of different treatments according to Williams and Abdi (2010).

RESULTS AND DISCUSSION

       The results of the two studied factors and their interaction on the several characters of lettuce plants during 2019 and 2020 seasons can be presented below three titles as follow: 1- Vegetative growth characters and root characteristics.  2- Chemical contents. 3- Chlorophyll and protein contents.

Mean performances of vegetative growth characters and root characteristics of lettuce

         Data presented in Tables (2 and 3) indicated that all values of the tested parameters decreased as salinity levels increased. The highest values of the given parameters were obtained from control treatment, whereas that of 6.0 mS/cm salinity gave the lowest ones, in both seasons. At salinity of 6.0 mS/cm, the estimated percentage reductions, expressed as leaves number, head weight, total fresh weight, head dry weight, Plant dry weight, root length, root fresh weight and root dry weight, were (51.76 and 51.24 %), (59.12 and 59.41 %), (61.38 and 61.61 %), (60.36 and 60.62%), (61.11 and 61.15 %), (46.63and 46.28 %), (80.66 and 80.62%) and (64.60 and 63.66%) compared to the control treatment in the first and second season, respectively.  The adverse effects of high salinity on plants are connected to the subsequent factors: (1) low water potential of soil solution (water stress), (2) nutritional imbalance and disturbing ionic homeostasis (ionic stress), (3) specific ion effect (salt stress), (4) over-production of reactive oxygen species  (oxidative stress) (Parvaiz and Satyawati, 2008; Hasanuzzaman et al., 2013).Salinity stress is known to retard plant growth through its effect on several dynamic factors of plant metabolism, including osmotic adjustment (Sakr and El-Metwally, 2009) nutrient uptake, protein and nucleic acid synthesis, photosynthesis (Zaibunnisa, et al., 2002), organic solute accumulation, enzyme activity, hormonal balance and reduced water availability at the cell level and then reduced plant growth and finally reduced yield. Meanwhile, the effect of soil salinity on lettuce yield was significant. Maximum yield was obtained from the control treatment, and lettuce yield decreased as soil salinity increased (Ünlükara et al., 2008, and Silva et al.,2019). Also, in spinach, the tested characters of plant height, plant fresh weight, plant dry weight, number of leaves per plant, root length, root fresh weight and root dry weight decreased with increasing salinity levels. The reduction rate on any character varied depending on the imposed level of salinity stress (Gabr et al.,2022). Furthermore, in tomato, increasing salinity was accompanied by significant reductions in shoot weight, root length, and root surface area per plant (Mohammad et al., 1998). In spinach, the fresh shoot and dry matter of spinach was affected negatively by salinity (Sheikhi and Ronaghi., 2012; and Ünlükara et al., 2017). Likewise, Kaya et al (2001) found that salinity significantly decreased spinach shoots fresh weight and dry weight, leaf relative water content, and specific leaf area compared to control treatment. The growth reduction was produced by the osmotic effect of salt outside the roots, and following growth reduction was caused by the inability to prevent salt from reaching toxic levels in transpiring leaves (Hniličková, et al., 2019).

Concerning the main effect of the protection treatments (humic acid and mycorrhiza) on the leaves number, head weight, total fresh weight, head dry weight , Plant dry weight,  root length, root fresh weight and root dry weight of lettuce plants, results existing in Tables (2 and 3) revealed that addition of humic acid and mycorrhiza showed significant effect in most studied characters, except root dry weight on  humic acid at 1.5 g in the first season only, compared to the control treatment, in both seasons. For example, application of humic acid at 3.0 g / L + mycorrhiza recorded, generally, the highest average values of leaves number, head weight, total fresh weight, head dry weight, Plant dry weight, root length, root fresh weight and root dry weight compared to the other treatments, in both seasons. However, the differences between the three treatments (mycorrhiza), (humic acid at 1.5 g/ L + mycorrhiza) and (humic acid at 3.0 g / L + mycorrhiza) were not significant in head weight, total fresh weight and head dry weight in the first season, in addition leaves number and head dry weight in the second season. Moreover, the differences between humic acid at 3.0 g/L + mycorrhiza and both (humic acid at 1.5 g + mycorrhiza) and mycorrhiza in most cases were not significant.  This specific treatment (humic acid at 3.0 g / L + mycorrhiza) the estimated percentages increase in leaves number, head weight, total fresh weight, head dry weight, Plant dry weight, root length, root fresh weight and root dry weight were (42.92+39.37 %), (22.81+22.27 %), (23.03+22.85 %), (25.57+25.67 %), (27.35 and 28.22%), (35.61 and 39.86 %), (25.57 and 29.62 %) and (36.56 and 42.05%) compared to the control treatment in the first and second season, respectively. The current results could be attributed to the role of each ameliorative material. In this respect, Humic acids are rich in mineral nutrients like potassium, calcium, magnesium, zinc, iron, copper and organic acids (Tahir et al., 2011; Canellas et al., 2015).  Humic compounds have been shown to stimulate plant growth in terms of increasing plant height and dry or fresh weight as well as enhancing nutrient uptake (Malan, 2015). Humic substances can improve nutrient applications, increase chlorophyll production, improve seed germination, enhance fertilizers, and ultimately strengthening plants (Kandil et al., 2020). Thus, the plants that grow in soils containing adequate amounts of humic acid are less stressed because humic substances are an important part of soil organic matter and shows anti-stress effects (Hanafy et al, 2010). Humic acid application as well significantly increased the head weight of lettuce (Türkmen et al., 2004). Sandepogu et al., (2019) found that humic acid increased fresh and dry weight of lettuce and spinach plants.  El-Hamdi et al. (2016) indicated that humic acid exhibited a protective effect against salinity stress. Applications of humic acid produced significant increases in shoot length, root length, number of leaves, fresh weights of stems and roots and dry weights of stem and root of pepper (Capsicum annuum) plants grown under salt stress as compared with untreated plants (Akladious and Mohamed 2018). The humic acid advanced nutrient uptake, photosynthetic pigment concentrations and yield of chicory (Gholami et al., 2019).

Mycorrhization, has been shown to improve the host plant's fitness by increasing its growth and biomass. Arbuscular Mycorrhizal Fungi -inoculated plants develop better than non-inoculated plants under salt stress, according to several studies. (Al-Karaki, 2000; Cantrell and Linderman, 2001; Giri et al., 2003; Sannazzaro et al., 2007; Zuccarini and Okurowska, 2008).  The large hyphae of the fungus allow them to explore greater soil volume than non-mycorrhizal plants, allowing them to raise P concentration in plants by enhancing its uptake (Ruiz-Lozano and Azco'n, 2000). Increased P uptake by AMF in saline-grown plants may reduce the negative effects of Na+ and Cl^- ions by maintaining vacuolar and selective ion intake (Rinaldelli and Mancuso, 1996), preventing ions from interfering with growth metabolic pathways (Cantrell and Lindermann, 2001). According to Al-Karaki (2000), a mycorrhizal tomato plant had higher shoot and root dry weight, fresh fruit yield, fruit weight, and fruit quantity than a non mycorrhizal tomato plants. When Cucurbita pepo plants colonized with Glomus intraradices were exposed to salinity stress, Colla et al. (2008) found better growth, yield, hydration status, nutrient content, and quality of fruits. Cantrell and Linderman (2001) reported that dry shoot masses of VAM lettuce and onion plants were significantly greater than those of non-VAM plants. In addition, Santander et al. (2019) in lettuce, reported that the mycorrhizal plants had higher biomass production, increased synthesis of N uptake, and clear changes in ionic relations, particularly reduced accumulation of Na⁺, than those non-mycorrhizal plants under stress conditions.

Concerning the interaction effect between salinity levels and the ameliorative treatments (humic acid and mycorrhiza) on leaves number, head weight, total fresh weight, head dry weight, Plant dry weight root length, root fresh weight and root dry weight of lettuce plants, results presented in Tables (2 and 3) revealed significant differences among the means of their interactions between both variables. In general, the combination between zero salinity and humic acid at 3.0 g / L + mycorrhiza inoculation reached the highest average values of the above aforementioned characters in both seasons compared to other tested treatments.

Table (2): Mean values of lettuce vegetative growth characters as affected by salinity levels and soil application of humic acid and mycorrhiza and their interaction during winter seasons of 2019 and 2020.

*Means having the same alphabetical letter (s) in column, within a comparable group of means, do not significantly differ, using the revised L.S.D. test at p = 0.05 level of probability.

**H₁=Humic acid at 1.5 g/L, H₂= Humic acid at 3.0 g/L, M= Mycorrhiza.

Table (3): Mean values of lettuce root characteristics as affected by salinity levels and soil application of humic acid and mycorrhiza and their interaction during winter seasons of 2019 and 2020.

Means having the same alphabetical letter (s) in column, within a comparable group of means, do not significantly differ, using the revised L.S.D. test at p = 0.05 level of probability.

**H₁=Humic acid at 1.5 g/L, H₂= Humic acid at 3.0 g/L, M= Mycorrhiza.

Mean performances of chemical contents of lettuce

        Concerning the main effect of salinity levels on the percentages of nitrogen, phosphorus, potassium, calcium, sodium, potassium/sodium ratio, results are presented in Table (4) shown that most tested parameters decreased as salinity levels increased, except for sodium percentages which increased as salinity levels increased. The reduction rate on any character varied depending on the level of imposed salinity stress. The highest values of the given parameters (except sodium) were obtained from control treatment, while that of 6.0 mS/cm salinity gave the lowest ones, in both seasons. At salinity of 6.0 mS/cm, the estimated percentage reductions, expressed as nitrogen, phosphorus, potassium and calcium, were (24.35 and 19.33%), (40.00 and 36.21 %), (26.11 and 23.32%) and (24.22 and 24.26 %) for the first and second seasons, respectively and relative to the control treatment. The current results were in harmony with several investigators (Rogers et al. 2003; Hu and Schmidhalter 2005) who reported that nutritional disorders may result from the effect of salinity on nutrient availability, competitive uptake, transport, or distribution within the plant. Also, numerous reports indicated that salinity reduces nutrient uptake and accumulation of nutrients into the plants and it can differently affect the nutrient concentrations in plants depending upon crop species and salinity levels (Oertli, 1991). Salinity can decrease N accumulation in plants (Feigin et al., 1991; Pessarakli, 1991and Al-Rawahy et al., 1992). Similarly, salinity could reduce nitrogen accumulation in plants. Decreased N uptake under saline situations occurs due to interaction between Na⁺ and NH⁴⁺ and/or between Cl⁻ and NO³⁻ that ultimately reduce the growth and yield of the crop. This reduction in NO₃⁻ uptake is connected with Cl⁻ antagonism or reduced water uptake under saline conditions (Lea-Cox and Syvertsen, 1993; Rozeff, 1995; Bar et al., 1997). Examples of such an effect have been found in cucumber, (Martinez and Cerda, 1989), melon, (Feigin et al., 1987), tomato (Martinez and Cerda, 1989). Spanish (Gabr et al. 2022).  Many attributed this reduction to Cl⁻ antagonism of NO₃⁻ uptake (Bar et al., 1997; Feigin et al., 1987; Kafkafi et al., 1982) while others attributed the response to salinity's effect on reduced water uptake (Lea-Cox and Syvertsen, 1993). High salinity can increase the uptake of Na⁺ and Cl^- from the soil, therefore suppressing the transport of other essential nutrients such as N, P, K, and Ca (Shrivastava and Kumar, 2015; Safdar et al., 2019). Kim et al., (2021) found that increasing NaCl concentration decreased amounts of minerals (K⁺, Ca²⁺, and Fe²⁺) in leaves of spinach and changed ratios of Na⁺: K⁺ and Na⁺: Ca²⁺.  Qadir and Schubert (2002) showed that availability of phosphorous is reduced in saline soil due to (a) ionic strength effects that reduced the activity of PO₄³⁻, (b) phosphate concentrations in soil solution was tightly controlled by sorption processes, and (c) low solubility of Ca-P minerals. Hence, it is noteworthy that phosphate concentration in agronomic crops decreases as salinity increases.

       Under saline conditions, external Na⁺ not only interfere with K⁺ acquisition by the roots, but also may disrupt the integrity of root membranes and alter their selectivity. The selectivity of the root system for K⁺ over Na⁺ must be sufficient to meet the levels of K⁺ required for metabolic processes, for the regulation of ion transport, and for osmotic adjustment (Marschner, 1995). However, potassium concentrations in salt-stressed plants depend on whether the source of nitrogen fertilization is NH₄⁺ or NO₃⁻ as K⁺ uptake by cucumber seedlings salinized with NaCl was inhibited by the combination of both NH₄ and NO₃ but stimulated by NO₃⁻ alone; this response may be primarily associated with the well-documented competition between K and NH₄ (Martinez and Cerda, 1989). Also, salt stress induced Na accumulation in New Zealand spinach and potassium content decreased with increasing salinity in New Zealand spinach (yousif et al., 2010). High K⁺/Na⁺ selectivity in plants under saline conditions has been suggested as an important selection criterion for salt tolerance (Ashraf, 2002; Wei et al., 2003). While, Sheikhi and Ronaghi (2012) demonstrated that NaCl decreased potassium, iron and magnesium in spinach aerial parts but increased concentrations of N, phosphorus, sodium and chlorine.

Data presented in Table (4) established that application of humic acid and mycorrhiza revealed significant effect on the percentages of nitrogen, phosphorus, potassium, calcium and potassium/sodium ratio compared to control treatment in both seasons, except potassium in humic acid at 1.5 g/L, in the first season only. The obtained results indicated also that soil application treatments of humic acid and mycorrhiza, generally, decreased sodium contents. It is clear that addition of humic acid at 3.0 g/L + mycorrhiza gave the highest mean values of nitrogen, phosphorus, potassium, calcium and potassium/sodium ratio compared to the other treatments, in both seasons. However, the differences between (humic acid 1.5 g/l and mycorrhiza) and (humic acid at 3.0 g/L + mycorrhiza) in phosphorus percentage, were not significant, in the first season only. At humic acid of 3.0 g/L + mycorrhiza, the estimated percentage increase expressed as nitrogen, phosphorus, potassium and calcium, were (9.65 and 9.45%), (21.85 and 27.50 %), (7.42 and 7.36 %) and (16.46 and 13.61 %) in the first and second seasons, respectively and compare to the control treatment. The current results could be attributed to the role of each ameliorative material. In this respect, humic acid enhanced uptake of N and NO₃^- and accelerated N metabolism by improving nitrate reeducates activity, which resulted in production of protein in lettuce leaves (Haghighi et al., 2012). Humic acid under different salt stress levels, slightly increased the content of N, P, K. while, Na content was decreased in pepper plants (El-Sarkassy et al., 2017). Applications of humic acid to pepper plants grown under salt stress and normal conditions caused significant increases in N, P and K contents. (Akladious and Mohamed 2018). Aydin et al. (2012) showed that humic acid added to saline soil significantly increased plant nitrate, nitrogen and phosphorus content in bean (Phaseolus vulgaris L.).

Concerning mycorrhiza, Cantrell and Linderman (2001) found that inoculation of lettuce, grown under salt stress, with VA mycorrhizal fungi increased Ca, P, Zn, B, Cu and Mg than non-VAM plants at all salt levels. Similarly, Vicente-Sánchez et al. (2013) indicated that mycorrhization of plants increased the ability to acquire N, Ca, and K from both non-saline and saline media. Mycorrhiza enhanced the contents of macronutrients such as N, P, K, Ca, and Mg of Antirrhinum majus under drought (Bati et al., 2015).   Mycorrhiza improve the uptake of almost all essential nutrients and contrarily decrease the uptake of Na and Cl, leading to growth stimulation (Evelin et al., 2012). The interaction of salinity stress and AMF significantly affects the concentrations of P and N and the N:P ratio in plant shoots (Wang et al., 2018). Concentrations of total P, Ca²⁺, N, Mg²⁺, and K⁺ were higher in the AMF-treated Cucumis sativus plants compared with those in the uninoculated plants under salt stress conditions (Hashem et al., 2018).

The combined treatment between zero salinity and humic acid at 3.0 g/L + mycorrhiza, generally, achieved the highest average values of nitrogen, phosphorus, potassium and calcium and minimize the hazard effect of sodium in both seasons compared to the other treatments. Humic acid and mycorohiza either alone or in combination under different salt stress levels, slightly increased the content of proline, N, P, K and photosynthetic pigments. while, Na content was decreased in pepper plants (El-Sarkassy et al., 2017).

Table (4): Mean values of lettuce chemical contents as affected by salinity levels and soil application of humic acid and mycorrhiza and their interaction during winter seasons of 2019 and 2020.

Means having the same alphabetical letter (s) in column, within a comparable group of means, do not significantly differ, using the revised L.S.D. test at p = 0.05 level of probability.

**H₁=Humic acid at 1.5 g/L, H₂= Humic acid at 3.0 g/L, M= Mycorrhiza.

Mean performances of chlorophyll and protein contents of lettuce

Regarding the main effect of salinity levels on the total chlorophyll and protein percentage, data offered in Table (5) indicated that both tested parameters decreased as salinity levels increased in both seasons.  The reduction rate on total chlorophyll and protein percentage varied depending on the level of imposed salinity stress. The highest values of total chlorophyll and protein percentage were obtained from control treatment, while that of 6.0 mS/cm salinity gave the lowest ones, in both seasons. At salinity of 6.0 mS/cm, the estimated percentage reductions, expressed as total chlorophyll and protein percentage, were (24.73 and 22.10 %), and (24.32 and 19.38 %) for the first and second seasons, respectively and relative to the control treatment. The reduction in photosynthetic rates in plants under salt stress is mainly due to the reduction in water potential (Chutipaijit et al., 2011). Khan et al. (2013) indicated that total leaf chlorophyll contents significantly decreased with an increasing in NaCl levels of cucumber plants. Likewise, Brengi (2019) found that increasing salinity levels from 2 to 4 dsm^-1 reduced significantly chlorophyll contents in cucumber plants. Also, with increasing salinity levels, total chlorophyll in pepper leaves significantly decreased, this reduction may be related to enhanced activity of the chlorophyll-degrading enzyme, chlorophyllase. Moreover, increased salt content also interferes with protein synthesis and influences the structural component of chlorophyll (Jaleel et al., 2008). Similarly, in spinach, Seven and Sağlam (2020) reported that chlorophyll and protein contents were reduced as affected by salinity. Furthermore, increased salt content also delayed protein synthesis and influences the structural component of chlorophyll (Jaleel et al., 2008). Recently, Gabr et al. (2022) in spinach plants, found that increasing salinity reduced total chlorophyll and protein percentage.

Table (5): Mean values of lettuce chlorophyll and protein percentage as affected by salinity levels and soil application of humic acid and mycorrhiza and their interaction during winter seasons of 2019 and 2020.

Means having the same alphabetical letter (s) in column, within a comparable group of means, do not significantly differ, using the revised L.S.D. test at p = 0.05 level of probability.

**H₁=Humic acid at 1.5 g/L, H₂= Humic acid at 3.0 g/L, M= Mycorrhiza.