Growth and Yield of Spinach As Affected by Silicon and Fulvic Acid Under Salt Stress

INTRODUCTION

     Spinach plants (Spinacia oleracea L.) belongs to the family Amaranthacea. Spinach is originated from south western and central Asia (Avşar, 2011). China is the largest spinach producer followed by United States and Japan (FAOSTAT, 2017). Fresh spinach is rich in many nutrients ( protein, Ca ,Mg, Na, P, Fe,  vitamins C, B-carotene, vitamins E, and vitamin A). However , spinach leaves also, contains high concentration of oxalates and phytates  (Heaney et al., 1988 and McConn  and   Nakata 2004).  Spinach is a moderately salt-tolerant glycophyte in the winter, but sensitive to moderately-sensitive if cultivated in the spring and summer (Ferreira et al., 2020).  Agriculture sustainability is threated by increased soil salinization, which reduces both the productivity and availability of land for agriculture (Shrivastava and Kumar, 2015). Soil salinity is one of the major abiotic stresses that hinder crop growth and productivity worldwide. It has been reported 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% annually 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. Thus, living with salinity is the only way of sustaining agricultural production in the salt affected soil. So that, it is must to find the best management to alleviate salt hazard (Al-Rawahy et al. 2011).

       In recent years, exogenous protectants such as osmoprotectants, phytohormones, humic compounds, antioxidants and various elements such  as silicon have been found useful to alleviate the salt-induced damages (Khan et al., 2017). The development of methods and strategies to ameliorate the deleterious effects of salt stress on plants has received considerable attention (Senaratna et al., 2000). In this respect, application of fulvic acid enhanced transport of minerals, improved plant hormone activity, modified enzyme activities, promoted photosynthesis, solubilization of micro and macro elements, protein synthesis, and reduction of active levels of toxic minerals (Aiken et al., 1985; Khang, 2011; Billard et al., 2014; Kandil et al., 2020). Moreover, the use of silicon can stimulate greater physical performance and better quality because of the positive effects of Si, Ca, Mg, and K absorption (Ferreira et al., 2010). Also, silicon mediated decreased uptake and transport of Na⁺ and increased uptake and transport of K⁺ (Tuna et al., 2008; Hashemi et al., 2010 and Farshidi et al., 2012), from roots to shoots under salt stress. Therefore, the objective of this study was to investigate the role of fulvic acid and silicon in alleviating the negative impacts of salt stress and to evaluate the expected outcomes that may have on its growth and chemical characteristics on spinach plants irrigated with water in different salinity levels.

MATERIALS AND METHODS

           Two pots experiments were conducted at Abu Hummus, EL- Beheira Governorate, north Egypt, during the successive winter seasons of 2019 and 2020 to investigate the effect of fulvic acid and silicon in elevating the negative effect of salinity on vegetative growth, yield and chemical composition of spinach (Spinacia oleracea L. cv. Balady) under different salinity levels.  Soil physical and chemical properties were analyzed at the Agricultural Directorate Lab of Damanhur city, El-Behera Governorate, Egypt. Properties of the selected soil are shown in Table (1).

Table( 1):Chemical and physical properties of the experimental soil.

       The spinach seeds, cv. Balady, purchased from a local seeds market,  were sown in plastic pots (35 cm inner diameter, and 30 cm height),  each was filled with 12 kg of soil (Table 1) , and placed in the open field. The seeds were planted on 15^th and 10^th of November in 2019 and 2020, respectively. Each treatment composed of five replicated pots with four plants in each pot. Each experiment includes 20 treatments which were the combinations between four salinity levels (Tap water, 1500 , 3000 and 4500 ppm) and soil application treatments of fulvic acid  (  1.5 and 3.0 gm / L) in form potassium fulvate , silicon (1.5 and 3.0 mM) in form  potassium silicate and  distilled water as the control treatment. The recommended concentrations of soil application treatments were applied as a drench to the spinach plants. The control plants were treated with tap water. Each soil application treatment was applied three times after planting. The first application was conducted in the two specific leaves phase (15 days) after sowing and the others were applied with one week intervals (Smolen and Sady 2012; Fouda, 2016). Harvesting was done after 50 days of planting in  both seasons (Barkat et. al. 2018).

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 spinach.

Plant measurements

vegetative growth parameters

Spinach plants were harvested after 50 days and the measurement of vegetative growth parameters was performed immediately. Ten spinach plants from each treatment were randomly taken to measure:

Plant height (cm);it was measured with the help of measuring scale from the surface of the soil to the growing tip of the selected plants and then the average was calculated.

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

Plant dry weight (gm); the collected 10 plants were oven dried at 70 C˚ 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. in a forced-oven at 70 C˚till the weights became constant., then the dry matter was weighted.

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

Root length (cm) ;it was measured for 10 plants  randomly taken, and the average root length (cm) was calculated.

Root fresh weight (gm) ; the whole fresh root for 10 plants was weighted and the average weight (gm) was calculated

Root dry weight (gm) ; the collected fresh root for 10 plants were oven dried at 70 C^º in a forced air oven till obtaining a constant weight to obtain roots dry weigh (gm).

Leaf area per plant (cm²): leaves area / plant was calculated using the weight method as used by Fayed (1997). The leaves from the plant samples (three plants) were cleaned from dust and weighted. then, twenty random disks were taken from the leaves, using a circular puncher and weighted.

Where          20 = number of random disks

                     3   = number of plant sample

                              area of disk =   πr²

Chemical measurements

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

Total nitrogen, phosphorus ,  potassium, sodium and chloride; leaves samples were oven dried at 70ºC till obtaining a constant weight for 48 hours and ground in a mill with stainless steel blades. Wet digestion procedure was performed according to Chapman and Pratt (1978). Nitrogen percentage in leaves was determined by micro kjeldahl method as described by Page et al. (1982). Phosphorus percentage was determined calorimetrically as reported by Jackson (1973). Potassium and sodium were determined by atomic absorption Spectrophotometry methods (Bhowmik et al. 2012). Chloride was determined according to the method described by Jackson and Brown (1955).

Vitamin C and nitrate contents; vitamin C (mg100 g^-1) and nitrate (ppm) were determined according to the method described by Singh (1988).

Total oxalate ; total oxalate (mg 100g^-1) were determined according to the method described by Mazumdar and Majumder (2003).

Statistical analysis

     The experimental design was split plots in a randomized complete block design, whereas the salinity levels arranged in the main plots and the soil application treatments of  fulvic and silicon were randomly placed in the sub-plots. 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 effects of salinity levels, soil applications of fulvic acid , silicon and their interaction on vegetative growth of spinach plants are presented in Tables (2 and 3). Concerning the main effect of salinity levels on plant height ,  plant  fresh weight, plant  dry weight, number of leaves /plant, leaves area, root length, root fresh weight and root dry weight, results presented in Tables (2 and 3) revealed that all tested parameters decreased by increasing salinity levels. The reduction rate on any character varied depending on the level of imposed salinity stress. The highest values of the given parameters were obtained from the control treatment, while that the rate of 4500 ppm salinity recorded the lowest ones, in both seasons. At salinity of 4500 ppm, the estimated percentage reductions, expressed as plant height, plant  fresh weight ,  plant  dry weight, number of leaves , leaves area, root length, root fresh weight and root dry weight of the two seasons, were (26.42 and 31.18 %), (26.59 and 27.59 %), (26.16 and 26.77 %), (17.69 and 16.67% ), (20.01 and 29.04 %), ( 35.98 and 40.32 %), ( 40.70 and 40.17 %) and ( 23.07 and 22.79 % ) as compared to the control treatment in the first and second season, respectively. The adverse effects of high salinity on plants are related to the following 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).

These results were in harmony with those reported by Mohammad et al.(1998) on tomato, Shereen et al. (2005) on radish , Gama et al.(2007) on common bean, Céccoli et al.( 2011) and  Siddikee et al.(2011) on sweet pepper, Brengi (2019) on cucumber, Ors and Suarez (2017) , Seven  and Sağlam (2020), Fayed, et al. (2021) and Kim et al., (2021) on spanish who reported, generally, that vegetative growth parameters, decreased with increasing salinity rates.

     Regarding the main effect of the ameliorative treatments (fulvic acid and silicon) on the plant height ,  plant  fresh weight and  plant  dry weight, number of leaves , leaves area, root length, root fresh weight and root dry weight of spinach plants, results presented in Tables (2 and 3) exhibited that adding FA and Si in all concentrations showed significant effect in improving all the studied traits as compared with the control treatment, in both seasons. For instance, application of silicon at the highest level (i.e. 3 mM) recorded, generally, the highest values of plant height,  plant  fresh weight , plant  dry weight , number of leaves, root length, root fresh weight and root dry weight compared to the other treatments, in both seasons . However, leaves area reached its maximum when plants were treated with FA at the rate of 1.5 gm /l in both seasons. The  particular treatment  of Si at the rate of 3 mM  the resulted estimated percentages increase in plant height, plant fresh weight , plant dry weight, root length, root fresh weight and root dry weight   of 34.84 and 35.59 %), (25.19 and 27.34 %) , (26.19 and 28.65), (37.04 and 33.99 %) , (23.48 and 14.36 %) and (45.25 and 34.77%)  comparing to the control treatment in the first and second season, respectively. The positive effects of Si could plays different roles in plant growth and development, improve plant resistance to diseases and pests, increase photosynthesis, regulate respiration and increase the tolerance of the plant to elements toxicity (Zargar  et al., 2019). Moreover, Si fertilizer application can alleviate the adverse effects of various abiotic (e.g., drought, salt and metal toxicity) and biotic (pests and plant diseases) stresses on plants (Ma et al. 2004). Silicon seems to affect acquisition of other essential nutrients such as nitrogen, phosphorus and calcium and other micronutrients as well (Liang et al., 2003 and Farshidi et al., 2012), thereby improving the growth of plants and the generally tolerance against salt stress

Table (2): Plant height, plant fresh weight , plant dry weight , Number of leaves and Leaves area of spinach plants as affected by salinity and soil application of both fulvic acid and silicon in both seasons of 2019and 2020.

         *Means having the same letter (s) within the same column are not significantly different according to LSD for all-pairwise comparisons test at 5% level of probability.

Table (3): Root length, root fresh weight and root dry weight of spinach plants as affected by salinity and soil application of both fulvic acid and silicon during in both seasons of 2019and 2020.

*Means having the same letter (s) within the same column are not significantly different according to LSD for all-pairwise comparisons test at 5% level of probability.

Regarding the interaction effect between salinity levels and the ameliorative treatments (fulvic acid and silicon) on plant height ,  plant  fresh weight ,  plant  dry weight, number of leaves , leaves area, root length, root fresh weight and root dry weight of spinach plants, whereas results presented in Tables (2 and 3) showed significant interactions between both variables. The combined treatment between zero salinity and silicon at 3 mM accomplished , generally,  the highest values of aforementioned characters, in both seasons compared to other treatments.

Percentages of nitrogen, phosphor, potassium ,sodium and chloride in leaves

 Regarding the main effect of salinity levels on the percentages of nitrogen, phosphor potassium, sodium and chloride in plant leaves, result presented in Table (4) revealed that nitrogen, phosphor , potassium decreased as salinity levels increased. However sodium and chloride percentages increased as salinity levels increased. The highest values of nitrogen, phosphor and potassium were obtained from control treatment, while that of 4500 ppm salinity gave the lowest ones, in both seasons. At salinity of 4500 ppm, the estimated percent reductions, for  nitrogen, phosphor and potassium, were (22.63 and 30.61%), (11.11 and 22.80 %) and (29.90 and 26.58 %) in the first and second seasons, respectively and relative to the control treatment. Also, The highest values of sodium and chloride percentages were obtained from salinity at 4500 ppm treatment, while that of control treatment gave the lowest ones, in both seasons.

The nutritional disorders may result from the effect of salinity on nutrient availability, competitive uptake, transport, or distribution within the plant. Numerous reports indicated that salinity reduces nutrient uptake and accumulation of nutrients into the plants (Rogers et al. 2003; Hu and Schmidhalter 2005). A number of laboratory and greenhouse studies have shown that salinity can reduce N, P and K accumulation in plants (Feigin et al., 1991; Pessarakli, 1991; Al-Rawahy et al., 1992). This is not surprising since an increase in Cl uptake and accumulation is often accompanied by a decrease in shoot-NO₃ concentration. Examples of such an effect have been found in cucumber (Martinez and Cerda, 1989), eggplant (Savvas and Lenz, 1996), melon (Feigin et al., 1987), and tomato (Kafkafi et al., 1982; Feigin et al., 1987; Martinez and CerdaÂ, 1989). In addition, Salinity stress decreases the uptake and concentration of P in plant tissues. Thus, plants exhibit reduced and stunted growth, dark green coloration of the leaves, production of slender stems, and death of older leaves (Taiz and Zeiger, 2006). Under saline-sodic or sodic conditions, high levels of 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 (Martinez and Cerda (1989).

Concerning the main effect of the soil application treatments (fulvic acid and silicon) on the percentages of nitrogen, phosphor, potassium, sodium and chloride results presented in Tables (4)  showed that application of fulvic acid and silicon exhibited significant effect on the percentages of nitrogen, phosphorus and potassium as compared with the control treatment in both seasons. However, the differences between the two concentrations of either fulvic acid (1.5 and 3.0 g/l) or silicon (1.5 and 3.0 mM) were not significant. Whereas, soil application of fulvic acid and silicon differed in their effect on the contents of Na. The highest mean value of sodium was obtained with fulvic acid at 3 gm/l, where the lowest value was achieved with silicon at 3mM, in both seasons. So, fulvic acid activated the absorption of sodium, while silicon reduced it. Silicon at the rate of 3.0 mM reduced  chloride ,compared to the other treatments, in both seasons.  At silicon concentration of 3.0 mM, the estimated percent increase in nitrogen, phosphor and potassium, were (7.48 and 7.37%), (15.21 and 8.33 %) and (8.44 and 17.10 %)  in the first and second seasons, respectively relative to the control treatment. Meanwhile, for silicon at 3.0 mM, the estimated percent decrease  in sodium and chloride  were (23.23 and 22.08%) and (20.87and 20.53 %) in the first and second seasons, respectively in relative to the control treatment.

The positive effect of silicon could be mediated decrease in the uptake and transport of Na⁺ and increased uptake and transport of K⁺ (Tuna et al., 2008;  Hashemi et al., 2010 and Farshidi et al., 2012), from roots to shoots under salt stress. Silicon seems to affect acquisition of other essential nutrients such as nitrogen, phosphorus and calcium and other micronutrients as well (Liang et al., 2003 and Farshidi et al., 2012), thereby improving the growth of plants and generally the tolerance against salt stress. Moreover, P  concentration and total P contents were increased by adding silicon under saline conditions. The possible causes for this may be associated with both Si-stimulated root activity showed by root dehydrogenase activity and Si-improved P bioavailability in soils due to the chemical competition between H₂PO₄⁻ and silicate (H₃SiO₄⁻) anions for the sorption sites. ( Liang et al. ,1999) .

Pertaining the interaction effect between salinity levels and protection treatments (fulvic acid and silicon) on the percentages of nitrogen, phosphor, potassium, sodium and chloride in spinach leaves, results offered in Table (4) indicated significant differences among the interactions between both variables. The combined treatment between zero salinity and silicon at the rate of 3 mM  achieved , generally, the highest values of N, P and K percentages in both seasons compared to other treatments, except nitrogen in the second season and potassium in both seasons. However, the combination between 4500 ppm salinity level and fulvic acid at 3 gm/L achieved the highest values of sodium , in both seasons.Moreover, the combination between 4500 ppm salinity level and control treatment reached, generally, the highest values of chloride , in both seasons.

It is vital to note that silicon reduced the risk effect of either sodium or chloride because it plays different roles in plant growth and development, improve plant resistance to diseases and pests, increase photosynthesis, regulate respiration and increase the tolerance of the plant to elements toxicity (Deshmukh et al., 2017). Roshdy and Brengi (2016) found that silicon treatment resulted in significant decrease in leaves Na and Cl but increased K/Na ratio in snap bean leaves under salt stress condition.

Table (4): Percentages of nitrogen (N), protein, and phosphor (P) in leaves of spinach plants as affected by salinity and soil application of both fulvic acid and silicon in both seasons of 2019and 2020.

*  Means having the same letter (s) within the same column are not significantly different according to LSD for all-pairwise comparisons test at 5% level of probability.

Total chlorophyll, protein,ascorbic acid, nitrate and total oxalate contents

Regarding the main effect of salinity levels on the total chlorophyll, protein, ascorbic acid, nitrate and total oxalate contents  , data presented in Table (5) revealed that the total chlorophyll, protein and nitrate  decreased by salinity levels increased, whereas, ascorbic acid and total oxalate increased with increasing salinity levels , in the two seasons.  The reduction rate on total chlorophyll ,protein and nitrate varied depending on the level of imposed salinity stress. The highest values of total chlorophyll ,protein and nitrate content were obtained from  the control treatment, while that 4500 ppm of salinity gave the lowest ones, in both seasons. However, the highest values of ascorbic acid and total oxalate were attained from 4500 ppm salinity, although that zero salinity reached the maximum values, in both seasons. At salinity of 4500 ppm, the estimated percent reductions, in total chlorophyll ,protein and nitrate were (13.72 and 14.37%), (22.71 and 30.68 %) and (30.75 and 30.24 %),  in the first and second seasons, respectively relative to the control treatment .However, at salinity of 4500 ppm, the estimated percent increase in ascorbic acid and total oxalate were (10.11 and 9.53%) and (45.53 and 40.17%) in the first and second seasons, respectively relative to the control treatment . The present results are in agreement with those of Parida et al. (2005) who stated that salt stress has been shown to change the photosynthesis, osmoregulation, mineral ion contents, and chlorophyll content of spinach treated with 0–200 mmol L^–1 NaCl and that salt stress showed toxic effects on plants and lead to metabolic changes, like loss of chloroplast activity and decreased photosynthetic rate. Also, the same conclusion were obtained by Khan et al.( 2013) and Berengi (2019) in cucumber. The decrease in chlorophyll content under stress is a commonly reported phenomenon, and in various studies, this may be due to different reasons, one of them is related to membrane deterioration (Mane et al., 2010). 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, as suggested by Mishra and Sharma, (1994) who indicated that increasing saline increased oxidation of chlorophyll leading to its decreased concentration. Moreover, other investigators indicated that during water stress brought about by salt stress, generation of reactive oxygen species (ROS) are thought to play important roles in inhibiting photosynthesis and H₂O₂ and OH- are responsible for injurious effect of salt stress on chlorophyll content and chloroplast ultra-structure (Yamane et al., 2004). Also, in spinach, Seven  and Sağlam(2020) found that chlorophyll and total protein content in spinach leaves  were reduced as salinity increased. Furthermore, increased salt content also interfered with protein synthesis and influences the structural component of chlorophyll (Jalee et al., 2008).Vaidyanathan et al. (2003) reported that the non-enzymatic antioxidants such as ascorbic acid, glutathione, α-tocopherol, and flavonoids, showed an accumulation in root tissues in rice plants subjected to salt stress.

            Concerning nitrate contents, our results were in agreement with those obtained by Bian et al.(2020) who reported that chloride showed an opposite trend to nitrate as it is well-known that salinity can reduce nitrate accumulation in leafy vegetables due to antagonism between nitrate and chloride for the same root anion channel. A linear decrease in nitrate concentration has been reported in romaine lettuce baby-leaf grown in high salinity solution (Scuderi et al., 2011 ; Barbieri et al., 2011 ; Bonasia et al., 2017) ). The increasing  in EC resulted in a reduction in nitrate concentration along with a Cl⁻ rise in soilless-grown wild rocket (Bonasia et al., 2017). Moreover, it is known that chloride ions inhibit the activity of the enzymes involved in the N metabolism and consequently N assimilation (Barber et al., 1989; Debouba et al., 2006, 2007). Oxalic acid  in lettuce   leaves increased by increasing NaCl treatments (Tarakcioglu and Inal ,2002).

       Regarding, the main effect of the soil application of fulvic acid and silicon  on the total chlorophyll, protein, ascorbic acid, nitrate and total oxalate contents , results presented in Tables (5)  demonstrated that soil application of fulvic acid and silicon revealed significant effect on total chlorophyll and protein, in both seasons, compared to control treatment. However, ascorbic acid contents reached the maximum values when plants received control treatment, in both seasons. Nevertheless, the differences between the two levels of both fulvic acid and silicon in total chlorophyll and protein percentage, generally, were not significant, in both seasons. Nitrate contents reached its maximum when plants  were treated with fulvic acid followed by silicon at low concentration. Also, the differences between the high concentration of silicon (3 mM) and the control treatment were not significant, in both seasons. The highest values of total oxalate contents were obtained from control treatment, followed by the fulvic acid treatments, while that of silicon  treatments recorded the lowest ones, in both seasons. So, silicon reduce the hazard effect of oxalate.

  These results were in agreement with those of Lobato et al. (2009) who, documented that silicon encouraged a progressive increase in total chlorophyll in (Capsicum annuum L.) under water

stress compared to control. Also, Li et al. (2015) indicated that chlorophyll contents were increased as a results of adding Si application to tomato seedlings under salt stress. Moreover, in spinach plants, exogenous application of Si increased chlorophyll concentration under salinity stress (Eraslan et al., 2008).  Application of fulvic acid enhanced transport of minerals, improved plant hormone activity, modified enzyme activities, promoted photosynthesis, solubilization of micro and macro elements, protein synthesis, and reduction of active levels of toxic minerals (Aiken et al., 1985; Khang, 2011; Billard et al., 2014; Kandil et al., 2020).

Regarding, the interaction effect between salinity levels and protection treatments (fulvic acid and silicon) on total chlorophyll, protein, ascorbic acid, nitrate and total oxalate contents   of spinach plants, results in Table (5)  indicated  that the combined treatment between salinity level at 4500 ppm and control gave , generally, the lowest   chlorophyll and protein contents. The combination between 4500 ppm salinity level and fulvic acid at 3 gm/L achieved the highest values of ascorbic acid contents , in both seasons. Moreover, the combination between zero salinity and fulvic acid at 1.5 gm/l achieved the highest values of nitrate in both seasons compared to other treatments. However, the combined treatment between salinity level of 4500 ppm and control treatment attained , generally, the highest values of total oxalate contents, compared to other treatments.

Table (5) Total chlorophyll, protein, ascorbic acid and total oxalate contents of spinach plants as affected by salinity and soil application of both fulvic acid and silicon in both seasons of 2019and 2020.

*  Means having the same letter (s) within the same column are not significantly different according to LSD for all-pairwise comparisons test at 5% level of probability.