Effects of Different Fertilizer Types on Pigment Content and Some Stress Molecules in Perennial Ryegrass (Lolium perenne L.)

Plant material and experimental design
 
In March 2024, the growth of perennial ryegrass (Lolium perenne L.) under various fertilization conditions was studied in a greenhouse in Pazar, Rize, Turkey. Grass was grown in 16 cm diameter, 13 cm deep plastic pots filled with sterile peat (pH 6.0, 1.0 g/l fertilizer). The experiment used a factorial design with three replications. Seeding was done by broadcasting, followed by pressing and covering with peat (Fig 1). Thirty days after seeding, different fertilizer solutions (liquid seaweed, microbial organic, vermicompost, chicken manure, bat guano and 32 micro trace elements) were applied at concentrations of 0, 2, 4, 6 and 8%. Fertilizers were applied uniformly (500 ml) as aqueous solutions (Fig 2). The study measured Chlorophyll-a, Chlorophyll-b, Total Chlorophyll, Chlorophyll a/b, Carotenoid, Proline, MDA (Lipid Peroxidation), APX (Ascorbate Peroxidase)and Catalase. Fertilizer contents are as follows (Fig 3).



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Fertilizer types and contents
 
Liquid seaweed fertilizer
 
Organic Matter: 6% (w/w), pH: 3-5, Water-Soluble Potassium Oxide (K₂O): 1% (w/w), Maximum EC (Electrical Conductivity): 41 dS/m (Fig 4).

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Microbial liquid organic fertilizer
 
Names of live microorganisms: Bacillus subtilis, Bacillus megaterium, Bacillus amyloliquefaciens. Total Live Organism Count: 1 X 10⁸  CFU/ml (colony-forming units/ml) (Fig 4).
 
Organic liquid vermicompost
 
Organic Matter: 7%, Organic Nitrogen: 0.3%, Maximum EC (Electrical Conductivity): 0.5 dS/m, pH Range: 4.5-6.5 (Fig 4).
 
Chicken manure
 
It contains 100% organic matter. It is derived from chicken manure extract. (Chicken Manure Extract) (Fig 4).
 
Bat Guano
 
Organic matter: 25% (w/w), Organic carbon: 7% (w/w), Organic nitrogen (N): 2% (w/w), Water-soluble potassium oxide (K₂O): 5% (w/w), Total phosphorus pentoxide (P₂O₅): 1% (w/w), Total Humic+Fulvic Acid: 3% (w/w), pH: 5-7, Water-soluble calcium oxide (CaO): 1% (w/w), Maximum EC (dS/m): 17% (w/w)
 
Fertilizer with 32 Micro trace elements
 
Total Nitrogen (N): 32% (w/w), Ammonium Nitrogen (N-NH₄): 8% (w/w), Nitrate Nitrogen (N-NO₃): 16% (w/w), Urea Nitrogen (N-NH₂): 0.01% (w/w), Boron (B): 0.01% (w/w), Copper (Cu): 0.02% (w/w), Iron (Fe): 0.02% (w/w), Manganese (Mn): 0.01% (w/w), Molybdenum (Mo): 0.001% (w/w), Zinc (Zn): 0.002% (w/w)
 
 
Pigment analysis
 
The total chlorophyll and carotenoid content were determined using the Arnon, 1949 methodand the measured absorbance values were converted to chlorophyll a, chlorophyll b, total chlorophylland total carotenoid amounts using the formulas below (Fig 5):

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Total chlorophyll (C) = [20.2 x Δ645 + 8.02 x Δ663] x ml/1000
Chlorophyll a (Ca) = [12.7 x Δ663 - 2.69 x Δ645] x ml/100
Chlorophyll b (Cb) = [22.9 x Δ645 - 4.68 x Δ663] x ml/1000
Total Carotenoid = [1000 Δ470 - 1.90 x Ca - 63.14 x Cb] / 214] x ml/1000
Note: Absorbance measurements at 645 nm, 663 nmand 470 nm wavelengths were performed using a spectrophotometer.
 
Biochemical analysis
 
Proline (µmol/ml)
 
Proline analysis was conducted following Bates (1973). Leaf tissue (50 mg) was homogenized with 5 ml of 3% sulfosalicylic acid and centrifuged at 5000 g for 10 minutes. The supernatant was mixed with 2 ml acid-ninhydrin and 2 ml acetic acid, boiled for 1 hour at 100°C, cooledand extracted with 5 ml of toluene. The pink upper phase’s absorbance was measured at 520 nmand proline content was calculated using a standard curve. Proline content is expressed as µmol/g fresh weight. (Şekil 5).
 
Lipid peroxidation activity (MDA) (nmol/g)
 
Using the method of Çakmak and Horst (1991), samples stored at -80°C were utilized. 0.2 g of plant tissue was homogenized with 1 ml of 0.1% TCA at +4°C for 10 minutes and centrifuged at 12,000 g for 20 minutes. The supernatant (250 µl) was mixed with 1 ml of 20% TCA containing 0.6% TBA, incubated at 95°C for 30 minutes, then rapidly cooled on ice. After centrifuging at 12,000 g for 10 minutes, absorbance was measured at 532 and 600 nm. Lipid peroxidation was calculated as Δ(532-600)/1.56 x 10⁵  and expressed in nmol/L.
 
Ascorbate peroxidase (APX) (EU/ml)
 
APX activity was measured following Nakano and Asada (1981). Leaf tissue (0.5 g) was homogenized with extraction buffer (0.1 mM EDTA, 2 mM ascorbate, 2% PVPP, 50 mM PBS, pH 7) at +4°C and centrifuged at 14,000 rpm for 30 minutes. The supernatant was used for APX activity measurement, calculated as ΔAbs (290 nm)/(min x mg protein) and expressed in U/mg protein.
 
Catalase (EU/ml)
 
Catalase activity was determined according to Cho et al., (2000). A reaction mixture of phosphate buffer, H‚ O and water was prepared and incubated at 30°C for 3 minutes. Using the measurement buffer as a blank, absorbance was measured at 240 nm after adding 37.5 µl of supernatant to a quartz cuvette. Catalase activity was calculated as ΔAbs (240 nm)/(min x mg protein) and expressed as EU/mg protein.
 
Statistical analysis
 
Statistical analyses were performed using JMP software. Multiple comparisons were made using the Tukey test to determine the statistical significance of differences between groups.

Chlorophyll a is a critical pigment that determines the photosynthetic capacity of plants, playing a vital role in plant growth and productivity. In this study, the effects of different treatments on chlorophyll a content were found to be significant (p≤0.001: ***). The I-2 treatment had the highest chlorophyll a content at 3.68, indicating that I-2 supports chlorophyll a synthesis in plants (Table 1). The O-6 (2.56) and M-6 (2.37) treatments also showed high chlorophyll a levels, but no statistically significant difference was found among the other treatments. The control group (0.54) and S-4 (0.35) treatments had the lowest chlorophyll a values, indicating a negative effect on its synthesis. The coefficient of variation (CV) was 38.37%, indicating moderate variability in chlorophyll a levels. Statistically significant differences in chlorophyll b levels were observed among treatments (p≤0.001: ***). The I-2 treatment had the highest chlorophyll b level (2.07), supporting chlorophyll b synthesis, while M-4 (1.38) and O-6 (1.10) also increased chlorophyll b content. In contrast, the control (0.29) and S-4 (0.22) treatments showed the lowest chlorophyll b levels. I-2 also had the highest total chlorophyll content (5.75), suggesting it promotes overall chlorophyll synthesis, while the control (0.83) and S-4 (0.56) treatments had the lowest total chlorophyll values (Table 1). Olszewska et al., (2022) reported that high nitrogen levels and biostimulants positively influenced chlorophyll synthesis in Lolium perenne L. These findings are consistent with our observations that certain treatments (e.g., I-2, O-6, M-6) increased chlorophyll a and b levels. Xu et al., 2021 aimed to evaluate the effects of high TN (Total Nitrogen) + TP (Total phosphorus) levels on seed germination, plant growth, antioxidant responseand N and P assimilation rates in perennial grass. Yuan et al., (2021) found that high TN + TP levels negatively impacted plant growth and photosynthesis, similar to our findings where the control group and S-4 treatments reduced chlorophyll levels due to adverse conditions. They also reported that silicon (Si) and potassium (K) applications improved chlorophyll synthesis in Lolium perenne L. under salt-alkaline stress, partially aligning with our results. The O-4 (2.65) and T-4 (2.63) treatments had the highest chlorophyll a/b ratios, suggesting improved light use efficiency. The control (1.87) showed a moderate ratio, while M-4 (1.46) and S-4 (1.59) had the lowest. The coefficient of variation (CV) was 14.72%, indicating low variability in the a/b ratio (Table 1).



Carotenoid content varied significantly among treatments (p≤0.001: ***). The I-2 treatment had the highest carotenoid content (1.01), indicating a potential increase in antioxidant capacity, while O-6 (0.73) and O-4 (0.69) also had high levels. The control (0.18) and S-4 (0.14) treatments had the lowest carotenoid levels. The coefficient of variation (CV) was 30.62%, showing moderate variability (Table 1). Ascorbate peroxidase activity, an indicator of stress response, was highest in O-2 (29.02). The control (22.70) had moderate activityand I-2 (0.67) showed the lowest, suggesting a negative effect on stress response. The CV for ascorbate peroxidase activity was 0%, indicating no variability (Table 2).



Zuo et al., (2022) investigated how different nitrate levels affect the regrowth of Lolium perenne after grazing or defoliation, finding that medium nitrate levels enhanced photosynthetic pigments and efficiency. Our study aligns partially with these findings. Asghar et al., (2021) studied two wheat cultivars, showing that N+P fertilizer (80-60 kg/ha and 120-90 kg/ha) significantly improved yield-related traits and biochemical parameters. Foliar application of Proline and H₂O₂ boosted yield traits, with H₂O₂ enhancing ascorbate peroxidase (APX) and ascorbic acid (AsA) activities, while proline increased phenolic compounds. These results were similar to those of the O-2 fertilizer in our study, but not consistent with the I-2 treatment.

Catalase is a crucial antioxidant enzyme that helps plants detoxify harmful reactive oxygen species (ROS) generated under stress conditions, thereby protecting cells from oxidative damage. Increased catalase activity is often associated with enhanced stress tolerance in plants. Catalase activity also showed significant differences among the treatments (p≤0.001: *). S-2 treatment showed the highest catalase activity (175.80), with I-2 (172.96) and Y-8 (163.42) also high. The control group (89.97) had moderate activity, while M-2 had the lowest (11.54), possibly negatively affecting oxidative stress response. Proline content was highest in T-4 (48.05), indicating enhanced stress response; the control (19.17) was moderateand Y-2 had the lowest (1.15). Lipid peroxidase activity, indicating oxidative damage, was highest in T-2 (1.50), suggesting increased membrane damage; the control (0.90) was moderateand S-4 had the lowest (0.36). Overall, findings align with similar studies (Zuo et al., 2022; Asghar et al., 2021; Loudari et al., 2023; Safari et al., 2023; Wei et al., 2023; Aalipour et al., 2020; Azizi et al., 2021; Hossain et al., 2022; Zhang et al., 2023; Yildirim et al., 2023; Yıldırım et al., 2024). However, negative effects in some treatments highlight the need for further research on their impact on plant stress responses and antioxidant defenses.
 

This study examined the effects of different fertilizer treatments on chlorophyll, carotenoid, ascorbate peroxidase, catalase, prolineand lipid peroxidase levels in plants. The results revealed that the treatments had significant and statistically meaningful effects on the physiological and biochemical responses of the plants. The I-2 treatment supported chlorophyll synthesis in plants, resulting in the highest chlorophyll a and b levels. Total chlorophyll content also reached its highest level with the I-2 treatment, indicating an increase in the plant’s overall photosynthetic capacity. The O-4 and T-4 treatments stood out in terms of the chlorophyll a/b ratio. In stress response ascorbate peroxidase activity, enhancing the plant’s ability to cope with stress. The S-2 treatment had the highest catalase activity, supporting the plant’s oxidative stress response. Proline accumulation was highest with the T-4 treatment, indicating increased resistance to environmental stresses, while lipid peroxidase activity was highest with the T-2 treatment, suggesting that this could lead to increased cellular membrane damage. In conclusion, high levels of biochemical components such as proline, catalaseand ascorbate peroxidase play a crucial role in the plant’s ability to adapt to stress conditions and maintain healthy green growth. These levels can be optimized through the appropriate use of fertilizers. Fertilizer applications that support chlorophyll synthesis, enhance the plant’s oxidative stress responseand increase resistance to environmental stresses contribute to both the physiological and biochemical resilience of the plant. By balancing these components, fertilizer use becomes a critical factor in optimizing plant health and ensuring successful green cover establishment.
 
Disclaimers
 

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.