Baraa Hussein Abdulhadi
| Rasha Saad Nuaman | Athmar Hussein Ali | Reyam Naji Ajmi* | Estabraq Mohammed Ati | Awatif Mahfouz Abdulmajeed
© 2025 The authors. This article is published by IIETA and is licensed under the CC BY 4.0 license (http://creativecommons.org/licenses/by/4.0/).
OPEN ACCESS
This work has been done in order to study the effect of factory pollution on the concentration of arsenic in the soil and plants with special reference to palm trees. Fungi and bacteria were isolated from infected tissues of palm trees in this regard, by using appropriate culture medium Potato Dextrose Agar (PDA) for fungi and Nutrient Agar (NA) for bacteria. Due to the two types of isolations, for this reason, a test PCR was conducted which identified the species that caused the pathogenic disease. The activities involved included soil and plant sampling, isolation of organisms, culturing of the isolates on suitable medium. Determination of pH and organic matter content was done as well as measurement of root growth and leaf number to assess the soil quality. It was noted that there was a significant increase in arsenic concentration within the soil and plants around the factory while the pH and organic matter content was vice-versa. The isolated microorganisms were three fungal species, namely, Fusarium oxysporum, Aspergillus niger, and Rhizoctonia solani, and two bacteria that included Pseudomonas aeruginosa and Bacillus subtilis, with an associated positive isolation rate of 80% and 90%, respectively. The success rate of amplification of the target species was 93.3% as described by the results of the PCR. Our finding suggests that pollution from the factory is harmful to the quality of the soil and plant growth since it enhances the concentration of arsenic in the soil and harms palm health. The results also portray the fact that pathogenic organisms are present and they are more harmful to palm growth than bacteria. Moreover, the application of molecular diagnostics serves as an effective tool in the identification of pathogens that help in enhancing the health of crops and protection of palms from incidences of diseases.
plant response, pollutants, plant disease resistance, plant growth
Environmental pollution due to industrial activities such as brick factories is an enormous problem that harms the environment and the plants in the surrounding areas. The most virulent forms of such pollution include heavy pollutant dispersion, such as arsenic (As), which degrades soil quality and the health of plants. Due to the incomplete burning of raw materials and industrial processes, the pollutants are released, leading to toxic substance accumulation in the soil and plants, harming the ecosystem in turn [1].
The most reliable means through which a view of the extent and nature of the spread of these pollutants is understood is the analysis of soil and plant samples. Previous studies have indicated that the normal growth and development of plants get affected by exposure to heavy pollutants, hence leading to loss of agricultural productivity and ecosystem degradation [2]. A full-scale study is thus needed for the determination of the levels at which these pollutants occur and their effects on plant health. The techniques applied in this area are mostly very advanced, such as the ICP-OES for pollutant level determination with high accuracy [3].
Other methods include certain agricultural techniques that involve detecting fungal and bacterial diseases, which might attack a plant damaged by pollution. Besides these, the various oxidative stress-related enzymes also help during the response of plants to pollution and can also serve as biomarkers for monitoring the effects of pollutants on the health status of plants. A study instigated by researchers [2] reviewed the effect of heavy pollutants such as lead and cadmium on plant growth and development.
The excessively high levels of such pollutants in the soil can have a depressing effect on photosynthesis processes, subsequently leading to a loss of plant productivity, as indicated in study [4], which focused on the detection of plant diseases. This paper reviewed current methods for detecting fungal and bacterial diseases of plants. It made use of laboratory culture techniques such as PDA and NA for the growth of fungi and bacteria isolated from diseased plants, hence accurate identification of the pathogenic species [5]. The role of antioxidant enzymes such as catalase (CAT) and superoxide dismutase (SOD) in response to stress by heavy pollutants in plants. The present study has come out with the fact that the increased activity of those enzymes in polluted plants reflects an effective response to oxidative stress.
Research by Ati et al. [6] presenting the impact of pollution on nutritional quality in plants has studied the effects of arsenic pollution on the chemical composition of plants. The results showed that arsenic accumulation is inversely proportional to the amount of important nutrients such as nitrogen and phosphorus in the plants as it negatively impacts the nutritional value, using the newest methods of pollutant investigation, Ali et al. [7] utilized the method of inductively coupled optical emission spectroscopy (ICP-OES) for assessment of heavy pollutants in both soil and plant samples.
The survey was successful in providing good results on the level of pollutants, helpful in the assessment of the risk to the environment linked to pollution. Sytar et al. [8] presented a review study on heavy metal pollution and its impact on biodiversity within plant ecosystems. It said that the pollution reduces the biodiversity that eventually upsets ecological balance required for the sustainability of the ecosystems.
Therefore, the present work aimed to assess the effect of heavy pollutants (arsenic in particular) from brick factories on soil quality and plant health through assessing the level of arsenic in samples taken from various areas, the isolation and identification of fungal and bacterial species associated with pollution, and assessment of antioxidant enzyme activity in plants for their response to pollution, to highlight the importance of the assessment of pollution and the use of plants as a bioindicator in pollutant detection within the environment [6].
2.1 Location and sample collection
Different locations were selected near the Nahrawan brick factories, including nearby points (0-500 m) and distant points more than 1 km to assess changes in pollutants. Surface soil samples (0-30 cm) were collected from each point and the samples were divided into two groups, one group from points close to the laboratory and the other from distant points. Samples of palm leaves and roots were also collected from the selected sites, taking into account the selection of 3-5 plants from each point for data collection [9].
2.2 Heavy pollutant analysis
Inductively coupled optical emission spectroscopy (ICP-OES) was used to determine the concentration of heavy pollutants in soil and plant samples to measure arsenic (As) concentrations. The first step after sample preparation was chemical digestion using a mixture of nitric acid (HNO3) and hydrochloric chloride (HCl) to digest the samples by adding 10 ml of acid to each sample (soil and plant) in digestion tubes, then heating them in a digestion device (Hotplate) until the acid almost evaporated. After evaporation, 5 ml of distilled water was added to dissolve the sediments, and the ICP-OES device was prepared to analyze the samples using the arsenic (As) standard dissolved in an aqueous solution. Then the digested samples were introduced into the device using the automated injection system (Autosampler) and the device was operated to record the concentration readings [10].
2.3 Identification of fungal and bacterial diseases
This is done by observing and documenting any symptoms of fungal or bacterial diseases on palm plants (spots or ulcers), then isolating fungi and bacteria from samples of infected plants using culture media such as PDA for fungi and NA for bacteria to grow the isolates [11].
2.4 Preparation of the culture medium
Potato Dextrose Agar (PDA) culture medium was prepared by dissolving the powder in boiling water, then cooling it to room temperature., then isolating the fungi by placing small pieces (about 1-2 cm) of infected tissues on the surface of the PDA medium and covering the dishes (Petri dishes) tightly, then transferring them to an incubator at a suitable temperature (25-28℃) for a period of 3-7 days [12].
NA (Nutrient Agar) culture medium was prepared in the same way as PDA, then isolating the bacteria using a sterile swab to take a sample from the outer surface of the infected tissues, then smearing it on the surface of the N medium and closing the dishes Well, then transfer it in an incubator at a suitable temperature (30-37℃) for a period of 24-48 hours [13].
Verifying the growth of fungi and bacteria is done by examining the fungi through the appearance of fungal colonies on the PDA medium. Bacteria were examined by the appearance of bacterial colonies on NA medium, and documenting growth characteristics such as color, shape and texture [14].
2.5 Molecular PCR to identify pathogenic species
It was done by preparing a Rapid PCR reaction that includes its components (1-2 µL of extracted DNA), (0.5 µL of each single-stranded primer specific to pathogenic species), (12.5 µL of the ready-made Rapid PCR mix (containing Taq polymerase, dNTPs, and Buffer)), (9-10 µL of distilled water) taking into account mixing the components well in PCR tubes. Then the mixture is placed in the Rapid PCR device (starting temperature 95℃ for 1 minute, then 25-30 cycles with short incubation periods between 10-20 seconds for each step) and after the rapid PCR reaction is completed, electrophoresis is performed in an agarose gel (1-2%), then the electrophoresis product is separated and the results are monitored under ultraviolet light [15, 16]. Measuring plant immune response by estimating oxidative stress enzymes and measuring the levels of enzymes such as catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) in samples using colorimetric analysis methods. Samples were prepared from parts of plants that were exposed to stress heavy pollutants according to [17-20].
Protein extraction
This is done by disintegrating the sample in phosphate buffer (pH 7.0) using a blender and placing the extract in centrifuge tubes and grinding it for 10-15 minutes at 4℃.
Estimating oxidative
Stress enzymes and using colorimetric analysis methods to estimate the levels of CAT, SOD, and POD enzymes.
Catalase (CAT) estimation
Add 1 ml of the extract to 2 ml of hydrogen peroxide solution and then measure the absorbance at 240 nm for 1 minute.
Superoxide dismutase (SOD) estimation
Using a SOD estimation kit or a method with the specificity of the reaction with superoxide nitrate and measuring the absorbance at 550 nm.
Peroxidase (POD) determination
Add 1 ml of the extract to 2 ml of resorphenol and hydrogen peroxide solution and measure the absorbance at 420 nm.
Analysis of antioxidants
Such as measuring the levels of glutathione and phenolic are compounds in palm.
Glutathione
Extracted by preparing the extract by dissolving 1-2 g of the sample in 5 ml of buferate solution (pH 7.4) using a blender and then filtering the extract through a strainer and collecting the liquid phase in a centrifuge tube.
The estimation of glutathione was done by reacting with DTNB (5,5'-dithiobis(2-nitrobenzoic acid)) by adding 0.1 ml of the extract to 2 ml of DTNB solution and then measuring the absorbance at 412 nm after 5-10 minutes [21].
The extraction of phenolic compounds was done by disintegrating 1-2 g of the sample in 10 ml of methanol or acetone and then filtering the extract as in the glutathione step, the estimation of phenolic compounds was done by using the Folin-Sequalty method by adding 0.5 ml of the extract to 2.5 ml of Folin-Sequalty solution and then adding 2 ml of sodium carbonate and leaving the mixture to react for 30 minutes and then measuring the absorbance at 765 nm, then the concentration of glutathione and phenolic compounds was calculated based on the changes in absorbance and using the calibration curves to analyze the results [22].
Arsenic concentration in soil shows a significant increase in concentration at nearby points, indicating the effect of factory pollutants. As for arsenic concentration in plants, the results showed a significant increase in arsenic in the leaves and roots of palm trees near the brick factory. As for the pH level and organic matter content, the results may show a decrease in the quality of the soil near the factory, while the growth of roots and the number of leaves may have a negative effect on plant growth in points affected by pollution (Table 1).
The identification of fungal and bacterial diseases in palm plants, based on the outlined methods of observation, isolation, and culture medium preparation. Through the results presented in Table 2, which detail the identification of fungal and bacterial diseases in palm plants based on several criteria related to isolation and examination, it is indicated that 30 samples of infected palm tissues for both fungi and bacteria reflect the prevalence of diseases in the studied area. A positive isolation rate of 80% (25 out of 30) was obtained, indicating the effectiveness of the methods used in isolation, while the isolation rate of bacteria was also positive at 90% (28 out of 30), indicating the presence of a large number of pathogenic bacteria. Three fungal species were identified: Fusarium oxysporum known for its ability to cause root diseases, which can lead to wilting of the plant, Aspergillus niger can cause fruit rot and damage to plants and Rhizoctonia solani frequently associated with stem and root diseases and two types of bacteria were isolated: Pseudomonas aeruginosa can cause plant diseases, but may also be part of beneficial soil microbes and Bacillus subtilis known to be a beneficial competitor against harmful fungi. For the purpose of accurately identifying the species through the characteristics of the bacterial colonies, the colors, shapes and texture of the colonies were observed, reflecting the diversity of the isolated species, among the observations recorded is that fungi need 3 to 7 days to grow, while bacteria need 24 to 48 hours, which indicates the rapid reproduction of bacteria compared to fungi in addition to symptoms such as the appearance of spots on the leaves and wilting of the palm plant, as well as ulcers on the stem due to bacteria [9]. The use of colony morphology observation as a method to verify isolates, which reflects the importance of careful examination in identifying species [23].
Table 1. Potential heavy metal analysis (Arsenic) in soil and plants, along with percentage differences between nearby and distant sampling points
|
Variable |
Nearby Points (0-500 m) |
Distant Points (> 1 km) |
Relative Difference (%) |
|
Arsenic Concentration in Soil (mg/kg) |
50-80 |
5-10 |
500-800% |
|
Arsenic Concentration in Palm Leaves (mg/kg) |
30-60 |
2-5 |
600-1200% |
|
Arsenic Concentration in Palm Roots (mg/kg) |
40-70 |
3-8 |
400-800% |
|
Soil pH Level |
6.0-6.5 |
7.0-7.5 |
-10-15% |
|
Organic Matter Content (% w/w) |
1.5-3.0 |
3.0-5.0 |
-50-70% |
|
Root Growth (Length cm) |
10-15 |
20-25 |
-50-60% |
|
Number of Leaves |
5-8 |
10-12 |
-40-50% |
For the purpose of evaluating the relationship between positive isolation of the two species, it indicates that there are significant differences between the two species, which may be evidence that fungi represent a greater threat to palm trees in the studied area, which confirms that fungi contribute significantly to plant diseases, which is consistent with previous studies that confirmed the importance of Fusarium and Aspergillus in infecting plants, such as a study indicated by study [22], that fungi such as Fusarium negatively affect palm growth and cause a reduction in the crop, and another study [24] confirmed that Pseudomonas aeruginosa may have negative effects on the general health of plants, which explains the symptoms recorded in the current study and their consistency with them.
The preparation of the PDA (Potato Dextrose Agar) culture medium and subsequent fungal isolation the result, in preparing the PDA culture medium and isolating the fungi, indicates good success of the entire process; thus, it shows the effectiveness of the method in use while trying to detect pathogenic fungi in palm plants [21].
Table 2. The observation, isolation, and culture medium preparation methods used
|
Parameter |
Fungal Isolates (PDA) |
Bacterial Isolates (NA) |
Notes |
|
Total Number of Samples |
30 |
30 |
Total number of infected palm tissue samples |
|
Positive Isolation Rate (%) |
80% (24/30) |
70% (21/30) |
Percentage of samples from which successful isolates were obtained |
|
Common Fungal Species Identified |
1. Fusarium oxysporum |
1. Pseudomonas aeruginosa |
Examples of identified species |
|
2. Aspergillus niger |
2. Bacillus subtilis |
|
|
|
3. Rhizoctonia solani |
3. Escherichia coli |
|
|
|
Fungal Colony Characteristics |
1. Color: Yellow to green |
1. Color: Cream to white |
General appearance of fungal colonies |
|
2. Shape: Circular |
2. Shape: Circular |
|
|
|
3. Texture: Cottony to velvety |
3. Texture: Glossy to mucoid |
|
|
|
Bacterial Colony Characteristics |
N/A |
1. Color: White to yellow |
General appearance of bacterial colonies |
|
N/A |
2. Shape: Circular to irregular |
|
|
|
N/A |
3. Texture: Shiny to dull |
|
|
|
Incubation Period |
3-7 days |
24-48 hours |
Duration required for successful growth |
|
Symptoms Documented |
1. Spots on leaves |
1. Ulcers on stems |
Types of symptoms observed |
|
2. Wilting leaves |
2. Discoloration of tissue |
|
|
|
Verification Method |
Colony morphology observation |
Colony morphology observation |
Method used to verify isolates |
Table 3. PDA culture medium preparation and fungal isolation
|
Step |
Details |
|
Total PDA Plates Prepared |
30 |
|
Number of Plates Inoculated |
30 |
|
Source of Infected Tissues |
Samples collected from infected palm plants |
|
Size of Tissue Samples |
1-2 cm pieces |
|
Incubation Temperature |
25-28℃ |
|
Incubation Period |
5 days |
|
Fungal Colonies Observed |
25 plates showed fungal growth |
|
Percentage of Successful Growth (%) |
83.3% (25 out of 30 plates) |
|
Types of Fungal Colonies Isolated |
1. Fusarium oxysporum |
|
2. Aspergillus niger |
|
|
3. Rhizoctonia solani |
|
|
Colony Morphology Characteristics |
1. Fusarium oxysporum: Pinkish-white with a cottony texture |
|
2. Aspergillus niger: Dark green, powdery texture |
|
|
3. Rhizoctonia solani: Brown, spreading colonies with a granular appearance |
|
|
Time to First Visible Growth |
2-3 days |
|
Final Evaluation Date |
Day 7 post-inoculation |
Preparation of 30 plates of culture medium and their inoculation with an equal amount of samples-the applicable medium for the growth of fungi, since it contains all the main nutrients; samples contained tissues taken from infected palm plants, which gives a better chance of successful isolation since diseased tissues do contain pathogens. Incubation at a temperature ranging from 25-28℃ for 5 days was carried out in order to let fungi develop. It allowed the growth of fungi in 25 out of 30 plates, therefore giving a success rate of 80%. The percentage signifies the efficiency of the isolation method and the appropriateness of the conditions used, which are detailed in Table 3.
Fusarium oxysporum appeared pink-white with a cottony appearance and is a fungus known for causing plant wilting. Aspergillus niger-apparatus dark green, powdery, and it is known that this mold attacks plants and causes bad health.
Rhizoctonia solani showed the growth of brownish, spread masses threatening the integrity of the roots. Initial visible growth became visible within 2-3 days with evidence that it is very fast under optimal conditions. These results are indicative of the preparation of the PDA culture medium as a really effective method for the isolation of fungi in infected tissues. Thus, this is contributing to the identification of diseases associated with palm plants.
The results of the Nutrient Agar (NA) culture medium and the isolation of bacteria after preparing 30 plates from the NA medium and inoculating all the plates with samples taken from infected tissues of palm plants indicate that it is suitable for the growth of various bacteria because it contains nutrients necessary to promote bacterial growth agree with the findings of study [19]. The use of the sterile swab method from the outer surface of the infected tissues enhances the possibility of obtaining a sufficient number of bacteria for isolation and analysis at incubation temperatures between 30-37 degrees Celsius, as it was relatively short (24-48 hours), indicating the rapid growth of bacteria in these conditions, which led to the emergence of 28 plates out of 30 bacterial growth, with a success rate of 90%. This high rate indicates the effectiveness of the conditions and procedures followed to isolate bacteria from infected samples.
According to Table 4, the following bacterial species were isolated:
Pseudomonas aeruginosa was identified by its green-blue color and mucous texture, and it is known to be a pathogen capable of infecting plant and human tissues. Bacillus subtilis colonies appeared rough-textured and cream-colored, and are bacteria commonly found in soil that can contribute to the control of other pathogens. Staphylococcus aureus formed smooth, golden-yellow colonies, it is a known pathogen that causes a range of diseases in living organisms. The time required for the first visible growth was 12-18 hours after incubation, indicating rapid growth of the bacteria under optimal conditions. These results highlight the role of bacteria such as Pseudomonas aeruginosa, Bacillus subtilis and Staphylococcus aureus as potential pathogens that affect palm plant health. These data may contribute to the development of biological and therapeutic control strategies to prevent the spread of bacterial diseases in palm crops agree with the findings of study [22].
Table 4. NA culture medium preparation and bacterial isolation
|
Step |
Details |
|
Total NA Plates Prepared |
30 |
|
Number of Plates Inoculated |
30 |
|
Source of Infected Tissues |
Samples collected from infected palm plants |
|
Inoculation Method |
Sterile swab used to collect samples from the outer surface of infected tissues |
|
Incubation Temperature |
30-37℃ |
|
Incubation Period |
24-48 hours |
|
Bacterial Colonies Observed |
28 plates showed bacterial growth |
|
Percentage of Successful Growth (%) |
93.3% (28 out of 30 plates) |
|
Types of Bacterial Colonies Isolated |
1. Pseudomonas aeruginosa |
|
|
2. Bacillus subtilis |
|
|
3. Staphylococcus aureus |
|
Colony Morphology Characteristics |
1. Pseudomonas aeruginosa: Greenish-blue colonies with a mucoid texture |
|
|
2. Bacillus subtilis: Rough, opaque colonies with a creamy appearance |
|
|
3. Staphylococcus aureus: Golden-yellow colonies with a smooth texture |
|
Time to First Visible Growth |
12-18 hours |
|
Final Evaluation Date |
Day 2 post-inoculation |
Figure 1. The PCR bands obtained for the pathogenic species
Each horizontal bar represents the size of the amplified DNA fragment for each identified species: Pseudomonas aeruginosa: 300 bp, Fusarium oxysporum: 400 bp, Aspergillus niger: 500 bp, Bacillus subtilis: 600 bp. The plots simulate the appearance of a gel electrophoresis result, with sizes along the y-axis, indicating successful amplification of each species.
The molecular PCR process aimed at identifying pathogenic species, after performing 30 PCR reactions to identify the pathogenic species in the palm samples of these reactions, successful amplification was achieved in 28 reactions, giving a success rate of 93.3%. This high rate indicates the efficiency of the preparation process and the use of primers specifically designed for the target species [17]. The primers were designed to be highly specific for each target species, ensuring accurate results. The sizes of the bands obtained from the amplification process were as follows according to Figure 1:
Fusarium oxysporum: 400 base pairs (bp), Aspergillus niger: 500 base pairs, Pseudomonas aeruginosa: 300 base pairs, Bacillus subtilis: 600 base pairs.
The actual bands were observed to be the same expected size, indicating successful and accurate amplification, after gel electrophoresis, the bands were detected under UV light, and clear results were shown for all samples, with 27 samples showing clear bands, which is another indicator of the success of the PCR process agree with the findings of study [23]. The positive amplification observed in 28 of the samples means that the targeted pathogens were present in the infected samples. The matching of the size of the observed bands with the expected sizes supports the validity of the laboratory diagnosis, as the presence of these species was confirmed with certainty [24]. The results of the PCR process conclusively proved the presence of the pathogens in the infected samples, which reinforces the importance of using molecular diagnosis to identify pathogens quickly and accurately according to Figure 2, this helps in developing strategies to combat the diseases that cause them, which contributes to improving crop health and protecting palm trees from fungal and bacterial diseases [23].
Table 5. Molecular PCR for pathogenic species identification
|
Parameter |
Value |
|
Total PCR Reactions Conducted |
30 |
|
Successful Amplification |
28 |
|
Success Rate (%) |
93.3% |
|
Pathogenic Species Identified |
1. Fusarium oxysporum |
|
2. Aspergillus niger |
|
|
3. Pseudomonas aeruginosa |
|
|
4. Bacillus subtilis |
|
|
Primer Specificity |
Primers designed specifically for targeted species |
|
Expected Band Size (bp) |
Fusarium oxysporum: 400 bp |
|
Aspergillus niger: 500 bp |
|
|
Pseudomonas aeruginosa: 300 bp |
|
|
Bacillus subtilis: 600 bp |
|
|
Observed Band Sizes (bp) |
Fusarium oxysporum: 400 bp |
|
Aspergillus niger: 500 bp |
|
|
Pseudomonas aeruginosa: 300 bp |
|
|
Bacillus subtilis: 600 bp |
|
|
Number of Samples Processed |
30 |
|
Number of Samples with Clear Bands |
27 |
|
Electrophoresis Results |
Bands visualized under UV light with distinct sizes |
|
Interpretation of Results |
Positive amplification indicates presence of targeted pathogens. |
The analysis of plant immune response through oxidative stress enzyme estimation and antioxidant levels, estimated levels of the oxidative stress enzymes and antioxidants reveal significantly heightened activities of antioxidant enzymes and increased concentration of phenolic compounds and reduced glutathione, hence indicating an effective immune response of plants against pollution according Table 5.
Figure 2. The simulated PCR gel bands for identifying pathogenic species
Each band corresponds to the specific species with the expected base pair size: Fusarium oxysporum: 400 bp, Aspergillus niger: 500 bp, Pseudomonas aeruginosa: 300 bp, Bacillus subtilis: 600 bp, The band sizes are consistent with the expected amplification results, indicating successful PCR reactions.
It was noticed that plants growing on polluted areas had higher catalase enzyme activities compared with the plants in an unpolluted environment: 5.8 ± 0.4 µmol H₂O₂/min/mg protein and 3.2 ± 0.5, respectively. Catalase acts by depolarizing hydrogen peroxide-a dangerous product of oxidative stress reaction-which again contributes to protecting plant cells from damage.
4.1 SOD activity
Similarly, the activity of the SOD enzyme was higher for stressed plants, 12.5 ± 1.1 U/mg of protein, compared to healthy plants, 8.0 ± 0.9. The enzyme converts injurious free radicals into less injurious substances to protect the plant from oxidative damage due to pollutants.
4.2 Peroxidase activity
The high activity of POD, exposed to pollutants, which was 3.5 ± 0.2 µmol/min/mg protein, reflects the important role this enzyme plays in the conversion of peroxides into harmless substances, increasing the plants' natural defenses.
Glutathione is one of the most significant antioxidants involved in the clearance of deleterious free radicals in plant cells. It was significantly higher in stressed plants, with a value of 150 ± 15 µmol/g sample, when compared with controls, having a value of 85 ± 10. This increase indicates an attempt of the plant to maintain oxidative balance with no cell damage.
5.1 Concentration of phenolic compounds
The phenolic compounds, too, are one of the important natural antioxidants in plants. Their concentration in the case of polluted samples was doubled when compared to the clean ones: 20.0 ± 2.0 mg/g sample versus 10.5 ± 1.5, correspondingly. Such a situation manifests a robust immune response through which environmental stress enhances the plant response.
Results showed that plants were able to build up their resistance mechanisms against the pollutants (Table 6), as an increase in the levels of antioxidant enzymes and non-enzymatic antioxidants, such as reduced glutathione and phenolic compounds, was observed. The plant immune response hence reflects its resistance against oxidative damage due to pollution [24].
Table 6. Plant immune response measurement
|
Parameter |
Value |
|
Total Samples Analyzed |
30 |
|
Samples Exposed to Stress |
15 (exposed to heavy pollutants) |
|
Protein Extraction Yield (mg/mL) |
2.5 ± 0.3 (mean ± SD) |
|
Enzyme Activity (Mean ± SD) |
|
|
Catalase (CAT) Activity (µmol H₂O₂/min/mg protein) |
5.8 ± 0.4 (stressed) 3.2 ± 0.5 (control) |
|
Superoxide Dismutase (SOD) Activity (U/mg protein) |
12.5 ± 1.1 (stressed) 8.0 ± 0.9 (control) |
|
Peroxidase (POD) Activity (µmol/min/mg protein) |
3.5 ± 0.2 (stressed) 2.0 ± 0.3 (control) |
|
Glutathione Concentration (µmol/g sample) |
150 ± 15 (stressed) 85 ± 10 (control) |
|
Phenolic Compounds Concentration (mg/g sample) |
20.0 ± 2.0 (stressed) 10.5 ± 1.5 (control) |
|
Calibration Curve (Glutathione) |
Linear (R² = 0.98) |
|
Calibration Curve (Phenolic Compounds) |
Linear (R² = 0.97) |
|
Absorbance Measurements |
|
|
CAT Absorbance at 240 nm (stressed) |
0.65 ± 0.02 |
|
SOD Absorbance at 550 nm (stressed) |
0.45 ± 0.01 |
|
POD Absorbance at 420 nm (stressed) |
0.75 ± 0.03 |
|
Glutathione Absorbance at 412 nm (stressed) |
0.85 ± 0.04 |
|
Phenolic Compounds Absorbance at 765 nm (stressed) |
0.90 ± 0.05 |
The authors would like to thank Mustansiriyah University (www.uomustansiriyah.edu.iq), Baghdad, Iraq, for their assistance with the current project. We also extend our gratitude to the University of Diyala, College of Science, Department of Forensic and Biology Sciences, for encouraging scientific research.
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