Heavy metal pollutants i.e. lead and nickel are introduced to cultivable land through many sources and get accumulated in the soil profiles and contaminate water bodies which affect the growth and production of several crops. Seaweeds are rich in nutrients and have the ability to improve soil fertility by adding nutrients and organic contents to soil. In this study, S. ilicifolium was used with and without plant growth promoting rhizobacteria (PGPR) P. aeruginosa which showed heavy metal biodegradation capacity of 99.06% for lead and 98.8% for nickel against said metal stress. The individual and combined treatment of Sargassum ilicifolium as soil amendment and P. aeruginosa improved sunflower growth under 0.5 mM lead (Pb) and nickel (Ni) contaminations. The morphological characteristics of plants including shoot and root length and fresh weight were improved under stress and non-stressed condition. The biochemical analysis of proline, total phenols, peroxidase (POD), superoxide dismutase (SOD) and DPPH-radical scavenging activity also indicated that plant tolerance was improved under heavy metal stress.
Research Article
The impact of heavy metal tolerant bacterium and Sargassum ilicifolium as organic fertilizer on growth and antioxidant enzymes of sunflower under lead (Pb) and nickel (Ni) stress
https://doi.org/10.21203/rs.3.rs-3556534/v1
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Seaweed
S. ilicifolium
Heavy metals
Endophytic bacteria
P. aeruginosa
Sunflower
Pakistan, being maritime nation with extensive coastline, is home to several economically important marine algal species. Seaweeds have been drawn attention of researchers and consumers for their nutritional richness including minerals, proteins, vitamins, polyunsaturated fatty acids, and antioxidants like carotenoids and polyphenols which may vary species wise and with environmental conditions (Nazarudin et al. 2021). Sargassum sp. are known to have higher proteins, carbohydrates, fibers andhave metal accumulating capability (Sarker et al. 2021).Another salient characteristic of S. ilicifolium which make them an excellent organic fertilizer is their association to several bacterial communities, including PGPR, in a symbiotic relationship (Oh et al. 2021). P. aeruginosa produces vital plant growth promoting substances, such as IAA (indole-3-acetic acid), siderophores, and 1-aminocyclopropane-1-carboxylate (ACC) deaminase (Karnwal et al. 2023). The metallic ions bio-sorption efficiency of algae and bacteria is due to the presence of metal binding polypeptides such as phytochelatins (PCs) and metallothioneins (MTs) on their large surface-volume as physical adsorption whose synthesis is reported to be enhanced with increasing concentration of metals (Cd2+, Zn2+, Pb2+, Hg2+) in the environment (Balzano, et al. 2020; Tufail et al.2022). These molecules have great surface adhering properties and are biodegradable as well as less toxic when compared to artificial surfactants (Abo-Alkasem et al. 2023). Subsequently, these metal ions move gradually into the cytoplasm where they are utilized via converting into nutrients through enzymatic processes, called chemisorptions (Yan 2022). This makes them a competent biological representative for bioremediation and metal immobilization (Znad et al, 2022). However, the symbiotic interaction seaweeds with bacteria need further investigations at molecular and anatomical levels to understand the mechanism.
Sunflower is a major source of oil, biomass production, and is commercially utilized for chemical, energy and industrial purposes (Bayat et al. 2021). The major source of heavy metals incorporation into plant body and seed oil is the contaminated soil and irrigation water (Laila et al. 2022). The accumulation of these metals causes a substantial decline in agricultural yield and productivity while the consumers experience serious health issues (Pruteanu et al. 2022). Lead (Pb) and nickel (Ni)are non-essential elements whose contamination in the soil and water is becoming an escalating ecological and economical issue in Pakistan. Accumulation of these metallic ions in root hair hinders the absorption of essential minerals and causes efflux of vital ions (Collin et al. 2022). The prolonged exposure to Pb and Ni generally damages plasma membranes, causes swelling of mitochondria, interferes with enzyme activities, oxidative stress, and damages other cellular organelles. (Słota et al. 2022). Elevated amount of heavy metals strictly retards seed germination by downregulating the activities of ribonucleases, proteases and amylases which affects the absorption and mobilization of the stored food in germinating seeds (Begum et al. 2022). Consequently, metal toxicity in plants declines photosynthetic activity, disturbs nitrogen assimilation, trims down antioxidant activity and eventually lowers the yield and quality of agricultural products (Mustafa et al. 2023).
The use of bio-fertilizers is one of the eco-friendliest and cost effective ways to improve plant growth under heavy metal stress (Khan et al. 2023).Therefore, this research is to examine the potential capability of seaweed Sargassumilicifolium with and without PGPR Pseudomonas aeruginosa in alleviating the heavy metal toxicity of Pb and Ni and improving plant growth.
Seaweed collection and soil amendment
Brown seaweed Sargassum ilicifolium was collected at low tide from the sandy seashore of Buleji, Karachi. After cleaning of unwanted algae and washed thoroughly by means of faucet water,S. ilicifolium samples were dried out in the shade. Dried seaweeds were crushed into fine powder and preserved in polyethylene bags at room temperature to be used later in the experiments.
Heavy metal biodegradability assay
The heavy metals degradation capacity of P. aeruginosa was determined using the method reported by Marzan et al. (2017). The bacterial isolate P. aeruginosa was cultured in LB (Luria Bertani) broth medium containing 200 mg/L Pb(NO3)2 and NiSO4 salt, incubated for 24 hours in a rotary shaker at 150 rpm (pH 7.0 at 37°C). Afterwards, bacterial cultures were removed via centrifugation for 15 min at 5000 rpm and the metallic ions in supernatants were acid digestedusing concentrated nitric acid and heated at 100°C on a hot plate. The Pb and Ni ions concentration was quantified by atomic absorption spectrophotometer and was calculated using the following formula.
Screen house experiments
Healthy seeds of Helianthus annuus L. were obtained from the local market. The mercuric chloride (1%)was used for surface sterilization of seeds. Afterwards, ten seeds were sown in each earthen pot (10 cm diameter) having 1 kg of soil (sandy loam, pH = 7.2, EC = 4 mS/m, 50% water holding capacity). For soil amendment, 1% w/w of dried seaweed S. ilicifolium powder was mixed with the soil properly, seven to ten days prior to sowing and was kept moist.The Pseudomonas aeruginosa obtained from Agricultural Biotechnology and Phytopathology Laboratory, Department of Botany was cultured in King’s Bbroth. At the time of sowing, 25 mL P. aeruginosa suspension (10 days old broth, 8x108cfu mL− 1) was poured in each pot. At four leaf stage, the pots were thinned to four seedlings and were subjected to the heavy metal treatment via irrigation using aqueous solution of Pb(NO3)2 (0.5mM) and NiSO4 (0.5mM) as the source of Pb and Ni. Control plants were irrigated with tap water.
The field experiments were carried out at Biological Research Center Institute, University of Karachi. Each treatment was completely randomized and each field plot (area 2×2 meters and 4 rows) had thirty plants in a row with four replicates. The P. aeruginosabacterial suspension (250 mL/plot) and seaweed powder 70 g/plotwere used in treatment plots. The heavy metal treatments were started via irrigation when the seedlings were ten days old, thrice a week.
Morphological data
All the plants were harvested forty-five days after treatment to observe the effects of Pb and Ni concentrations on shoot length, root length, shoot weight and root weight in all treatments.
Biochemical data
Proline Content
The total proline content was assessed using protocol of Bates et al.(1973). Leaf material (0.25g) was crushed in 5 mL of 3% sulphosalicylic acid and the filterate was used for the estimation of proline. The 1 ml aliquot, 1 ml of glacial acetic acid and 1 mL of working ninhydrin solution were mixed and heated in a water bath for approximately 30–60 minutes at 100°C. Afterwards, 2 mL toluene was added and vortexed. The chromophore was read at 520 nm against reagent blank (toluene).
Total phenols
The total phenols were estimated by following the procedure proposed by Rahman et al. (2017). The leaf extract (0.2 mL) was combined with 2 mL of freshly prepared (2%) sodium carbonate and after keeping at room temperature for 2 minutes, 50% folin-ciocalteau phenol reagent (0.1 mL) was mixed and the reaction mixture was incubated in the dark for 30 minutes. The absorbance of samples was obtained at 720 nm.
Peroxidase (POD) assay
Peroxidase activity (POD) was estimated using Reddy et al. (1995) method. The enzymes were extracted by crushing 100 mg/mL fresh leaf material in ice cold phosphate buffer (0.1 M, pH 6.5), centrifuged at 5000 rpm for 15 min. The sample extract (0.1 mL) was mixed with pyrogallol solution (3 mL, 0.05M) and the spectrophotometer was adjusted to zero at 430 nm. Afterwards, 0.5 mL of H2O2 solution (1%) was added and the change in optical density was noted up to three minutes.
Superoxide dismutase (SOD) assay:
The superoxide dismutase (SOD) activity was quantified as described by Kono (1978). The reaction mixture contained 50 mM sodium carbonate buffer (pH 10.0), 96 µM nitrobluetetrazolium (NBT), and 0.6% Triton X-100. The reaction was started by the addition of 0.1 mL hydroxylamine-HCl (20 mM) and after two minutes, leaf extract (0.07 mL) was added. The reduction of NBT was observed as a result of increase in absorbance at 540 nm.
DPPH -free radical scavenging activity assay
DPPH free radical scavenging activity was estimated by using the method described by Rahman et al.(2016). The aliquot (0.2 mL) was mixed in 0.8 mL (100 mM, pH 7.4) Tris-HCl buffer and 1 mL working solution of DPPH (30 µM) in the reaction tube and vortex for 2–3 seconds. A solution of DPPH (1 mL) with ethanol (1 mL) reserved as a control. Absorbance of the samples was recorded at 517 nm at 0 minute against blank (ethanol) and after incubating in dark for 30 minutes. The scavenging activity in percentage was determined by using the equation:
Statistical analysis
All the data obtained from potted as well as filed trials was statistically analyzed using one-way ANOVA (analysis of variance) and comparison of group means was estimated by Duncan’s Multiple Range Test (DMRT) at a probability level of P < 0.05.
Heavy metal biodegradation capability
Heavy metal biodegradation ability was determined by atomic absorption spectroscopy. P. aeruginosa was inoculated in Luria Bertani broth having lead (Pb) and nickel (Ni) 200 ppm concentration. The results showed that the lead (Pb) and nickel (Ni) degrading percentage of P. aeruginosa was 99.06% and 98.8% as shown in Table 1.
Screen house experiment
Morphological growth parameters
The plant height and biomass production was substantially lowered in plants that were irrigated with Pb and Ni containing water (Fig. 1). The results showed that the soil amended with powder of driedS. ilicifolium and P. aeruginosa inoculation caused significant (p < 0.05) improvement in plant growth and weight under heavy metal contamination. It was also noticed that individual S. ilicifolium application was more effective for plant growth promotion in comparison with P. aeruginosa treatment.
Proline content
Maximum amount of proline content was quantified in those plants which were irrigated with 0.5 mM Pb and Ni incorporated water (Figure. 2). InS. Ilicifolium application, proline content was lowered while in stressed plants, its effect was more prominent when co-applied withP. aeruginosa.
Total phenols
The application of S. ilicifolium and P. aeruginosa increased total phenolic content under non-stressed conditions. The plants grown under stressed conditions had higher levels of phenols and the effect of individual application of S. ilicifolium in stressed plants remained non-significant (P < 0.05). However, phenols were increased in P. aeruginosa applications, with the highest levels noted in P. aeruginosa co-applied with S. ilicifolium (Fig. 2).
Peroxidase (POD) activity
The highest peroxidase activity was observed in the Pb and Ni stressed plants. Whereas, both the S. ilicifoliumamendment and bacterium inoculation reduced the peroxidase activity under heavy metals stress. The lowest POD activity was found in S. ilicifoliumamendment against Pb stress while in Ni contamination, the combination of S. ilicifoliumandP. aeruginosa showed the least POD activity.
Superoxide dismutase (SOD) activity
The P. aeruginosa caused a decrease in superoxide dismutase activity in stressed plants in individual application as well as combined with S. ilicifolium. However, the solo application of S. ilicifolium showed maximum SOD activity under both metals treatment (Fig. 2).
DPPH –radical scavenging activity (%)
Results demonstrated that the combined application of S. ilicifolium and P. aeruginosa successfully quenched the DPPH –radical at 0 min and after 30-minutes incubation in Pb and Ni contaminated and healthy plants. All the treatments showed better scavenging activity, but the S. ilicifolium amended plants had the highest inhibition percentage at 0–30 minutes in healthy and unhealthy soil. Standard antioxidant butylated hydroxytoluene (BHT) was examined for a maximum scavenging activity including all treatments. However, the combined treatment of S. ilicifolium + P. aeruginosa showed better results than their solo application (Fig. 2).
| Bacterial strain | Heavy metal (HM) | HM added to broth (ppm) | HM in culture (ppm) | Degradation % |
|---|---|---|---|---|
| P. aeruginosa | Pb | 200 | 1.87 | 99.06 |
| Ni | 200 | 2.4 | 98.8 |
Field experiment
Morphological parameters of growth
The growth promoting effect of S. ilicifolium and P. aeruginosa was prominent in healthy and stressed sunflower plants in field conditions as well. Significantly (P < 0.05) greater plant length and plant weight was determined in response to S. ilicifolium and P. aeruginosa against Pb and Ni toxicity (figure. 3).
Proline content
The stress marker proline was prominently decreased in plants treated with P. aeruginosa and S. ilicifolium in healthy conditions as well as in response to heavy metal toxicity. Both the P. aeruginosa and S. ilicifolium were found to be more effective in individual applications than their co-application. The plants treated with only P. aeruginosa produced lowest amounts of proline in stressed conditions (Figure. 4).
Total phenols
The results illustrated that lead and nickel toxicity increased the production of total phenols. The amendment of S. ilicifolium in soil greatly encouraged the synthesis of total phenols in plants relative to all treatments. All individual and combined treatments of S. ilicifolium and endophytic bacterium significantly (P < 0.05) improved the phenols in plants with the exception of singly applied P. aeruginosa in Pb stress.
Peroxidase (POD) activity
Both theS. ilicifolium and P. aeruginosa treatments lowered the POD activity which was increased in response to heavy metal stress (Figure.4). The highest peroxidase activity was measured in plants that survived under Pb contamination followed by Ni stressed plants in contrast to healthy control. Comparatively, P. aeruginosa was found to be more effective in lowering POD levels than S. ilicifolium amendment.
Superoxide dismutase (SOD) activity
In healthy plants the superoxide dismutase (SOD) activity was the greatest which was decreased by S. ilicifolium and P. aeruginosa treatment. The heavy metal treatment reduced SOD levels and the Pb affected plants showed lower activity of superoxide dismutase than Ni stressed plants to combat with free radicals produced in stress. The application of S. ilicifolium with or without P. aeruginosa significantly (P < 0.05) reduced the SOD activity in Ni stressed plants. However, the single treatments of both (S. ilicifolium and P. aeruginosa) did not cause significant (p < 0.05) effect under Pb stress while their co-application decreased SOD activity.
DPPH –radical scavenging activity (%)
The DPPH radical scavenging activity explained the greater increment of inhibition percentage in plants that were grown in S. ilicifolium amended soil with inoculation of P. aeruginosa at 0–30 minute. The lower free radical scavenging ability of plants was displayed in Pb and Ni stressed plants which was improved by soil amendment and bacterial treatment. Among all positive treatments against stress, the highest scavenging percentage was recorded in S. ilicifolium treatment in contrast to P. aeruginosa and their co-application.
A number of studies have shown the pollution status of heavy metal contamination in the industrial parts of Pakistan (Bux et al. 2021). The present research showed that the application of S. ilicifolium seaweed with inoculation of heavy metal tolerant P. aeruginosa significantly improved the plant growth under toxicity of lead and nickel in screen house and field experiments. Morphological data showed that the S. ilicifolium amendment enhanced the plant height and biomass in control and heavy metal contaminated plants. The combined application of S. ilicifolium + P. aeruginosa significantly (P < 0.05) promoted the shoot length, shoot weight, root length, and root weight of sunflower under Pb and Ni stress plants. Many studies (Jayakumar et al. 2021; Mansor 2020; Truong et al. 2023; Barquilha et al. 2017) have been supported the utilization of different species of Sargassum sp. for adsorption and immobilization of heavy metals in contaminated medium. These seaweeds have a variety of chemical moieties on their cell surface like carboxyl, hydroxyl, phosphate, and amide, which serves as metal-binding sites for toxic contaminants (Foday et al. 2021) thus, reducing metal uptake by plants and alleviate toxicity. The algae, when used as a soil amendment, degrades easily and adds organic substances to the soil which nourishes beneficial microflora so they multiply and indirectly improve plant growth, yield and quality of crops (Kumari et al.2022). Our previous research (Rahman et al. 2016; 2017) revealed that Sargassumilicifolium has a potential source of natural biomolecules to significantly enhance growth parameters in cotton and soybean plants against biotic and abiotic stress. Due to their bioactive compounds, the use of S. ilicifolium as liquid fertilizer and soil supplementation has shown remarkable results on growth and physiological performance of tomato plants (Archana et al. 2022), P. zeylanica (Kakade et al. 2022) and peanut (Meng et al. 2022).Similar results regarding PGPR have been reported by Warwate et al.(2017) which suggested that the improvement in shoot and root length and plant weight is due to the potential of PGPR to produce several growth regulators within plants like indole acetic acid (IAA), gibberellic acid (GA), salicylic acid (SA) and also improve bioavailability of nutrients in the soil. In all experiments, the growth retardation was attributed in the plants which were grown in the lead and nickel contaminated soil at 0.5 mM concentration. Heavy metals accumulate in growing parts of plants and harmfully affect water balance, total chlorophyll content, photosynthesis and finally reduce plant biomass production via decreasing enzyme activity and may cause severe oxidative stress (Riyazuddin et al. 2021). The nickel (Ni) toxicity hinders sugar transportation, photosynthesis, and causes chlorosis and growth retardation in plants (Goyal et al. 2020). The results of heavy metal degradation by P. aeruginosa showed the removal percentage of Pb was about 90.06% and Ni was about 98.8%. Several genera of endophytic bacteria including Pseudomonas sp. are acknowledged to induce heavy metal resistance in plants by metal detoxification via biosurfactant production and stimulate plant growth via IAA synthesis, siderophore production, and ACC deaminase activity (Kumar 2022).
In the present research the biochemical evaluation of proline content indicated that it was highly increased in stressed plants in screen house and field experiments. While the individual and combined application of S. ilicifolium with P. aeruginosa significantly reduced the amounts of these stress markers. Plants cope with the oxidative stress by producing osmolytes and antioxidant molecules like ascorbic acid, cysteine, proline, and glutathione (Mujeeb et al. 2018) while their reduction indicates the alleviation of stress. Similar results were observed in PGPR treated Melissa officinalis plants (Gorgi et al.2022) and P. aeruginosa + S. ilicifolium treated soybean against abiotic stress (Rahman et al. 2020). The results of screen house and field trials showed that the S. ilicifolium and P. aeruginosa decreased peroxidase activity (POD) and superoxide dismutase (SOD) levels which were elevated in stressed plants while total phenols and DPPH were increased. Plants have certain defense mechanisms which comprise plant growth regulators and enzymatic and non-enzymatic antioxidant molecules (Rahim et al. 2022). The metal toxicity enhances levels of reactive oxygen species (ROS) and triggers destruction to membranes, DNA and prolonged exposure may lead to mutation which raises the need of ROS scavengers. Investigation of Saif and Khan (2018) revealed that metal tolerant P. aeruginosa reduced toxicity of chromium (Cr) and nickel (Ni) and turned down the level of proline, POD, SOD, CAT in chickpea. The inoculation of PGPR reduces membrane damage and ROS production caused by lead in plants and decreases the need and production of antioxidants like ascorbic acid, phenols and enzymes like peroxidase (POD) (Mesa-Marín et al. 2018). Another important defensive mechanism of plants is the production of secondary metabolites (phenolic content, alkaloids, terpenoids) which regulate normal metabolic processes linked to plant growth and development and they also act as stress protectant by scavenging the ROS (Maleki et al. 2017). Phenolic content increases in plants under metal stress as they have ability to chelate metals, scavenge ROS and stabilizing membranes (Chen et al. 2019). Pereira et al. (2021) and Remya et al. (2022) acknowledged that Sargassum sp. contain potential antioxidants and secondary metabolites like flavonoid, total phenols that play an important role in the defense mechanism of the host plant by having higher radical scavenging ability.
Present study showed the positive effects of S. ilicifolium seaweeds with heavy metal tolerant endophytic bacterium P. aeruginosa on the growth of sunflower plants under lead and nickel toxicity as it increased the vegetative growth of plant and improved physiological parameters such as proline content, total phenols and antioxidants like peroxidase (POD) activity, superoxide dismutase activity (SOD) and DPPH-radical scavenging activity under stress. This work would help the farmers in ameliorating the harmful effect of lead and nickel on crop plants and reduce the toxicity in the soil. Further investigation is required to understand the mechanism and association of various novel endophytic heavy metal tolerant bacteria along with different seaweeds to reduce toxicity and to improve plant growth attributes with their yield.
Corresponding author
Correspondence to Afshan Rahman
Ethics approval and consent to participate
Not applicable
Consent for publication
All authors are informed and agree to the study.
Funding
This research was funded by Higher education commission (HEC) Startup Research Grant Program (SRGP) having Award Letter No 21-2158.
Competing interests
The authors declare no competing interests
Author contributions
Afshan Rahman: conceptualization, methodology, formal analysis, resources, visualization, supervision, and project administration.
Nawal Gul Shaikh: investigation and prepared the first draft of the manuscript.
Fouzia Naseer: executed editing and review the draft of the manuscript.
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