Effect of Plant Growth Promoting Rhizobacteria (PGPR) and Plant Growth Regulators (PGR) on Antioxidant Properties of Chickpea (Cicer arietinium L.) in Presence of Toxic Effect of Thiamethoxam

Chickpea (Cicer arietinum L.) is a leguminous crop, belonging to Fabaceae family. It is a self-pollinated with chromosomal number 2n = 14. India has a total area of 9.55 million hectares dedicated to chickpea production, with a production of 9.94 million tons and productivity of 806 kg/ha. The productivity of chickpea in Punjab is 700 kg/ha and in Rajasthan, it is 680 kg/ha. Chickpea seeds are composed of carbohydrates (50-58%), protein (15-22%), moisture (7-8%), fat (3.8-10.20%) and micronutrients (<1%) (Tutlani et al., 2023). Chickpea is a vital legume crop valued for its nutritional benefits and economic importance worldwide. As the demand for increased food production grows, it is crucial to develop strategies that not only enhance crop yields but also promote environmental sustainability and human health. Recent studies in agriculture have focused on the role of Plant Growth-Promoting Rhizobacteria (PGPR) in mitigating the negative impacts of pesticides on crops (Zahedi  and Abbasi, 2015). PGPR, a diverse group of beneficial soil bacteria, establishes symbiotic relationships with plants, influencing various aspects of plant growth and stress tolerance (Resmi et al., 2024). They enhance plant health through mechanisms like improved nutrient uptake, systemic resistance induction and regulation of plant hormones (Bhattacharyya et al., 2012).
       
The drive for higher crop yields in modern agriculture has led to the extensive use of chemical pesticides, with Thiamethoxam being particularly prominent due to its efficacy against numerous pests. Introduced as a second- generation neonicotinoid, thiamethoxam is effective in controlling a wide array of pests, including lepidopterans, thrips, hoppers, flea beetles, aphids and whiteflies. However, increasing crutiny has been placed on thiamethoxam because of its persistence in oil and accumulation in plants, posing risks to non-target species (Barnes et al., 1992; Wang et al., 2020). Its success in managing plant diseases like powdery mildew, rusts and fungal rots has further solidified its usage in pre-and post-harvest treatments (Kaur et al., 2022). The continuous use of such pesticides raises concerns about their long-term environmental impacts, necessitating a reassessment of sustainable pest management practices.
       
At the same time, melatonin, traditionally recognized for its role in regulating circadian rhythms in animals, has emerged as a versatile plant growth regulator (PGR). Recent research has demonstrated its significant role in plant stress responses, including oxidative stress reduction, gene expression control and enhancing overall resilience to environmental stresses. This study aims to explore the potential synergistic effects of PGPR and melatonin in aiding the degradation of carbendazim and thiamethoxam in chickpea plants. Microorganisms, including soil biota, are known to degrade various pesticides and certain bacteria use pesticides as their sole carbon source, providing opportunities for bioremediation (Qiu et al., 2007).
               
PGPR, by altering the rhizosphere, boosting plant biomass and improving nutrient uptake, helps plants adapt to harsh conditions (Backer et al., 2018). Specifically, the root microbiome Pseudomonas putida plays a critical role in protecting plants from stress by minimizing ROS-induced cellular damage (Srivastava et al., 2017). P. putida has also been shown to degrade thiamethoxam without producing harmful metabolites (Rana et al., 2011) and it exhibits high resistance to and degradation capacity for this pesticide (Jan et al., 2021). Therefore, supplementing chickpea plants with exogenous P. putida may alleviate pesticide-induced stress. By investigating the interactions between beneficial microbes, plant regulators and pesticide degradation pathways, this study aims to contribute to sustainable pest management practices that align with ecological balance and agricultural resilience. Building on previous research that highlights the complex interplay between PGPR, melatonin and pesticide breakdown, we will explore these relationships in the specific context of chickpea to advance sustainable agricultural practices. The study aimed to evaluate the impact of plant growth promoting rhizobacteria (PGPR) and plant growth regulators (PGR) on the antioxidant properties of chickpea (Cicer arietinum L.). It specifically assessed their effectiveness in mitigating the toxic effects of the insecticide thiamethoxam. The objective was to identify treatments that enhance plant defense and antioxidant potential under chemical stress.

Collection of microbial culture (PGPR)
 
The IDMT-CC3314 accession of the lyophilized strain of Pseudomonas putida was obtained from CSIR- IMTECH in Mohali, India. It was cultivated for 48 hours at 28^oC in 50 millilitres of fresh nutrient broth (NB) media. The culture was centrifuged (Plasto Crafts, Rota 4R-V/Fm) for 20 minutes at 4^oC to extract the pellet. After that, the water was double-distilled and the bacterial population was raised to 109 cells/ml by re-suspending (Jan et al., 2020).
 
Plant growth regulators (PGR)
 
Melatonin and strigolactone
 
Melatonin and Strigolactone was purchased from Himedia laboratories Pvt. Ltd, Mumbai, India. A stock solution of 1 mM was prepared by dissolving melatonin in analytical grade methanol. Different concentrations of melatonin (50 µM and 100 µM) were prepared by serial dilution of stock. For current study, the concentration of melatonin was selected on the basis of effective concentration. Accordingly, 50 µM concentrations was chosen for the experimental work.
 
Plant growth promoting rhizobacteria (PGPR)
 
Pseudomonas putida (MTCC-1194), a plant growth-promoting rhizobacterium (PGPR), was obtained from CSIR-IMTECH in Mohali, Punjab, India. 50 milliliters of sterile nutrient broth medium (NB media; 13 gL^-1) were used to cultivate the lyophilized strain. For the purpose of proliferation, the culture flask was maintained at 28^oC (24 to 48 hours) in a BOD incubator (Calton Deluxe Automatic, New Delhi, India). Using 50 mL of NB media and 1 mL of growing culture, the experiments were conducted at 28^oC for 24 to 48 hours in a BOD incubator. In order to collect pellets, it was centrifuged (Plasto Crafts, Rota 4R-V/Fm) for 20 minutes at 10000 rpm and 4^oC. Pellet wasre-suspended to acquire 10⁹ cells/ml.
       
In vitro germination of seedlings
 
Surface sterilized seeds were immersed in freshly prepared melatonin solution (50 µM) for 7 hours. The melatonin and Strigolactone dosed seeds were swilled with double distilled water, blotted dried and placed in dark at room temperature, until returning to their initial weight (over-night). Autoclaved petri-plates were layered with Whatman (Grade 1) filter paper and added with thiamethoxam solution (0.6 mM). Subsequently, primed seeds were sown in thiamethoxam supplemented petri-plates and simultaneously microbial suspension (10⁹ cells/ml) was inoculated into petri-plates containing seeds. The petri-plates were kept in seed germinator (Caltan, NSW 191-192) under controlled condition (light intensity-175 µmolm^-2s^-1; temperature-25±0.5^oC, photo-period-16 hours). After 10 days sowing, the seedlings were harvested for further analysis. Fig 1 showed the seedling growth after applying percentage treatment of thiamethoxam in chickpea.


 
Photosynthetic parameter evaluation
 
The content of anthocyanin, total flavonoid, carotenoid, chlorophyll-a and chlorophyll-b was determined using a Shimadzu UV-1800 UV-Vis Spectrophotometer.
 
Estimation of chlorophyll and carotenoid content
 
After homogenizing 0.2 g of fresh plant material in a cooled pestle-motor with 4 mL of 80% acetone, the sample was centrifuged at 12,000 RPM for 20 minutes at 4^oC and the absorbance was recorded. The following wave lengths: 480 and 510 nm for carotenoid concentration and 645 and 663 nm for chlorophyll. The procedure was adhered to in order to determine the content (Arnon et al., 1949; Maclachlan et al., 1963).
 
Anthocyanin content estimation
 
In order to estimate the anthocyanin concentration, 0.35 g of fresh plant tissue was ground up in a iced pestle-motor using a 3 ml extraction mixture that contained 0.03 mL, 2.37 mL, 0.6 mL and 0.03 mL of methanol, HCl and D.H₂O, respectively. The absorbance measurements were taken at 657 and 530 nm (Mancinelli et al., 1984).
 
Total flavonoid content estimation
 
Using a chilled pestle-motor, a fresh plant sample weighing 0.35 g was crushed in 3 mL of absolute methanol. After centrifugation at 4^oC, 12,000 rpm and 20 minutes, the resulting supernatant was diluted by adding 0.3 mL, 0.3 mL and 4 mL of NaNO₂, AlCl₃ and D.H₂O, respectively. Following the development of a pink hue during incubation, 2 milliliters of NaOH were added and the optical density at 510 nm was measured (Kim et al., 2021).
 
Osmolyte content estimation
 
Trehalose content
 
After crushing 10 mg of oven-dried plant material in 80% ethanol, the mixture was centrifuged at 4^oC, 5000 rpm and for 15 minutes. Four milliliters and two milliliters of TCA anthrone reagent were added to the 0.1 milliliter supernatant, resulting in the production of a yellow complex with an absorbance measured at 620 nm (Trevelyan et al., 1956). Trehalose was measured using D-glucose as a reference and the result was represented in mg/gm DW.
 
Glycine-betaine content
 
10 mg oven dried plant material was crushed in 5 mLD.H₂O containing 0.05% of toluene followed by filtration after incubation (24 hours). Following an ice-cold treatment, 10 mL and 2 mL of 1, 2-dichloromethane and D.H₂O, respectively, were added to the mixture containing 0.1 mL potassium tri-iodide, 1 mL 2N HCl and 0.5 mL filtrate. Thorough mixing of reaction tubes was done till two separate layers were formed. Upper layer was removed and absorbance of pink coloured lower layer was recorded at 365 nm. Standard curve of betaine hydrochloride was plotted and used for assessing the glycine-betaine content (Grieve et al., 1983).
 
Proline content
 
A 250 mg plant sample was ground up in 10 mL of sulpho salicylic acid (3%) and centrifuged at 4^oC for 10 min at 10,000 rpm. Two milliliters of ninhydrin and glacial acetic acid were then added to two milliliters of supernatant. This was treated with a water bath for one hour at a temperature of 100^oC. The reaction was then terminated by moving it to an ice bath. In addition, 4 mL of toluene was added and agitated for 50-60 seconds. After the toluene layer was removed, absorbance was measured at 520 nm and quantified in mg/gm using a standard plot of L-proline (Bates et al., 1982).
 
Estimation of oxidative stress markers
 
Quantification of superoxide anion (O.-2) content
 
After homogenizing 500 mg of fresh plant tissue in 4 ml of phosphate buffer (65 mM, pH-7.8) with 1% PVP, the mixture was centrifuged at 4^oC for 15 minutes at 12,000 rpm. The supernatant was then combined with 0.1 mL and 0.5 mL of hydroxylamine hydrochloride and phosphate buffer, respectively. At room temperature, the amalgam was incubated for thirty minutes. After incubating the combination containing 1 mL of 1-napthylamine and 1 mL of 3-amino benzene sulphonic acid, absorbance was finally measured at 520 nm. By using sodium nitrate, the quantification was given in ìmole/g FW (Wu et al., 2010).
 
Hydrogen peroxide (H₂O₂) content
 
Fresh plant material (500 mg) was pulverized in 2 mL trichloroacetic acid (1M) and subjected to centrifugation (Time= 15 min; Temperature= 4^oC rpm= 5000) followed by addition of 0.5 mL of KI (Molarity=1M) and 1mL of potassium phosphate buffer (PPB) (Molarity= 10 mM) into 0.5 mL supernatant. The absorbance (wavelength = 390 nm) was recorded for quantification in mmole/g FW by taking hydrogen peroxide (Velikova et al., 2011).

The results offer a comparative evaluation of several biochemical and physiological parameters across various treatment combinations. Each comparison is classified as highly significant (HS), moderately significant (MS), low significant (LS), or non-significant (NS). The key comparisons focus on treatments involving TMX (thiamethoxam), MEL (melatonin), PGPR (plant growth-promoting rhizobacteria) and Strigolactone, both in relation to the control group and between the different treatment combinations. (Table 1 and 2).




       
Thiamethoxam elevates oxidative stress, whereas the application of melatonin and PGPR mitigates its levels in chickpea seedlings exposed to thiamethoxam. The TMX+MEL+PGPR treatment results in the highest overall chlorophyll content, while the inclusion of STGR significantly reduces total chlorophyll (T-Chl) levels. However, the combination of TMX+STGR+MEL+PGPR leads to the highest chlorophyll-a (Chl-a) production, suggesting a positive interaction between these components. TMX+MEL alone exhibits the greatest impact on chlorophyll-b (Chl-b), indicating that melatonin may have a specific role in boosting Chl-b levels, though the addition of STGR diminishes this effect. These findings indicate that different treatments influence chlorophyll synthesis in distinct ways, with TMX+MEL and TMX+STGR combinations having different impacts on chlorophyll type balance (Fig 2).


       
A highly significant increase in total chlorophyll is observed with TMX alone compared to the control and when MEL or PGPR is added, chlorophyll levels rise further, highlighting their supportive role in boosting chlorophyll content. However, the addition of strigolactone causes a highly significant decrease, indicating a possible antagonistic effect between STRG and other components in chlorophyll synthesis. Similar trends are seen with chlorophyll-a, where the addition of STRG shows no significant improvement. Previous research has shown that melatonin can counteract the decline in total chlorophyll synthesis, potentially due to increased uptake of magnesium and nitrogen, as well as reduced chlorophyll degradation (Alharbi et al., 2021). Additionally, melatonin has been linked to enhanced photosynthesis and PSII activity (Kaya et al., 2019).
       
The treatments with TMX+MEL and TMX+PGPR result in significantly higher anthocyanin levels compared to the control, indicating these combinations may enhance the plant’s stress defense mechanisms. However, the combination of TMX+MEL+PGPR or TMX+STGR+MEL+ PGPR leads to a decrease in anthocyanin content, which could suggest an interaction that negatively affects anthocyanin production. A substantial reduction in anthocyanin levels observed with TMX+MEL suggests that melatonin may inhibit its production. Similarly, the comparison between TMX+PGPR+MEL and TMX+PGPR+ MEL+STRG shows a notable decrease, indicating that STRG may interfere with the anthocyanin-enhancing effects of PGPR and MEL (Fig 2).
       
P. putida treatment upregulates the antioxidant enzymes activity in TMX dosed (Cicer arietinium L.) seedlings while strigolactones have less effect to reduce toxicity of thiamethoxam. The TMX+MEL treatment results in the highest flavonoid content, suggesting asynergistic interaction between melatonin and thiamethoxam. In contrast, the TMX+PGPR combination leads to are duction in flavonoid levels, though adding MEL to TMX+PGPR brings the levels closer to those seen in the TMX+MEL treatment. The TMX+STGR+MEL+PGPR combination significantly reduces flavonoid content, indicating potential antagonistic effects (Fig 2). TMX+MEL increases trehalose levels more than the control and other treatments, indicating melatonin’s role in enhancing stress tolerance via trehalose accumulation. While TMX+PGPR also elevate trehalose levels, the TMX+MEL+PGPR combination shows a slight decrease in comparison (Fig 2).
       
In the pursuit of protecting and enhancing crop yields, pesticides are often used excessively (Jan et al., 2020). While thiamethoxam is effective in controlling insect populations, it also leaves behind significant toxic residuesin crops, posing potential risks to living organisms (Saran et al., 2018 and Riascos-Flores et al., 2021). In recent times, microorganisms have been successfully introduced into plants to aid in the remediation of pesticides (Nurzhanova et al., 2021 and Jan et al., 2023). Various studies have shown that PGPRs (plant growth-promoting rhizobacteria) enhance plant growth by activating defense mechanisms under stress conditions (Seth et al., 2021; Bibi et al., 2024 and Zahedi and Abbasi, 2015). This study explores the ability of Pseudomonas putida to alleviate the adverse effects of thiamethoxam in chickpea seedlings. Morphological analysis indicated that thiamethoxam negatively affected seedlings, resulting in reduced seedling length, fresh weight and dry weight. Our findings align with earlier research, which reported significant growth inhibition in germination rate, root length and weight in Allium cepa seedlings treated with thiamethoxam (Cavuşoğlu et al., 2012). The reduction in plant biomass and growth can be attributed to disruptions in chlorophyll synthesis, photosynthesis, nutrient absorption and imbalances in hormones and water regulation (Jan et al., 2020).
       
The exposure of thiamethoxam leads to decrease in chlorophyll content as well as pigments like anthocyanins, carotenoids and flavonoids, whereas all these parameters showed a marked increase in P. putida-treated seedlings exposed to thiamethoxam. The reduction in chlorophyll content may be due to increased chlorophyllase activity, chloroplast degradation and chlorophyll oxidation caused by reactive oxygen species (ROS) (Harpaz-Saad et al., 2007). Additionally, previous research has documented that thiamethoxam reduces chlorophyll levels in algae (Al-Badri et al., 2020) and negatively affects photosynthetic pigments and the functionality of the photosynthetic apparatus in Zea mays (Todorenko et al., 2020).
       
Abiotic stressors, including drought, heat, ultraviolet rays and cold temperatures, significantly hinder plant growth through various mechanisms, leading to reduced crop yields (Yilmaz and Kulaz, 2018). Among these factors, drought is particularly complex, as its recurring natured adversely impacts agriculture, the economy, water resources and ecosystems (Zhou et al., 2021). Similarly, addition of pesticides results in water deficiency, protein degradation and a decrease in the accumulation of organic compounds within plants. These changes lead to osmotic stress, which negatively affects plant growth, development, biomass and chlorophyll production. Consequently, the present study demonstrates that co-inoculation of Pseudomonas putida and melatonin significantly enhances growth and development, increases biomass and chlorophyll content and lowers reactive oxygen species (ROS) levels to mitigate pesticide stress (Rangecroft et al., 2019 and Huseynova et al., 2016).

The current study reveals that thiamethoxam negatively impacts the growth characteristics and photosynthetic pigment levels in seedlings, leading to excessive reactive oxygen species (ROS) production and significant redox imbalance. However, the addition of Pseudomonas putida and plant growth regulators (PGRs) such as melatonin and strigolactone enhances the plant’s defense mechanisms by increasing the activity of ROS- scavenging antioxidative enzymes and antioxidants. Thiamethoxam also promotes the accumulation of osmolytes, which is further amplified by P. putida inoculation, helping to alleviate oxidative stress in seedlings. In conclusion, P. putida demonstrates strong potential to mitigate the stress caused by thiamethoxam. Thus, applying P. putida externally in combination with PGR could be a promising strategy for managing pesticide-related stress from an agronomic perspective. Given the concerns surrounding pesticide-induced toxicity in crops, it is crucial to develop effective strategies to address this issue. Further research utilizing biotechnological and genetic approaches could uncover the mechanisms behind P. putida-induced tolerance to thiamethoxam.

The authors would like to express their gratitude to the CSIR-IMTECH in Mohali, Punjab, India for providing Pseudomonas putida (MTCC-1194), a plant growth-promoting rhizobacterium (PGPR) and Lovely Professional University (LPU) for providing the necessary research facilities to conduct this work.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

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.