Plant growth-promoting rhizobacteria improved growth and physio-biochemical properties of geranium (Pelargonium graveolens L.) under salinity stress
Subject Areas : Phytochemistry
Qolamreza Mirzakhani
1
,
Marzieh Ghanbari Jahromi
2
*
,
Vahid Abdossi
3
1 - Department of Horticultural Science and Agronomy, Science and Research Branch, Islamic Azad University, Tehran, Iran
2 - Department of Horticultural Science and Agronomy, Science and Research Branch, Islamic Azad University, Tehran, Iran
3 - Department of Horticultural Science and Agronomy, Science and Research Branch, Islamic Azad University, Tehran, Iran
Keywords: Azotobacter, Pseudomonas, Growth regulation, Salt stress, Secondary metabolites ,
Abstract :
The use of plant growth-promoting bacteria (PGPRs) to modulate salinity is of utmost importance for enhancing plant growth and adaptation in saline environments. This study aimed to investigate the effects of Azotobacter chroococcum and Pseudomonas putida on the growth and biochemical characteristics of rose-scented geranium under salinity stress. The experiment was conducted using a factorial design with four levels of PGPR treatment (control, Azotobacter, Pseudomonas, and Azotobacter + Pseudomonas) and three levels of salt at 0, 60, and 120 mM NaCl. The results indicate that salinity stress resulted in a decrease in plant yield, with the high est reduction observed at 120 mM salinity. This led to reductions in plant weight (31%), root weight (37%), total chlorophyll (33%), relative water content (RWC, 19%), essential oil yield (25%), as well as increases in malondialdehyde (MDA, 45%), catalase activity (179%), and superoxide dismutase activity (100%). However, the inoculation of geranium plants with PGPRs, particularly the simultaneous application of Azotobacter and Pseudomonas, resulted in stress mitigation. This was evident through an increase in biomass, photosynthetic rate, RWC, as well as a reduction in the activity of antioxidant enzymes and MDA in the leaves. Among the different treatments, the combined application of Azotobacter and Pseudomonas, along with a salinity stress level of 60 mM, resulted in the highest production of secondary metabolites, including total phenols, flavonoids, and essential oil content. In conclusion, the combined treatment of Azotobacter and Pseudomonas is recommended as an effective approach to mitigate salinity stress and increase plant yield in rose-scented geranium.
Abdel Latef, A. a. H., A. M. Omer, A. A. Badawy, M. S. Osman and M. M. Ragaey. 2021. Strategy of salt tolerance and interactive impact of Azotobacter chroococcum and/or Alcaligenes faecalis inoculation on canola (Brassica napus L.) plants grown in saline soil. Plants, 10, (1) 110.
Ahmad, B., H. Jaleel, Y. Sadiq, M. M. A. Khan and A. Shabbir. 2018. Response of exogenous salicylic acid on cadmium induced photosynthetic damage, antioxidant metabolism and essential oil production in peppermint. Plant Growth Regulation, 86, 273-286.
Alharbi, K., E. Rashwan, H. H. Mohamed, A. Awadalla, A. E.-D. Omara, E. M. Hafez and T. Alshaal. 2022. Application of silica nanoparticles in combination with two bacterial strains improves the growth, antioxidant capacity and production of barley irrigated with saline water in salt-affected soil. Plants, 11, (15) 2026.
Arnon, D. I. 1949. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant physiology, 24, (1) 1.
Aslani, Z., A. Hassani, B. A. Mandoulakani, M. Barin and R. Maleki. 2024. The symbiotic association with Piriformospora indica and Pseudomonas fluorescens improves salt tolerance in sage (Salvia officinalis) plants. Plant and Soil, 495, (1) 391-410.
Bergman, M. E., Á. Chávez, A. Ferrer and M. A. Phillips. 2020. Distinct metabolic pathways drive monoterpenoid biosynthesis in a natural population of Pelargonium graveolens. Journal of experimental botany, 71, (1) 258-271.
Cui, B., X. Wang, Y. Su, C. Gong, D. Zhang, Z. Ouyang and X. Wang. 2022. Responses of tree growth, leaf area and physiology to pavement in Ginkgo biloba and Platanus orientalis. Frontiers in Plant Science, 13, 1003266.
Darakeh, S. a. S. S., W. Weisany, M. Diyanat and R. Ebrahimi. 2021. Bio-organic fertilizers induce biochemical changes and affect seed oil fatty acids composition in black cumin (Nigella sativa Linn). Industrial Crops and Products, 164, 113383.
Del Rosario Cappellari, L., M. V. Santoro, A. Schmidt, J. Gershenzon and E. Banchio. 2019. Induction of essential oil production in Mentha x piperita by plant growth promoting bacteria was correlated with an increase in jasmonate and salicylate levels and a higher density of glandular trichomes. Plant Physiology and Biochemistry, 141, 142-153.
Diagne, N., M. Ndour, P. I. Djighaly, D. Ngom, M. C. N. Ngom, G. Ndong, S. Svistoonoff and H. Cherif-Silini. 2020. Effect of plant growth promoting rhizobacteria (PGPR) and arbuscular mycorrhizal fungi (AMF) on salt stress tolerance of Casuarina obesa (Miq.). Frontiers in Sustainable Food Systems, 4, 601004.
El-Beltagi, H. S., I. Ahmad, A. Basit, H. M. Abd El-Lateef, M. Yasir, S. Tanveer Shah, I. Ullah, M. Elsayed Mohamed Mohamed, I. Ali and F. Ali. 2022. Effect of azospirillum and azotobacter species on the performance of cherry tomato under different salinity levels. Gesunde Pflanzen, 74, (2) 487-499.
Feizi, H., R. Moradi, N. Pourghasemian and H. Sahabi. 2021. Assessing saffron response to salinity stress and alleviating potential of gamma amino butyric acid, salicylic acid and vermicompost extract on salt damage. South African Journal of Botany, 141, 330-343.
Ghassemi-Golezani, K. and S. Abdoli. 2023. Alleviation of salt stress in rapeseed (Brassica napus L.) plants by biochar-based rhizobacteria: new insights into the mechanisms regulating nutrient uptake, antioxidant activity, root growth and productivity. Archives of Agronomy and Soil Science, 69, (9) 1548-1565.
Hasanuzzaman, M., M. B. Bhuyan, F. Zulfiqar, A. Raza, S. M. Mohsin, J. A. Mahmud, M. Fujita and V. Fotopoulos. 2020. Reactive oxygen species and antioxidant defense in plants under abiotic stress: Revisiting the crucial role of a universal defense regulator. Antioxidants, 9, (8) 681.
Hasanuzzaman, M., M. R. H. Raihan, A. a. C. Masud, K. Rahman, F. Nowroz, M. Rahman, K. Nahar and M. Fujita. 2021. Regulation of reactive oxygen species and antioxidant defense in plants under salinity. International Journal of Molecular Sciences, 22, (17) 9326.
Hashemi, M., B. Behboodian, E. Karimi and E. Oskoueian. 2022. Enhancing biosynthesis and bioactivity of Trachyspermum ammi seed essential oil in response to drought and Azotobacter chroococcum stimulation. Chemical and Biological Technologies in Agriculture, 9, (1) 26.
Hazrati, S., A. Ertani and S. Nicola. 2022. Production and quality of medicinal and aromatic plants: Recent findings on stress effects, elicitors, harvesting and market development. p. 1109: MDPI
Heath, R. L. and L. Packer. 1968. Photoperoxidation in isolated chloroplasts: II. Role of electron transfer. Archives of biochemistry and biophysics, 125, (3) 850-857.
Jaradat, N., M. Hawash, M. Qadi, M. Abualhasan, A. Odetallah, G. Qasim, R. Awayssa, A. Akkawi, I. Abdullah and N. Al-Maharik. 2022. Chemical markers and pharmacological characters of Pelargonium graveolens essential oil from Palestine. Molecules, 27, (17) 5721.
Javardi, N. S. M., M. Pakravan, P. Panahi and R. Zarei. 2023. Assessment of the Relationships Between Leaf Characteristics with Air Pollutants: A Case Study on Oriental Plane (Platanus orientalis L.) and Caucasian Hackberry (Celtis caucasica Willd). Arboriculture & Urban Forestry (AUF), 49, (6) 329-342.
Khodadadi, R., R. Ghorbani Nasrabadi, M. Olamaee and S. Movahedi Naini. 2020. Effect of Azotobacter and Azospirillum on growth and physiological characteristics of barley (Hordeum vulgare) under salinity stress. Water and Soil, 34, (3) 649-660.
Mazeed, A., P. Maurya, D. Kumar, S. S. Yadav and P. Suryavanshi. 2022. Efficient nutrient management for rose scented geranium (Pelargonium graveolens L′ Herit ex Ait). Journal of Applied Research on Medicinal and Aromatic Plants, 31, 100409.
Minuț, M., M. Diaconu, M. Roșca, P. Cozma, L. Bulgariu and M. Gavrilescu. 2022. Screening of Azotobacter, Bacillus and Pseudomonas species as plant growth-promoting bacteria. Processes, 11, (1) 80.
Mirzaei, M., A. Ladan Moghadam, L. Hakimi and E. Danaee. 2020. Plant growth promoting rhizobacteria (PGPR) improve plant growth, antioxidant capacity, and essential oil properties of lemongrass (Cymbopogon citratus) under water stress. Iranian Journal of Plant Physiology, 10, (2) 3155-3166.
Nasirzadeh, L., B. Sorkhilaleloo, E. Majidi Hervan and F. Fatehi. 2021. Changes in antioxidant enzyme activities and gene expression profiles under drought stress in tolerant, intermediate, and susceptible wheat genotypes. Cereal Research Communications, 49, 83-89.
Nawaz, A., M. Shahbaz, Asadullah, A. Imran, M. U. Marghoob, M. Imtiaz and F. Mubeen. 2020a. Potential of salt tolerant PGPR in growth and yield augmentation of wheat (Triticum aestivum L.) under saline conditions. Frontiers in Microbiology, 11, 2019.
Nawaz, M., S. A. Anjum, U. Ashraf, F. Azeem and Z. Wang. 2020b. Antioxidant defense system and reactive oxygen species (ROS) interplay in plants under drought condition. Handbook of Climate Change Management: Research, Leadership, Transformation, 1-25.
Nehela, Y., Y. S. Mazrou, T. Alshaal, A. M. Rady, A. M. El-Sherif, A. E.-D. Omara, A. M. Abd El-Monem and E. M. Hafez. 2021. The integrated amendment of sodic-saline soils using biochar and plant growth-promoting rhizobacteria enhances maize (Zea mays L.) resilience to water salinity. Plants, 10, (9) 1960.
Neshat, M., A. Abbasi, A. Hosseinzadeh, M. R. Sarikhani, D. Dadashi Chavan and A. Rasoulnia. 2022. Plant growth promoting bacteria (PGPR) induce antioxidant tolerance against salinity stress through biochemical and physiological mechanisms. Physiology and Molecular Biology of Plants, 28, (2) 347-361.
Omer, A. M., M. S. Osman and A. A. Badawy. 2022. Inoculation with Azospirillum brasilense and/or Pseudomonas geniculata reinforces flax (Linum usitatissimum) growth by improving physiological activities under saline soil conditions. Botanical Studies, 63, (1) 15.
Pagán, E., J. Robles, A. Temnani, P. Berríos, P. Botía and A. Pérez-Pastor. 2022. Effects of water deficit and salinity stress on late mandarin trees. Science of the Total Environment, 803, 150109.
Rashmi, D., W. A. Ansari, N. Y. Kadoo, V. T. Barvkar, R. Deshmukh and A. B. Nadaf. 2023. Role of ions and their transporters in combating salt stress in Pandanus odorifer (Forssk.) Kuntze. Acta Physiologiae Plantarum, 45, (5) 66.
Ritchie, S. W., H. T. Nguyen and A. S. Holaday. 1990. Leaf water content and gas‐exchange parameters of two wheat genotypes differing in drought resistance. Crop science, 30, (1) 105-111.
Rossi, S., C. Chapman and B. Huang. 2020. Suppression of heat-induced leaf senescence by γ-aminobutyric acid, proline, and ammonium nitrate through regulation of chlorophyll degradation in creeping bentgrass. Environmental and Experimental Botany, 177, 104116.
Sapre, S., I. Gontia-Mishra and S. Tiwari. 2022. Plant growth-promoting rhizobacteria ameliorates salinity stress in pea (Pisum sativum). Journal of Plant Growth Regulation, 41, (2) 647-656.
Sefidkon, F., K. Abbasi and G. B. Khaniki. 2006. Influence of drying and extraction methods on yield and chemical composition of the essential oil of Satureja hortensis. Food chemistry, 99, (1) 19-23.
Shalaby, O. A. and M. E.-S. Ramadan. 2024. Mycorrhizal colonization and calcium spraying modulate physiological and antioxidant responses to improve pepper growth and yield under salinity stress. Rhizosphere, 29, 100852.
Ullah, S., A. Bano, A. Ullah, M. A. Shahid and N. Khan. 2022. A comparative study of plant growth promoting rhizobacteria (PGPR) and sowing methods on nutrient availability in wheat and rhizosphere soil under salinity stress. Rhizosphere, 23, 100571.
Xu, B. J. and S. K. Chang. 2007. A comparative study on phenolic profiles and antioxidant activities of legumes as affected by extraction solvents. Journal of food science, 72, (2) S159-S166.
Yaghoubian, I., S. Ghassemi, M. Nazari, Y. Raei and D. L. Smith. 2021. Response of physiological traits, antioxidant enzymes and nutrient uptake of soybean to Azotobacter chroococcum and zinc sulfate under salinity. South African Journal of Botany, 143, 42-51.
Zhishen, J., T. Mengcheng and W. Jianming. 1999. The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food chemistry, 64, (4) 555-559.
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Plant growth-promoting rhizobacteria improved growth and physio-biochemical properties of geranium (Pelargonium graveolens L.) under salinity stress
Qolamreza Mirzakhani, Marzieh Ghanbari Jahromi* and Vahid Abdossi
Department of Horticultural Science and Agronomy, Science and Research Branch, Islamic Azad University, Tehran, Iran.
_____________________________________________________________________________
Abstract
The use of plant growth-promoting bacteria (PGPRs) to modulate salinity is of utmost importance for enhancing plant growth and adaptation in saline environments. This study aimed to investigate the effects of Azotobacter chroococcum and Pseudomonas putida on the growth and biochemical characteristics of rose-scented geranium under salinity stress. The experiment was conducted using a factorial design with four levels of PGPR treatment (control, Azotobacter, Pseudomonas, and Azotobacter + Pseudomonas) and three levels of salt at 0, 60, and 120 mM NaCl. The results indicate that salinity stress resulted in a decrease in plant yield, with the high est reduction observed at 120 mM salinity. This led to reductions in plant weight (31%), root weight (37%), total chlorophyll (33%), relative water content (RWC, 19%), essential oil yield (25%), as well as increases in malondialdehyde (MDA, 45%), catalase activity (179%), and superoxide dismutase activity (100%). However, the inoculation of geranium plants with PGPRs, particularly the simultaneous application of Azotobacter and Pseudomonas, resulted in stress mitigation. This was evident through an increase in biomass, photosynthetic rate, RWC, as well as a reduction in the activity of antioxidant enzymes and MDA in the leaves. Among the different treatments, the combined application of Azotobacter and Pseudomonas, along with a salinity stress level of 60 mM, resulted in the highest production of secondary metabolites, including total phenols, flavonoids, and essential oil content. In conclusion, the combined treatment of Azotobacter and Pseudomonas is recommended as an effective approach to mitigate salinity stress and increase plant yield in rose-scented geranium.
Keywords: Azotobacter, Pseudomonas, Growth regulation, Salt stress, Secondary metabolites
Mirzakhani Q., M. Ghanbari Jahromi and V. Abdossi. 2025. Plant growth-promoting rhizobacteria improved growth and physio-biochemical properties of geranium (Pelargonium graveolens L.) under salinity stressIranian Journal of Plant Physiology 15(1), 5391-5405.
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____________________________________ * Corresponding Author E-mail Address: ghanbari@srbiau.ac.ir Received: February, 2024 Accepted: September, 2024
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Salinity stress negatively affects plant physiological processes such as photosynthesis and antioxidant systems. High salt concentrations disrupt photosynthetic efficiency, reducing carbon dioxide availability and energy production. Salinity stress also induces oxidative stress by producing reactive oxygen species (ROS) at a rate that exceeds the plant's antioxidant defense mechanisms (Sapre et al., 2022). This imbalance leads to damage to cellular structures and impairment of plant development. However, plants have adaptive mechanisms to restore photosynthetic activity and enhance antioxidant defenses. Understanding these effects is important for developing strategies to improve plant tolerance to salinity stress (Nawaz et al., 2020a). In recent years, researchers and farmers alike have shown an increasing interest in exploring alternative approaches to alleviate the negative impacts of salinity stress on plants. In recent times, there has been a growing enthusiasm among researchers and farmers to explore effective and environmentally friendly methods to mitigate the adverse effects of salinity stress on plants (Neshat et al., 2022; Sapre et al., 2022).
One promising strategy is the utilization of plant growth-promoting rhizobacteria (PGPR). These beneficial bacteria colonize the rhizosphere, the region surrounding the root system, and form a mutually beneficial relationship with plants. PGPR have been found to enhance plant growth and development through various mechanisms, including facilitating nutrient uptake, boosting stress tolerance, and promoting hormonal balance (Diagne et al., 2020). Azobacter and Pseudomonas are two types of bacteria that have shown potential in mitigating the negative impacts of salinity stress on plants. Azobacter is a nitrogen-fixing bacteria that can form a symbiotic relationship with certain plants, providing them with a source of fixed nitrogen and promoting growth (Ullah et al., 2022). Pseudomonas bacteria produce plant growth-promoting substances and enzymes that improve nutrient availability, water uptake, and antioxidant activity in plants. Both bacteria offer a natural and sustainable solution to enhance crop productivity in saline environments (Abdel Latef et al., 2021).
Geranium (Pelargonium graveolens L.) is a slow-growing perennial plant from the Geraniaceae family. Most plants in this family are herbaceous and rarely acquire a woody nature. Geraniums come in various types and are flowering plants that hold economic importance (Mazeed et al., 2022). The hydraulic extract derived from the aromatic geranium leaf is abundant in flavonoid compounds and essential fatty acids (Jaradat et al., 2022). It also contains multiple vitamins, including vitamin A, E, and coumarin. This extract plays a significant role in the breeding efforts of geraniums, aiming to produce different varieties and hybrids with distinct shapes, colors, and more beneficial compounds (Mazeed et al., 2022). The extraction of essential oils (EOs) is primarily performed using the leaves and aerial parts of the plant (Bergman et al., 2020).
The positive effects of PGPRs on mitigating salinity stress have been reported in various plants such as canola (Abdel Latef et al., 2021; Ghassemi-Golezani and Abdoli, 2023), Casuarina obesa (Diagne et al., 2020), and cherry tomato (El-Beltagi et al., 2022). However, limited information is available regarding the effect of co-applied Azotobacter and Pseudomonas species on modulating salinity in geranium. This research aimed to address this knowledge gap by exploring the potential of PGPR in enhancing geranium's tolerance to salinity stress by investigating physiological and biochemical attributes in geranium plants. The findings will provide valuable insights into the mechanisms by which PGPR promotes plant growth and physiological adaptations, contributing to the development of sustainable solutions for managing salinity stress in geranium and potentially other ornamental and medicinal species.
Materials and Methods
Plant Materials and Experimental Treatments
Cuttings of the geranium plant were initially grown in a culture medium containing sand. After one month, when rooting and leafing (4 leaves) were observed, they were transplanted into plastic pots filled with agricultural soil with an EC of 1.1 dS m⁻¹ and a pH of 7.1. The experimental design was conducted in a factorial arrangement based on a randomized complete block design (RCBD) with three levels of salinity and four levels of PGPRs in three replications. Four levels of PGPRs were applied to the plant roots by soaking: control (no bacteria), Azotobacter, Pseudomonas, and a combination of Azotobacter and Pseudomonas. Azotobacter chroococcum and Pseudomonas putida were used in a soluble form, isolated, and purified by the Soil Biological Research Department of the Soil and Water Research Institute, Karaj, Iran. The inoculum population used was approximately 10⁸ colony-forming units (CFU). One month after transplantation, salt stress was induced by applying sodium chloride (NaCl) at three levels: 0 (control), 60, and 120 mM. Each pot received 200 mL of the NaCl solution. To prevent salt accumulation, the pots were rinsed with water (without salt) after every four irrigations with salt water. The stress period lasted for 40 days, and the plants were harvested in the middle of the flowering stage. The total plant growth period was 100 days, during which no fertilizers or pesticides were applied.
Plant Weight
The fresh weight of shoots and roots after harvesting was measured with a digital scale accurate to 0.01 g. The aerial parts were cut from the collar with scissors, and all the aboveground parts (stem, flower, and leaf) were measured. The roots were gently separated from the soil and weighed with a digital scale.
Total Chlorophyll (Chl)
The Chl content assay was conducted using the method developed by Arnon (1949). A 0.1 g leaf sample was ground in a mortar with 3 mL of 80% acetone and diluted to a final volume of 15 mL. The extract was clarified by centrifugation at 5000 × g for 10 min. The absorbance was measured using a spectrophotometer (Shimadzu UV-160) at wavelengths of 645 nm and 663 nm. Total Chl (mg g⁻¹) contents were calculated using the equation:
[(20.2 × A645) + (8.02 × A663)] × V / 1000 × W
where A represents the absorbance at the specified wavelengths, V is the final volume of 80% acetone in mL, and W is the weight of the fresh leaf sample in g.
Relative Leaf Water Content (RWC) Measurement
RWC was measured by weighing fully developed leaves, hydrating them in distilled water for 4-5 hours, drying them with filter paper, and reweighing. The leaves were then dried at 70 °C for 48 hours to obtain the dry weight. RWC was calculated using the equation:
RWC = (FW - DW) / (TW - DW) × 100
where FW is the fresh weight, DW is the dry weight, and TW is the turgid weight (Ritchie et al., 1990).
Malondialdehyde (MDA) Concentration
MDA concentration was determined using the method of Heath and Packer (1968). A 0.5 g fresh leaf sample was ground with trichloroacetic acid (TCA), centrifuged, and the supernatant mixed with TBA. After boiling and cooling, absorbance was measured at 532 nm and 600 nm. The concentration was calculated in µmol g⁻¹ FW using calibration standards.
Enzyme Assay
Enzyme assays for catalase (CAT) and superoxide dismutase (SOD) activities were performed using fresh samples homogenized in potassium phosphate buffer with EDTA-Na₂ and ascorbate. CAT activity was measured by the decline in H₂O₂ concentration, and SOD activity by the reduction of NBT in the presence of riboflavin, with absorbance readings taken at room temperature (Nasirzadeh et al., 2021).
Determination of Total Phenolic Content (TPC)
TPC was measured using Folin–Ciocalteu reagent and spectrophotometry at 725 nm, with results expressed as mg Gallic acid (GA) g⁻¹ dry weight (Xu and Chang, 2007).
Determination of Total Flavonoid Content (TFC)
Fig. I. Shoot weight and root weight of geranium plants under salinity (a and b) and Azotobacter chroococcum (AC) and Pseudomonas putida (PP) inoculation (c and d). Different letters above the column show statistical significance (P≤0.05).
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Essential Oil (EO) Content and Yield
EO content was determined by hydro-distillation of 100 g dried plant material using a Clevenger-type apparatus, with yield calculated from EO content and plant dry weight (Sefidkon et al., 2006).
Data Analysis
Data were analyzed using SAS (version 9.3, SAS Institute, Cary, NC) with mean comparisons made using the LSD test at a 5% probability level (P ≤ 0.05).
Results
Plant Weight
The shoot weight and root weight of the plants decreased significantly with increasing levels of salinity stress. The lowest shoot weight and root weight were observed in the 120 mM salinity stress treatment. The shoot weight decreased by 12% and 31% in the 60 and 120 mM salinity stress treatments, respectively, compared to the control treatment (Fig. I a). The root weight decreased by 22% and 37% in the 60 and 120 mM salinity stress treatments, respectively, compared to the control treatment (Fig. I b). These results indicate that salinity stress negatively impacts plant growth, with higher levels of salinity stress resulting in greater reductions in shoot and root weight.
Fig. II. Total chlorophyll (Chl, a) and relative water content (RWC, b and c) of geranium plants under salinity and Azotobacter chroococcum (AC) and Pseudomonas putida (PP) inoculation. Different letters above the column show statistical significance (P≤0.05).
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Chl and RWC
The results indicated that the mutual effect of salinity stress and growth stimulants had a significant impact on the total Chl content of the plants. The salinity stress treatments led to a decrease in total Chl compared to the control treatment. However, when the growth stimulants were applied in combination with salinity stress, the total Chl content improved compared to the control treatment without growth stimulants. The highest total Chl content was observed in the control treatment and the combination of Azotobacter and Pseudomonas in the absence of salt stress. In the 120 mM salt stress treatment, the combination of Azotobacter and Pseudomonas resulted in a 17% increase in total Chl compared to the control treatment without growth stimulants. These findings suggest that the application of growth stimulants can mitigate the negative effects of salinity stress on Chl content and help improve the plant's photosynthetic capacity (Fig. II a).
Fig. III. Malondialdehyde (MDA, a), total phenolic content (TPC, b), and total flavonoid content (c and d) of geranium plants under salinity and Azotobacter chroococcum (AC) and Pseudomonas putida (PP) inoculation. Different letters above the column show statistical significance (P≤0.05).
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MDA, TPC, and TFC
The interaction between salinity stress and PGPRs had a significant effect on MDA levels. Salinity stress treatments increased the amount of MDA, indicating lipid peroxidation and oxidative stress. However, when PGPRs were applied in combination with salinity stress, the levels of MDA decreased compared to the control treatment. The highest amount of malondialdehyde was observed in the 120 mM salinity stress treatment and the control treatment without PGPRs. In the 120 mM salt stress treatment, the combination treatment of Azotobacter and Pseudomonas resulted in a significant decrease of 22% in MDA compared to the control treatment without PGPRs (Fig. III a). These findings suggest that the application of growth stimulants can mitigate oxidative stress caused by salinity stress and reduce lipid peroxidation in plants.
Fig. IV. Superoxide dismutase (SOD, a) and catalase (CAT, b) activity of geranium plants under salinity and Azotobacter chroococcum (AC) and Pseudomonas putida (PP) inoculation. Different letters above the column show statistical significance (P≤0.05).
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SOD and CAT Activity
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EO Content and EO Yield
Salinity stress increased the essential oil (EO) content compared to the control treatment, with no significant difference between the salinity at 60 and 120 mM. The highest EO content was observed in both the 60 and 120 mM salinity stress treatments (Fig. V a). Conversely, the control treatment had the lowest EO content. Additionally, salinity at 120 mM stress significantly decreased the EO yield compared to the control treatment, while the 60 mM treatment did not show a significant difference. The lowest yield of EO was observed in the 120 mM salinity, with a 26% decrease compared to the control treatment. Furthermore, the PGPRs significantly increased the EO yield compared to the control treatment. There was no significant difference in the yield of essential oil between the Azotobacter and Pseudomonas treatments. The combination treatment of Azotobacter and Pseudomonas showed the highest EO yield. Specifically, the EO yield increased by 30% in the combination treatment of Azotobacter and Pseudomonas compared to the control treatment (Fig. V b,c).
Discussion
Salinity had an extensive effect on the weight loss of plant shoots and roots. Faced with salinity stress, plants react to the stressful situation and try to adapt to harsh environmental conditions. One of the effects of salinity stress on the weight loss of aerial parts of plants is a significant reduction in the volume of water in the aerial parts. This causes the weight of the aerial parts of the plants to decrease (Nawaz et al., 2020a). Under salinity stress conditions, the rhizosphere system of the plant is also affected. In an effort to obtain more water and minerals, plants produce additional roots in selected tissues, such as exposed roots. This action causes the roots to grow more, resulting in an increase in root weight. Increasing the concentration of salt in the soil increases the external osmotic pressure and decreases the plant's capacity to absorb water. This causes the amount of water the plant can receive to decrease, leading to reduced plant vigor. Salt stress can disrupt the process of water secretion from leaves and other parts of the plant. This can lead to the accumulation of salts in plant tissues, further reducing plant vigor. Additionally, salinity can lead to disturbances in the activity of enzymes and metabolic processes of the plant (Shalaby and Ramadan, 2024). These disturbances cause a decrease in water secretion and an increase in evaporation from the surface of the leaves, which consequently affects the weight of the plant. In general, salinity stress causes a decrease in plant weight. However, its actual effect depends on factors such as plant type, plant age, salt concentration, duration of salinity stress, and environmental conditions.
Azotobacter and Pseudomonas are two genera of bacteria that are commonly known as beneficial plant bacteria. These bacteria can have a positive effect on the growth and weight of aerial parts of plants under salt stress conditions. These beneficial bacteria can help plants reduce the concentration of solutes in the soil through a cooperative relationship between the plant and the bacteria. This leads to an increase in the absorption of water and salts from the soil and, as a result, an increase in the growth and weight of the aerial parts of the plant. Beneficial bacteria can regulate the production of plant hormones and help regulate plant growth and development. This hormonal regulation can help increase the growth and weight of aerial parts of plants under salt stress conditions. Azotobacter and Pseudomonas can produce antioxidants that provide the plant with more resistance against oxidative stress, one of the effects of salinity stress (Minuț et al., 2022). Khodadadi et al. (2020) pointed out the positive role of Azotobacter in increasing the fresh and dry weight of barley plants under salinity stress conditions, which aligns with the results of Azar's research. Also, Abdel Latef et al. (2021)stated that plant inoculation with Azotobacter helped adjust to salinity stress, resulting in a significant increase in plant weight.
The decrease in the amount of photosynthetic pigments under salinity stress can mainly be due to the destruction of the chloroplast structure and photosynthetic apparatus, photooxidation of chlorophylls, their reaction with oxygen radicals, destruction of the precursors of chlorophyll synthesis, and inhibition of chlorophyll biosynthesis. New changes and activation of Chl-decomposing enzymes, including chlorophyllase, and hormonal disorders also play a role. Leaf Chl content is considered an important factor in determining leaf photosynthetic capacity. Reducing chlorophyll content as a non-porous factor can lead to a decrease in leaf photosynthetic capacity (Cui et al., 2022). In addition, salinity stress interferes with the absorption of essential elements such as iron and magnesium, which are essential for Chl synthesis (Rashmi et al., 2023). Salinity stress causes premature aging of leaves, chloroplast breakage, and reduction of chlorophyll. The reduction of chlorophyll leads to decreased photosynthesis, and plants that maintain more chlorophyll during stress have higher photosynthesis efficiency and are more resistant to stress (Feizi et al., 2021). Some growth regulators, such as abscisic acid and ethylene, whose levels increase under stress conditions, stimulate the activity of this enzyme. Also, the decrease in greenness may be due to changes in nitrogen metabolism in relation to the production of proline amino acid compounds, which are produced under stress conditions for osmotic regulation (Rossi et al., 2020). Azotobacter and Pseudomonas, as useful plant bacteria, can have a positive effect on leaf chlorophyll levels under salt stress conditions. Beneficial bacteria can help increase chlorophyll production by regulating the activity of enzymes related to Chl activity (Abdel Latef et al., 2021). These enzymes play a role in the processes of Chl secretion and biochemical reactions related to the production of Chl and other plant pigments. Salinity stress usually increases oxidative stress in plants, which can lead to a decrease in leaf chlorophyll levels. Beneficial bacteria can help protect the plant's antioxidant system against oxidative stress by producing antioxidants and enzymes (Yaghoubian et al., 2021). The important role of Azotobacter in increasing nitrogen and other elements needed by the plant under stress conditions to boost Chl production can be highlighted. Azotobacter and Pseudomonas can help produce plant hormones that may assist in better absorption and use of nutrients, thus leading to an increase in leaf Chl levels (Abdel Latef et al., 2021). Aslani et al. (2024)reported an increase in the Chl content of Salvia under salt stress conditions with the use of Pseudomonas.
The relative water content of plants reflects their water status and is determined by the relationship between water supply to the leaf tissue and transpiration rate (Javardi et al., 2023). Water is essential for all metabolic processes in plant cells, and the ability of plants to recover from stress and yield depends on their relative water content. Decreased water levels result in reduced turgor pressure, leading to cell damage, wilting, and hindered plant growth. Conversely, maintaining a relatively high water content helps plants counteract reactive oxygen species and osmotic stress caused by drought, potentially leading to higher yields (Nawaz et al., 2020b). In response to salinity stress, plants reduce stomatal conductivity through a decrease in relative water content. Osmotic regulation improves drought tolerance by enabling cell enlargement, promoting plant growth, and partially opening stomata to maintain CO2 fixation during severe water deficit. Wheat plants accumulate various organic and inorganic solutes in their cytosol to reduce osmotic pressure and maintain cell turgor. Salinity negatively affects photosynthesis by altering the internal structure of chloroplasts, mitochondria, and the levels of chlorophyll and minerals. Pagán et al. (2022) have reported a significant decrease in relative water content during salinity stress treatment. Pseudomonas and Azotobacter are effective in increasing the relative water content of leaves due to their ability to improve root system function, enhance water absorption by the leaves, and enhance leaf structure and function, particularly under stress conditions. They also improve stomatal function and reduce water evaporation, leading to higher relative water content (Nehela et al., 2021). Additionally, these bacteria can increase soil water levels through activities such as the hydrolysis and transformation of organic matter, further contributing to an increase in the relative water content of leaves. Bacteria present in the rhizosphere of plant roots can create a more favorable moisture environment by secreting enzymes and establishing positive interactions with the plant, resulting in a higher relative water content in leaves (Yaghoubian et al., 2021). Similarly, Nehela et al. (2021)demonstrated that PGPRs, along with biochar, mitigate salinity stress by increasing the RWC.
Salinity stress can increase the production of reactive oxygen species (ROS) and non-oxygenated free radicals (NROS), leading to lipid oxidation and the formation of MDA. This process damages plant cell membranes and allows for further penetration of oxygen and heavy metals, resulting in increased ROS production and lipid oxidation ((Hasanuzzaman et al., 2021). Additionally, salt stress can decrease the activity of antioxidants responsible for inhibiting and neutralizing ROS, further exacerbating oxidative damage. Enzymes such as superoxide dismutase, peroxidase, and catalase, which play a role in neutralizing ROS, may also have decreased activity under salt stress conditions, leading to increased MDA production (Hasanuzzaman et al., 2020). Azotobacter and Pseudomonas can positively impact the reduction of MDA, which serves as an indicator of plant performance and response to oxidative stress. These bacteria can secrete antioxidant enzymes like superoxide dismutase, peroxidase, and ascorbate peroxidase, contributing to a balance between free radical production and destruction in plants. As a result, oxidative damage can be reduced through the elimination of free radicals and a decrease in MDA production (Abdel Latef et al., 2021). Furthermore, bacteria can improve the physiological and morphological characteristics of plants, leading to a reduction in oxidative stress and subsequently a decrease in MDA production (Yaghoubian et al., 2021).
Moderate salinity and PGPRs led to increased TPC and TFC. PGPRs can have positive effects on increasing the TPC and TFC in plant leaves. The first point is that plant growth-stimulating bacteria can increase the production of certain phenols by increasing the absorption of minerals by plant roots. Nitrogen, phosphorus, and calcium are the materials required for the synthesis of phenols, and these bacteria can facilitate the improvement of the absorption of these elements and thus increase the production of TPC and TFC in the plant. Secondly, Azotobacter and Pseudomonas can directly stimulate the biochemical pathways related to the synthesis of phenols and flavonoids. These bacteria can affect the production of phenylalanine ammonia lyase (PAL) enzymes, which play an essential role in the synthesis of phenols (Darakeh et al., 2021). Also, these bacteria can improve the performance of the antioxidant system by creating positive effects on the antioxidant activity of the plant and thus increase the amount of phenols in the plant. Antioxidant enzymes can have great activity in reducing oxidative stress and preventing plant damage. In general, PGPRs can have positive effects on increasing the phenolic content in plant leaves by stimulating the absorption of minerals, stimulating the synthesis pathway of phenols, and improving the function of the plant's antioxidant system (Khan et al., 2023). In a similar study, an increase in TPC and TFC of black cumin was reported by Darakeh et al. (2021).
Salt stress showed to increase the activity of the antioxidant enzymes. Catalase is responsible for breaking down hydrogen peroxide (H2O2) into water and oxygen. Salt stress can lead to an increased production of H2O2 in cells, and in response, the activity of catalase increases to mitigate the risks associated with elevated H2O2 levels (Hasanuzzaman et al., 2020). Similarly, salt stress can also boost the activity of the superoxide dismutase enzyme, which aids in the decomposition of the superoxide free radical (O2•-) into water and oxygen. Studies indicate that salt stress can induce the production of superoxide in cells, prompting an increase in the activity of superoxide dismutase to neutralize this free radical and prevent oxidative stress (Hasanuzzaman et al., 2020). Several biochemical pathways contribute to the activation of catalase and superoxide dismutase under salinity stress. These pathways include the direct influence of salts on enzyme activity, the regulation of minor elements and their impact on genes associated with enzyme activity, and the physiological effects of salinity on signaling systems related to sulfur oxide and plant adaptation to limited water supply. These pathways play critical roles in controlling catalase and superoxide dismutase activity in response to salt stress, helping to maintain oxidant balance and manage oxidative stress by reducing reactive oxygen species (Alharbi et al., 2022). Plant growth-stimulating bacteria can have a significant impact on the activity of catalase and superoxide dismutase enzymes through biochemical pathways. These bacteria can enhance catalase activity by preventing the accumulation of substances like hydrogen peroxide and promoting the production of plant growth hormones and the absorption of mineral elements. Furthermore, plant growth-stimulating bacteria can increase superoxide dismutase activity in plants by regulating the absorption of mineral elements, producing cytokinins, and, in some cases, directly transferring the enzyme to the plants. These mechanisms help to boost the activity of superoxide dismutase, thereby preventing oxidative stress (Neshat et al., 2022).
Geranium EO plays an important role in cosmetics and pharmaceuticals. Also, the role of EO in stress conditions is to protect the plant. The increase in EO content of different plants is influenced by external stimuli caused by the change in the size and number of EO secreting glands per unit area, which causes a change in the amount of essential oil (del Rosario Cappellari et al., 2019). Omer et al. (2022)reported an increase in the percentage of essential oil under saline conditions in lemongrass plants under conditions of moderate salinity stress with the use of growth-promoting bacteria in flax, which confirms the results of the present research. Plant growth-stimulating bacteria may have a positive effect on increasing essential oil production in plants. Plant growth-stimulating bacteria can change the regulation of plant growth hormones by changing the structure and physiology of the plant. These changes can stimulate the secretory glands of plants and increase the production of EO. Azotobacter and Pseudomonas can increase the activity of secretory glands by regulating the absorption and distribution of mineral elements in the plant (Khodadadi et al., 2020). Some bacteria are able to supply the required elements to plants, and these mineral elements may affect the activity of essential oil secretory glands (Khodadadi et al., 2020). In a similar study, Mirzaei et al. (2020)reported an increase in the percentage of lemongrass EO with the use of Azotobacter and drought stress conditions. As in the present study, it has been reported that the EO content of coriander leaves increased under moderate cadmium stress; on the other hand, severe cadmium stress caused a significant decrease in essential oil yield (Ahmad et al., 2018), which could be due to significant weight loss. In a similar study on cilantro, a decrease in EO yield was reported under extreme salinity stress (more than 100 mM), which is in line with the results of the present study (Hazrati et al., 2022). In addition, Hashemi et al. (2022) showed that Azotobacter caused a significant increase in EO yield.
Conclusion
The research findings suggest that the application of plant growth-promoting rhizobacteria (PGPR) can be an effective strategy for enhancing the growth, physiological, and biochemical properties of geranium plants under salinity stress. The results demonstrate that the PGPR treatment significantly improved plant growth parameters, such as increased shoot length, root length, and biomass production. Additionally, the PGPR-treated plants exhibited enhanced physiological properties, including increased chlorophyll content, antioxidant enzyme activities, and osmolyte accumulation, which contributed to improved stress tolerance in the plants. These findings indicate that PGPR can be a valuable tool for mitigating the negative effects of salinity stress on geranium plants and enhancing their overall performance. Further studies are warranted to explore the underlying mechanisms of this beneficial interaction and determine optimal application methods and dosage for different plant species.
References
Abdel Latef, A. a. H., A. M. Omer, A. A. Badawy, M. S. Osman and M. M. Ragaey. 2021. Strategy of salt tolerance and interactive impact of Azotobacter chroococcum and/or Alcaligenes faecalis inoculation on canola (Brassica napus L.) plants grown in saline soil. Plants, 10, (1) 110.
Ahmad, B., H. Jaleel, Y. Sadiq, M. M. A. Khan and A. Shabbir. 2018. Response of exogenous salicylic acid on cadmium induced photosynthetic damage, antioxidant metabolism and essential oil production in peppermint. Plant Growth Regulation, 86, 273-286.
Alharbi, K., E. Rashwan, H. H. Mohamed, A. Awadalla, A. E.-D. Omara, E. M. Hafez and T. Alshaal. 2022. Application of silica nanoparticles in combination with two bacterial strains improves the growth, antioxidant capacity and production of barley irrigated with saline water in salt-affected soil. Plants, 11, (15) 2026.
Arnon, D. I. 1949. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant physiology, 24, (1) 1.
Aslani, Z., A. Hassani, B. A. Mandoulakani, M. Barin and R. Maleki. 2024. The symbiotic association with Piriformospora indica and Pseudomonas fluorescens improves salt tolerance in sage (Salvia officinalis) plants. Plant and Soil, 495, (1) 391-410.
Bergman, M. E., Á. Chávez, A. Ferrer and M. A. Phillips. 2020. Distinct metabolic pathways drive monoterpenoid biosynthesis in a natural population of Pelargonium graveolens. Journal of experimental botany, 71, (1) 258-271.
Cui, B., X. Wang, Y. Su, C. Gong, D. Zhang, Z. Ouyang and X. Wang. 2022. Responses of tree growth, leaf area and physiology to pavement in Ginkgo biloba and Platanus orientalis. Frontiers in Plant Science, 13, 1003266.
Darakeh, S. a. S. S., W. Weisany, M. Diyanat and R. Ebrahimi. 2021. Bio-organic fertilizers induce biochemical changes and affect seed oil fatty acids composition in black cumin (Nigella sativa Linn). Industrial Crops and Products, 164, 113383.
Del Rosario Cappellari, L., M. V. Santoro, A. Schmidt, J. Gershenzon and E. Banchio. 2019. Induction of essential oil production in Mentha x piperita by plant growth promoting bacteria was correlated with an increase in jasmonate and salicylate levels and a higher density of glandular trichomes. Plant Physiology and Biochemistry, 141, 142-153.
Diagne, N., M. Ndour, P. I. Djighaly, D. Ngom, M. C. N. Ngom, G. Ndong, S. Svistoonoff and H. Cherif-Silini. 2020. Effect of plant growth promoting rhizobacteria (PGPR) and arbuscular mycorrhizal fungi (AMF) on salt stress tolerance of Casuarina obesa (Miq.). Frontiers in Sustainable Food Systems, 4, 601004.
El-Beltagi, H. S., I. Ahmad, A. Basit, H. M. Abd El-Lateef, M. Yasir, S. Tanveer Shah, I. Ullah, M. Elsayed Mohamed Mohamed, I. Ali and F. Ali. 2022. Effect of azospirillum and azotobacter species on the performance of cherry tomato under different salinity levels. Gesunde Pflanzen, 74, (2) 487-499.
Feizi, H., R. Moradi, N. Pourghasemian and H. Sahabi. 2021. Assessing saffron response to salinity stress and alleviating potential of gamma amino butyric acid, salicylic acid and vermicompost extract on salt damage. South African Journal of Botany, 141, 330-343.
Ghassemi-Golezani, K. and S. Abdoli. 2023. Alleviation of salt stress in rapeseed (Brassica napus L.) plants by biochar-based rhizobacteria: new insights into the mechanisms regulating nutrient uptake, antioxidant activity, root growth and productivity. Archives of Agronomy and Soil Science, 69, (9) 1548-1565.
Hasanuzzaman, M., M. B. Bhuyan, F. Zulfiqar, A. Raza, S. M. Mohsin, J. A. Mahmud, M. Fujita and V. Fotopoulos. 2020. Reactive oxygen species and antioxidant defense in plants under abiotic stress: Revisiting the crucial role of a universal defense regulator. Antioxidants, 9, (8) 681.
Hasanuzzaman, M., M. R. H. Raihan, A. a. C. Masud, K. Rahman, F. Nowroz, M. Rahman, K. Nahar and M. Fujita. 2021. Regulation of reactive oxygen species and antioxidant defense in plants under salinity. International Journal of Molecular Sciences, 22, (17) 9326.
Hashemi, M., B. Behboodian, E. Karimi and E. Oskoueian. 2022. Enhancing biosynthesis and bioactivity of Trachyspermum ammi seed essential oil in response to drought and Azotobacter chroococcum stimulation. Chemical and Biological Technologies in Agriculture, 9, (1) 26.
Hazrati, S., A. Ertani and S. Nicola. 2022. Production and quality of medicinal and aromatic plants: Recent findings on stress effects, elicitors, harvesting and market development. p. 1109: MDPI
Heath, R. L. and L. Packer. 1968. Photoperoxidation in isolated chloroplasts: II. Role of electron transfer. Archives of biochemistry and biophysics, 125, (3) 850-857.
Jaradat, N., M. Hawash, M. Qadi, M. Abualhasan, A. Odetallah, G. Qasim, R. Awayssa, A. Akkawi, I. Abdullah and N. Al-Maharik. 2022. Chemical markers and pharmacological characters of Pelargonium graveolens essential oil from Palestine. Molecules, 27, (17) 5721.
Javardi, N. S. M., M. Pakravan, P. Panahi and R. Zarei. 2023. Assessment of the Relationships Between Leaf Characteristics with Air Pollutants: A Case Study on Oriental Plane (Platanus orientalis L.) and Caucasian Hackberry (Celtis caucasica Willd). Arboriculture & Urban Forestry (AUF), 49, (6) 329-342.
Khodadadi, R., R. Ghorbani Nasrabadi, M. Olamaee and S. Movahedi Naini. 2020. Effect of Azotobacter and Azospirillum on growth and physiological characteristics of barley (Hordeum vulgare) under salinity stress. Water and Soil, 34, (3) 649-660.
Mazeed, A., P. Maurya, D. Kumar, S. S. Yadav and P. Suryavanshi. 2022. Efficient nutrient management for rose scented geranium (Pelargonium graveolens L′ Herit ex Ait). Journal of Applied Research on Medicinal and Aromatic Plants, 31, 100409.
Minuț, M., M. Diaconu, M. Roșca, P. Cozma, L. Bulgariu and M. Gavrilescu. 2022. Screening of Azotobacter, Bacillus and Pseudomonas species as plant growth-promoting bacteria. Processes, 11, (1) 80.
Mirzaei, M., A. Ladan Moghadam, L. Hakimi and E. Danaee. 2020. Plant growth promoting rhizobacteria (PGPR) improve plant growth, antioxidant capacity, and essential oil properties of lemongrass (Cymbopogon citratus) under water stress. Iranian Journal of Plant Physiology, 10, (2) 3155-3166.
Nasirzadeh, L., B. Sorkhilaleloo, E. Majidi Hervan and F. Fatehi. 2021. Changes in antioxidant enzyme activities and gene expression profiles under drought stress in tolerant, intermediate, and susceptible wheat genotypes. Cereal Research Communications, 49, 83-89.
Nawaz, A., M. Shahbaz, Asadullah, A. Imran, M. U. Marghoob, M. Imtiaz and F. Mubeen. 2020a. Potential of salt tolerant PGPR in growth and yield augmentation of wheat (Triticum aestivum L.) under saline conditions. Frontiers in Microbiology, 11, 2019.
Nawaz, M., S. A. Anjum, U. Ashraf, F. Azeem and Z. Wang. 2020b. Antioxidant defense system and reactive oxygen species (ROS) interplay in plants under drought condition. Handbook of Climate Change Management: Research, Leadership, Transformation, 1-25.
Nehela, Y., Y. S. Mazrou, T. Alshaal, A. M. Rady, A. M. El-Sherif, A. E.-D. Omara, A. M. Abd El-Monem and E. M. Hafez. 2021. The integrated amendment of sodic-saline soils using biochar and plant growth-promoting rhizobacteria enhances maize (Zea mays L.) resilience to water salinity. Plants, 10, (9) 1960.
Neshat, M., A. Abbasi, A. Hosseinzadeh, M. R. Sarikhani, D. Dadashi Chavan and A. Rasoulnia. 2022. Plant growth promoting bacteria (PGPR) induce antioxidant tolerance against salinity stress through biochemical and physiological mechanisms. Physiology and Molecular Biology of Plants, 28, (2) 347-361.
Omer, A. M., M. S. Osman and A. A. Badawy. 2022. Inoculation with Azospirillum brasilense and/or Pseudomonas geniculata reinforces flax (Linum usitatissimum) growth by improving physiological activities under saline soil conditions. Botanical Studies, 63, (1) 15.
Pagán, E., J. Robles, A. Temnani, P. Berríos, P. Botía and A. Pérez-Pastor. 2022. Effects of water deficit and salinity stress on late mandarin trees. Science of the Total Environment, 803, 150109.
Rashmi, D., W. A. Ansari, N. Y. Kadoo, V. T. Barvkar, R. Deshmukh and A. B. Nadaf. 2023. Role of ions and their transporters in combating salt stress in Pandanus odorifer (Forssk.) Kuntze. Acta Physiologiae Plantarum, 45, (5) 66.
Ritchie, S. W., H. T. Nguyen and A. S. Holaday. 1990. Leaf water content and gas‐exchange parameters of two wheat genotypes differing in drought resistance. Crop science, 30, (1) 105-111.
Rossi, S., C. Chapman and B. Huang. 2020. Suppression of heat-induced leaf senescence by γ-aminobutyric acid, proline, and ammonium nitrate through regulation of chlorophyll degradation in creeping bentgrass. Environmental and Experimental Botany, 177, 104116.
Sapre, S., I. Gontia-Mishra and S. Tiwari. 2022. Plant growth-promoting rhizobacteria ameliorates salinity stress in pea (Pisum sativum). Journal of Plant Growth Regulation, 41, (2) 647-656.
Sefidkon, F., K. Abbasi and G. B. Khaniki. 2006. Influence of drying and extraction methods on yield and chemical composition of the essential oil of Satureja hortensis. Food chemistry, 99, (1) 19-23.
Shalaby, O. A. and M. E.-S. Ramadan. 2024. Mycorrhizal colonization and calcium spraying modulate physiological and antioxidant responses to improve pepper growth and yield under salinity stress. Rhizosphere, 29, 100852.
Ullah, S., A. Bano, A. Ullah, M. A. Shahid and N. Khan. 2022. A comparative study of plant growth promoting rhizobacteria (PGPR) and sowing methods on nutrient availability in wheat and rhizosphere soil under salinity stress. Rhizosphere, 23, 100571.
Xu, B. J. and S. K. Chang. 2007. A comparative study on phenolic profiles and antioxidant activities of legumes as affected by extraction solvents. Journal of food science, 72, (2) S159-S166.
Yaghoubian, I., S. Ghassemi, M. Nazari, Y. Raei and D. L. Smith. 2021. Response of physiological traits, antioxidant enzymes and nutrient uptake of soybean to Azotobacter chroococcum and zinc sulfate under salinity. South African Journal of Botany, 143, 42-51.
Zhishen, J., T. Mengcheng and W. Jianming. 1999. The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food chemistry, 64, (4) 555-559.