The Effects of Mycorrhizal Fungi and Plant Growth-Promoting Bacteria on Photosynthetic Pigments, Antioxidant Enzymes, and Yield of Wheat (Triticum aestivum L.) under Different Irrigation Regimes

Document Type : Research Article

Authors

A Tobeh

A Tobeh

S Farzaneh

Department of Genetics and Plant Breeding, Faculty of Agriculture and Natural Resources, University of Mohaghegh Ardabili, Ardabil, Iran

10.22067/jcesc.2026.96731.1435

Abstract

Introduction
Wheat (Triticum aestivum L.) is a strategic global cereal, providing over 20% of human calorie intake. However, climate change, increased drought frequency, and declining irrigation water resources seriously threaten sustainable wheat production. In many agricultural regions, including Iran, drought stress is a primary factor limiting wheat yield. Therefore, finding biological strategies to enhance wheat's tolerance to water stress is a research priority in sustainable agriculture. Utilizing low-cost and environmentally friendly approaches to increase plant resilience to environmental stresses is essential. Among these, employing beneficial rhizosymbionts like Arbuscular Mycorrhizal Fungi (AMF) and Plant Growth-Promoting Rhizobacteria (PGPR), such as Pseudomonas and Azospirillum, has garnered significant research interest. AMF form extensive hyphal networks in the soil, dramatically increasing the root absorption surface area and improving plant access to water and less mobile nutrients like phosphorus. PGPR enhance plant growth and abiotic stress tolerance through various mechanisms, including producing plant growth hormones (e.g., auxins), synthesizing siderophores, fixing nitrogen, and inducing systemic resistance. While many studies have focused on the individual effects of AMF or PGPR, research on their combined impact on wheat growth and performance under limited irrigation condition is scarce. This study aimed to evaluate the individual and combined effects of mycorrhizal fungi and plant growth-promoting bacteria on photosynthetic pigments, antioxidant enzymes, biochemical resistance traits, and ultimately, the yield of wheat under different irrigation regimes.

Materials and Methods
This research was conducted during the 2021-2022 growing season at a research farm in the Arshaq region, Meshgin Shahr County, Ardabil Province, Iran. The experiment was arranged as a factorial based on a Randomized Complete Block Design with three replications. The first factor was biofertilizer type at four levels: control, mycorrhizal fungi (Rhizophagus intraradices), PGPR (Pseudomonas fluorescens and Azospirillum brasilense), and a combination of both mycorrhizal fungi and PGPR. The second factor was irrigation regime at three levels: no irrigation (rainfed), irrigation at 50% of plant water requirement, and full irrigation (100% water requirement). Measured traits included root colonization percentage, chlorophyll a, b, and carotenoid content, activities of the antioxidant enzymes catalase (CAT), peroxidase (POX), and superoxide dismutase (SOD), number of grains per spike, thousand-kernel weight, and grain yield. Root colonization was assessed using staining and microscopic examination. Photosynthetic pigments were extracted with acetone and measured spectrophotometrically. Enzyme activities were determined using specific spectrophotometric assays: CAT activity by monitoring H₂O₂ decomposition at 240 nm, POX activity by monitoring guaiacol oxidation at 470 nm, and SOD activity by measuring the inhibition of nitroblue tetrazolium reduction at 560 nm.

Results and Discussion
The analysis of variance indicated that the main effects of biofertilizers and irrigation regimes, as well as their interaction, were significant for all measured traits. The highest root colonization (79.6%) was observed in the full irrigation × combined biofertilizer treatment. Drought stress reduces carbohydrate allocation to roots, limiting the carbon source for fungi and hindering colonization. Biofertilizers, especially the combination, provided direct inoculation and improved the root environment, facilitating higher colonization. The maximum chlorophyll a (2.55 mg g^-1), chlorophyll b (0.85 mg g^-1), and carotenoid (0.204 mg g^-1) contents were recorded in the full irrigation × combined biofertilizer treatment. The minimum values were under severe drought without biofertilizers. Drought degrades chloroplasts and activates chlorophyll-degrading enzymes. Biofertilizers, by improving water and nutrient (especially phosphorus) uptake, helped maintain pigment synthesis and protect the photosynthetic apparatus from oxidative damage. The highest activities of CAT (32.5 U mg^-1), POX (4.88 U mg^-1), and SOD (28.8 U mg^-1) were found in the no irrigation × no biofertilizer treatment. Conversely, the lowest enzyme activities were consistently observed in the full irrigation × combined biofertilizer treatment. Elevated antioxidant enzyme activity under drought stress without biofertilizers is a physiological response to high oxidative stress caused by increased Reactive Oxygen Species (ROS) production. The application of biofertilizers, particularly the combination, mitigated water and nutrient deficits, thereby reducing ROS generation and the need for high antioxidant enzyme activity, indicating better physiological equilibrium. Increased significantly from 2501.7 kg ha^-1 under severe drought without biofertilizers to 4891.6 kg ha^-1 under full irrigation with combined biofertilizers. This demonstrates that the synergistic effect of AMF and PGPR in improving water relations, nutrient status, and oxidative stress management translated into superior yield performance.

Conclusion
The findings of this study demonstrate that the combined application of mycorrhizal fungi and plant growth-promoting bacteria is an effective strategy for mitigating the adverse effects of drought stress and enhancing wheat yield. This biological combination acted synergistically to significantly increase root colonization, preserve the photosynthetic system by maintaining higher chlorophyll and carotenoid levels, and reduce oxidative stress by modulating antioxidant enzyme activities. These improvements ultimately led to a significant increase in yield components. Therefore, employing these bio-inputs can serve as a sustainable and eco-friendly approach, paving the way for reduced chemical fertilizer use, increased plant resilience to water scarcity, and enhanced food security.

Keywords

Carotenoid

Catalase

Chlorophyll

Colonization

Peroxidase

Subjects

• Environmental stresses

---

Authors retain the copyright. This is an open access article distributed under Creative Commons Attribution 4.0 International License (CC BY 4.0)

References

1.           Adel, S., & Carels, N. (2023). Plant tolerance to drought stress with emphasis on wheat. Plants, 12(11), 2170. https://doi.org/10.3390/plants12112170

2.           Allen, R. G., Pereira, L. S., Raes, D., & Smith, M. (1998). Crop evapotranspiration: Guidelines for computing crop water requirements. FAO Irrigation and Drainage Paper No. 56. Food and Agriculture Organization of the United Nations. http://www.fao.org/3/X0490E/X0490E00.htm

3.           Alscher, R. G., Erturk, N., & Heath, L. S. (2002). Role of superoxide dismutases (SODs) in controlling oxidative stress in plants. Journal of Experimental Botany, 53(372), 1331-1341. https://doi.org/10.1093/jexbot/53.372.1331

4.           Askari Foroshani, F., Rahnama, A., Meskarbashee, M., & Kamranfar, I. (2025). Effect of waterlogging duration at different growth stages on some photosynthetic characteristics, antioxidant activity, and yield of safflower (Carthamus tinctorius L.). Iranian Journal of Field Crops Research, 23(2), 171-186. (In Persian). https://doi.org/10.22067/jcesc.2024.88492.1335

5.           Backer, R., Rokem, J. S., Ilangumaran, G., Lamont, J., Praslickova, D., Ricci, E., Subramanian, S., & Smith, D. L. (2018). Plant growth-promoting rhizobacteria: Context, mechanisms of action, and roadmap to commercialization of biostimulants for sustainable agriculture. Frontiers in Plant Science, 9, 1473. https://doi.org/10.3389/fpls.2018.01473

6.           Battini, F., Grønlund, M., Agnolucci, M., Giovannetti, M., & Jakobsen, I. (2017). Facilitation of phosphorus uptake in maize plants by mycorrhizosphere bacteria. Scientific Reports, 7(1), 4686. https://doi.org/10.1038/s41598-017-04959-0

7.           Begum, N., Qin, C., Ahanger, M. A., Raza, S., Khan, M. I., Ashraf, M., & Zhang, L. (2019). Role of arbuscular mycorrhizal fungi in plant growth regulation: Implications in abiotic stress tolerance. Frontiers in Plant Science, 10, 1068. https://doi.org/10.3389/fpls.2019.01068

8.           Bitterlich, M., Sandmann, M., & Graefe, J. (2018). Arbuscular mycorrhiza alleviates restrictions to substrate water flow and delays transpiration limitation to stronger drought in tomato. Frontiers in Plant Science, 9, 154. https://doi.org/10.3389/fpls.2018.00154

9.           Chandra, D., Srivastava, R., Glick, B. R., & Sharma, A. K. (2018). Drought-tolerant Pseudomonas spp. improve the growth performance of finger millet (Eleusine coracana (L.) Gaertn.) under non-stressed and drought-stressed conditions. Pedosphere, 28(2), 227-240. https://doi.org/10.1016/S1002-0160(18)60013-X

10.        Chandrasekaran, M., Chanratana, M., Kim, K., Seshadri, S., & Sa, T. (2019). Impact of arbuscular mycorrhizal fungi on photosynthesis, water status, and gas exchange of plants under salt stress–a meta-analysis. Frontiers in Plant Science, 10, 457. https://doi.org/10.3389/fpls.2019.00457

11.        Choudhury, F. K., Rivero, R. M., Blumwald, E., & Mittler, R. (2017). Reactive oxygen species, abiotic stress and stress combination. The Plant Journal, 90(5), 856-867. https://doi.org/10.1111/tpj.13299

12.        Dobbelaere, S., Vanderleyden, J., & Okon, Y. (2003). Plant growth-promoting effects of diazotrophs in the rhizosphere. Critical Reviews in Plant Sciences, 22(2), 107–149. https://doi.org/10.1080/713610853

13.        Farooq, M., Wahid, A., Kobayashi, N., Fujita, D., & Basra, S. M. A. (2009). Plant drought stress: Effects, mechanisms and management. Agronomy for Sustainable Development, 29(1), 185–212. https://doi.org/10.1051/agro:2008021

14.        Fayazi, H., Abdali Mashhadi, A., Koochekzadeh, A., Papzan, A., & Arzanesh, M. H. (2018). Effect of organic and biological fertilizers on nitrogen, phosphorous and potassium contents, photosynthetic pigments and active ingredient in coneflower (Echinacea purpurea L.). Iranian Journal of Field Crops Research, 16(2), 283–298. (In Persian). https://doi.org/10.22067/gsc.v16i2.49182

15.        Galindo, F. S., Pagliari, P. H., Fernandes, G. C., Rodrigues, W. L., Boleta, E. H. M., Jalal, A., & Teixeira Filho, M. C. M. (2022). Improving sustainable field-grown wheat production with Azospirillum brasilense under tropical conditions: A potential tool for improving nitrogen management. Frontiers in Environmental Science, 10, 821628. https://doi.org/10.3389/fenvs.2022.821628

16.        Gholinezhad, E., & Darvishzadeh, R. (2015). Effect of mycorrhizal fungi on yield and yield components of sesame (Sesamum indicum L.) landraces under different irrigation levels. Journal of Agricultural Science and Sustainable Production, 25(3), 119–135. (In Persian).

17.        Giannopolitis, C. N., & Ries, S. K. (1977). Superoxide dismutases: I. Occurrence in higher plants. Plant Physiology, 59(2), 309–314. https://doi.org/10.1104/pp.59.2.309

18.        Gill, S. S., & Tuteja, N. (2010). Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiology and Biochemistry, 48(12), 909-930. https://doi.org/10.1016/j.plaphy.2010.08.016

19.        Gutjahr, C., & Parniske, M. (2013). Cell and developmental biology of arbuscular mycorrhiza symbiosis. Annual Review of Cell and Developmental Biology, 29, 593–617. https://doi.org/10.1146/annurev-cellbio-101512-122413

20.        Hadwan, M. H., & Abed, H. N. (2018). Simple spectrophotometric assay for measuring catalase activity in biological tissues. BMC Biochemistry, 19, 7. https://doi.org/10.1186/s12858-018-0097-5

21.        Hasanuzzaman, M., Bhuyan, M. H. M. B., Zulfiqar, F., Raza, A., Mohsin, S. M., Mahmud, J. A., Fujita, M., & Fotopoulos, V. (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. https://doi.org/10.3390/antiox9080681

22.        Hosseini, R., & Heidari, M. (2025). Impact of drought stress on biochemical and molecular responses in lavender (Lavandula angustifolia Mill.): Effects on essential oil composition and antibacterial activity. Frontiers in Plant Science, 16, 1506660. https://doi.org/10.3389/fpls.2025.1506660

23.        Igiehon, N. O., & Babalola, O. O. (2018). Rhizosphere microbiome modulators: contributions of nitrogen fixing bacteria towards sustainable agriculture. International Journal of Environmental Research and Public Health, 15(4), 574. https://doi.org/10.3390/ijerph15040574

24.        Jamil, M., Ahamd, M., Anwar, F., Zahir, Z. A., Kharal, M. A., & Nazli, F. (2018). Inducing drought tolerance in wheat through combined use of l-tryptophan and Pseudomonas fluorescens. Pakistan Journal of Agricultural Sciences, 55(2). https://doi.org/10.21162/PAKJAS/18.4980

25.        Kang, S. M., Khan, A. L., Waqas, M., You, Y. H., Kim, J. H., Kim, J. G., Hamayun, M., & Lee, I. J. (2014). Plant growth-promoting rhizobacteria reduce adverse effects of salinity and osmotic stress by regulating phytohormones and antioxidants in Cucumis sativus. Journal of Plant Interactions, 9(1), 673-682. https://doi.org/10.1080/17429145.2014.894587

26.        Karimi, E., Mohammadi, S., & Esfandyari, E. (2022). The effect of biofilm forming growth promoting bacterium and tryptophan on root characteristics of rye and their relationship with root cadmium accumulation. Journal of Agricultural Science and Sustainable Production, 32(2), 253–268. (In Persian). https://doi.org/10.22034/saps.2021.45695.2671

27.        Khodadadi, R., & Ghorbani Nasrabadi, R. (2020). Mechanisms of plant growth promoting rhizobacteria on growth indices, physiology, nutrient uptake and production of secondary metabolites in medicinal plants. Technology of Medicinal and Aromatic Plants of Iran, 2(2), 52–69. (In Persian). https://doi.org/10.22092/mpt.2020.127462.1047

28.        Kuan, K. B., Othman, R., Abdul Rahim, K., & Shamsuddin, Z. H. (2016). Plant growth-promoting rhizobacteria inoculation to enhance vegetative growth, nitrogen fixation and nitrogen remobilisation of maize under greenhouse conditions. Plos One, 11(3), e0152478. https://doi.org/10.1371/journal.pone.0152478

29.        Kudoyarova, G., Arkhipova, T., Korshunova, T., Bakaeva, M., Loginov, O., & Dodd, I. C. (2019). Phytohormone mediation of interactions between plants and non-symbiotic growth promoting bacteria under edaphic stresses. Frontiers in Plant Science, 10, 1368. https://doi.org/10.3389/fpls.2019.01368

30.        Laxa, M., Liebthal, M., Telman, W., Chibani, K., & Dietz, K. J. (2019). The role of the plant antioxidant system in drought tolerance. Antioxidants, 8(4), 94. https://doi.org/10.3390/antiox8040094

31.        Lehmann, A., Zheng, W., Ryo, M., Soutschek, K., Roy, J., Rongstock, R., & Rillig, M. C. (2020). Fungal traits important for soil aggregation. Frontiers in Microbiology, 10, 2904. https://doi.org/10.3389/fmicb.2019.02904

32.        Lichtenthaler, H. K., & Wellburn, A. R. (1983). Determinations of total carotenoids and chlorophylls a and b of leaf extracts in different solvents. Biochemical Society Transactions, 11(5), 591–592. https://doi.org/10.1042/bst0110591

33.        Ma, Y., Dias, M. C., & Freitas, H. (2020). Drought and salinity stress responses and microbe-induced tolerance in plants. Frontiers in Plant Science, 11, 591911. https://doi.org/10.3389/fpls.2020.591911

34.        Mathur, S., Sharma, M. P., & Jajoo, A. (2018). Improved photosynthetic efficacy of maize (Zea mays) plants with arbuscular mycorrhizal fungi (AMF) under high temperature stress. Journal of Photochemistry and Photobiology B: Biology, 180, 149–154. https://doi.org/10.1016/j.jphotobiol.2018.02.002

35.        Naseri, R., Mirzaei, A., & Abbasi, A. (2021). Effect of application of different fertilizer sources on physiological and biochemical traits of new cultivars of barley under dryland conditions. Iranian Journal of Field Crops Research, 19(2), 121-140. (In Persian). https://doi.org/10.22067/jcesc.2021.37177.0

36.        Nasrollahzade Asl, V., Moharramnejad, S., & Yusefi, M. (2017). Grain yield, chlorophyll content, osmolyte accumulation, total phenolics and catalase activity in maize (Zea mays L.) under drought stress. Journal of Plant Environmental Physiology, 12(46), 1-14. (In Persian).

37.        Nemati, A., Alilou, A., & Sedghi, M. (2021). Evaluation of yield and yield components of some rapeseed cultivars with endophyte P. indica and A. siccitolerans under drought stress. Journal of Crop Production, 13(4), 87–110. (In Persian). https://doi.org/10.22069/ejcp.2021.18408.2364

38.        Omidi Nasab, D., Meskarbashee, M., & Rahnama Ghahfarokhi, A. (2024). The effect of deficit irrigation on grain yield and some physiological indicators of sunflower (Helianthus annus L.) cultivars. Iranian Journal of Field Crops Research, 22(1), 71-88. (In Persian). https://doi.org/10.22067/jcesc.2023.83198.1258

39.        Roger, A., Colard, A., Angelard, C., & Sanders, I. R. (2013). Relatedness among arbuscular mycorrhizal fungi drives plant growth and intraspecific fungal coexistence. The ISME Journal, 7(11), 2137-2147. https://doi.org/10.1038/ismej.2013.112

40.        Rouphael, Y., & Colla, G. (2020). Toward a sustainable agriculture through plant biostimulants: From experimental data to practical applications. Agronomy, 10(10), 1461. https://doi.org/10.3390/agronomy10101461

41.        Ruiz‐Lozano, J. M., Aroca, R., Zamarreño, Á. M., Molina, S., Andreo‐Jiménez, B., Porcel, R., & López‐Ráez, J. A. (2016). Arbuscular mycorrhizal symbiosis induces strigolactone biosynthesis under drought and improves drought tolerance in lettuce and tomato. Plant, Cell & Environment, 39(2), 441-452. https://doi.org/10.1111/pce.12631

42.        Saha, M., Sarkar, S., Sarkar, B., Sharma, B. K., Bhattacharjee, S., & Tribedi, P. (2016). Microbial siderophores and their potential applications: A review. Environmental Science and Pollution Research, 23(5), 3984-3999. https://doi.org/10.1007/s11356-015-4294-0

43.        Saini, H. S., & Westgate, M. E. (2000). Reproductive development in grain crops during drought. Advances in Agronomy, 68, 59-96. https://doi.org/10.1016/S0065-2113(08)60843-3

44.        Sedaghati, E., Ahmadzadeh, M., Sabri-Rise, R., Rahimi, A., Hatami, N., & Mohammadi Mirik, A. (2021). The effect of application of arbuscular mycorrhizal fungi with some microorganisms and chemical compounds on the antioxidant enzymes activity and phenolic compounds of corn under drought stress. Journal of Plant Biological Sciences, 13(2), 53-76. (In Persian). https://doi.org/10.22108/ijpb.2022.129942.1258

45.        Singh, S., Tripathi, D. K., Singh, S., Sharma, S., Dubey, N. K., Chauhan, D. K., & Vaculík, M. (2017). Toxicity of aluminium on various levels of plant cells and organism: A review. Environmental and Experimental Botany, 137, 177–193. https://doi.org/10.1016/j.envexpbot.2017.01.005

46.        Smith, S. E., & Read, D. J. (2008). Mycorrhizal Symbiosis (3rd ed.). Academic Press. https://doi.org/10.1016/B978-0-12-370526-6.X5001-6

47.        Smith, S. E., & Smith, F. A. (2011). Roles of arbuscular mycorrhizas in plant nutrition and growth: New paradigms from cellular to ecosystem scales. Annual Review of Plant Biology, 62, 227–250. https://doi.org/10.1146/annurev-arplant-042110-103846

48.        Sofo, A., Scopa, A., Nuzzaci, M., & Vitti, A. (2015). Ascorbate peroxidase and catalase activities and their genetic regulation in plants subjected to drought and salinity stresses. International Journal of Molecular Sciences, 16(6), 13561-13578. https://doi.org/10.3390/ijms160613561

49.        Trouvelot, S., Bonneau, L., Redecker, D., Van Tuinen, D., Adrian, M., & Wipf, D. (2015). Arbuscular mycorrhiza symbiosis in viticulture: A review. Agronomy for Sustainable Development, 35(4), 1449–1467. https://doi.org/10.1007/s13593-015-0329-7

50.        Vurukonda, S. S. K. P., Vardharajula, S., Shrivastava, M., & SkZ, A. (2016). Enhancement of drought stress tolerance in crops by plant growth promoting rhizobacteria. Microbiological Research, 184, 13–24. https://doi.org/10.1016/j.micres.2015.12.003

51.        Zhang, H., Xie, X., Kim, M. S., Kornyeyev, D. A., Holaday, S., & Paré, P. W. (2019). Effects of mycorrhizal fungi and PGPR on drought tolerance of wheat. Journal of Plant Growth Regulation, 38(2), 558–571.

52.        Zhang, L., Feng, G., & Declerck, S. (2018). Signal beyond nutrient, fructose, exuded by an arbuscular mycorrhizal fungus triggers phytate mineralization by a phosphate solubilizing bacterium. The ISME Journal, 12(10), 1–12. https://doi.org/10.1038/s41396-018-0171-4