Effect of Some Biofertilizers on the Physiological Characteristics of Wheat Flag Leaves and Rhizosphere Enzyme Activities at Different Irrigation Levels

Background and objectives: Drought stress is a major limiting factor in global agricultural productivity, significantly affecting plant growth and yield by altering the plant’s morphological, physiological, and biochemical characteristics. These changes, including reductions in chlorophyll content, leaf water status, metabolite content, compromise plant development and crop output. One promising approach to mitigating the detrimental effects of drought stress is the use of biofertilizers, particularly arbuscular mycorrhizal (AM) fungi and plant growth-promoting rhizobacteria (PGPR). AM fungi establish symbiotic relationships with plants, improving water uptake and nutrient absorption, while PGPR enhance plant growth through various mechanisms, including the production of enzymes and stress-relieving compounds. Although numerous studies have demonstrated the efficacy of biofertilizers in improving plant resilience to drought, there is a significant knowledge gap regarding the comparative effectiveness of different types of biofertilizers. Specifically, it remains unclear whether fungal-based or bacterial-based biofertilizers are more effective, and whether liquid or powder formulations provide superior benefits. Moreover, there is limited understanding of the role of soil microbial processes, such as enzyme activities in the rhizosphere, in the plant’s adaptive response to drought conditions. This study aims to fill these gaps by investigating the effects of different biofertilizer types and formulations on drought tolerance in wheat, focusing on key physiological, biochemical, and microbial indicators.Materials and methods: For these purposes, a split-plot experiment was conducted based on a randomized complete block design with three replications under field conditions. The main plots consisted of three irrigation treatments: 100%, 85%, and 65% of the plant's water requirement, representing full irrigation, mild water stress, and severe water stress, respectively. The subplots included different biofertilizer treatments: no biofertilizer (F1), Pseudomonas fluorescens producing ACC-deaminase (F2), P. fluorescens without ACC-deaminase production (F3), AM fungus in liquid form (F4), and AM fungus in powder form (F5). Samples of flag leaves, roots, and rhizospheric soil were collected at the spike emergence stage. Several parameters were measured, including chlorophyll, carotenoid, proline, relative water content (RWC), membrane stability index (MSI) in the flag leaves, root colonization percentage, and rhizosphere enzyme activities such as acid phosphatase, alkaline phosphatase, and β-glucosidase. These indicators were chosen to evaluate both the direct effects of biofertilizers on plant drought tolerance and the associated microbial processes in the soil.Results: The results indicated that among the bacterial biofertilizers, only the application of F2 under severe water stress led to a 5% decrease in proline, a 6.5% increase in chlorophyll a, a 6% increase in total chlorophyll, a 16% rise in carotenoids, as well as a 10% increase in β-glucosidase activity and a 13% increase in acid phosphatase activity compared to the treatment without biofertilizer. The powder form of AM fungus (F5) proved to be the most effective in colonizing the roots of wheat. Specifically, root colonization with F5 was 13%, 19%, and 8% higher at irrigation levels of 65%, 85%, and 100% of the plant's water requirement, respectively, compared to the liquid form of AM fungus (F4). Overall, fungal biofertilizers outperformed bacterial biofertilizers in enhancing the physiological characteristics of wheat. For instance, under severe water stress, the F5 and F4 treatments increased RWC by 8.5% and 6%, MSI by 20% and 14%, chlorophyll a by 1% and 14%, and total chlorophyll by 12% and 10% compared to the treatment without biofertilizer, which were significantly higher than the bacterial biofertilizers. Among the fungal biofertilizer formulations, the powder form of AM fungus was more efficient than the liquid form in increasing wheat's drought tolerance. The powder form also improved β-glucosidase activity under both severe and mild water stress conditions and increased the activity of all the investigated enzymes under full irrigation. A stepwise linear regression model revealed that that among the biochemical and physiological characteristics of the flag leaf of the wheat, the amount of proline and carotenoid are the most important key variables affecting the β-glucosidase activity with relative importance index of 25.8% and 18.9%, respectively. Also, the amount of total chlorophyll and chlorophyll a had the greatest effect on the amount of alkaline and acid phosphatase activities.Conclusion: The findings of this study underscore the superior effectiveness of fungal biofertilizers, particularly in powder form, in enhancing drought tolerance in wheat. The powder form of AM fungi was more efficient than the liquid form in promoting root colonization and increasing enzyme activity in the rhizosphere, leading to improved physiological and biochemical traits in the wheat plants. This formulation likely contains a greater diversity of AM fungal species, which may contribute to its enhanced performance. The positive feedback loop observed between rhizospheric enzyme activities and plant physiological traits suggests that biofertilizers, particularly AM fungi, can play a crucial role in improving the drought resilience of crops in water-limited environments.