University of Isfahan Journal of Plant Biological Sciences 3041-9603 17 3 2025 09 23 The impact of nitrogen concentration on the physiological responses of rice seedlings after inoculation with Azospirillum brasilence The impact of nitrogen concentration on the physiological responses of rice seedlings after inoculation with Azospirillum brasilence 67 88 30080 10.22108/ijpb.2025.146865.1423 FA Mohammad Noori 1Department of Biology-Plant Physiology, Faculty of Science, Golestan University, Gorgan, Iran Mahnaz Aghdasi 1Department of Biology-Plant Physiology, Faculty of Science, Golestan University, Gorgan, Iran Mohammad Hossein Arzanesh Department of Soil and Water Research, Golestan's Agricultural and Natural Resources Research Center, Gorgan, Iran Journal Article 2025 09 30 Azospirillum is one of the most important plant growth-promoting bacteria, with the ability to fix nitrogen. The current study aimed to investigate the physiological responses of rice plants with different nitrogen concentrations when inoculated with Azospirillum brasilense. In this experiment, the growth and physiological responses of 21-day-old rice seedlings (Oryza sativa L., cv. Hashemi) were investigated after 4 weeks of treatment with different nitrogen levels, including complete nitrogen (N), half (N/2), fourth (N/4), and zero nitrogen (control) concentrations under two inoculated (AB) and non-inoculated (NI) conditions. The experiment was conducted in a completely randomized design with 4 replications. The current data showed that nitrogen concentration significantly affects all measured parameters in both AB and NI plants. The highest values of growth traits, biomass accumulation, photosynthetic pigments, soluble sugars, and antioxidant enzyme activities (catalase and peroxidase) were observed in plants treated with full nitrogen and inoculated with A. brasilense. These findings highlight the potential of A. brasilense to enhance nitrogen efficiency in agricultural crops. Meanwhile, our data showed that inoculation with A. brasilense can improve rice plant productivity, especially under optimal nitrogen conditions. Introduction Rice (Oryza sativa) is a monocotyledonous plant that belongs to the Poaceae family. Rice is a strategic crop and the primary food source for more than half of the world’s population. The increase in rice cultivation areas in our country and the widespread use of chemical fertilizers have led to the emergence of various diseases. Today, the use of Plant Growth Promoting Rhizobacteria (PGPR) is considered an alternative to chemical fertilizers. These bacteria are present in the rhizosphere of plants and enhance plant growth in various ways, including increasing nutrient absorption, producing plant hormones, and protecting plants against pathogens. These bacteria enhance mineral nutrient uptake by stimulating root system development. A group of PGPRs is called endophytes. Endophytes are non-pathogenic bacteria that spend part of their life cycle within plant tissues. So far, various types of nitrogen-fixing endophytes have been identified and reported in the stems, roots, and seeds of rice. Azospirillum is one of the most important plant growth-promoting bacteria, capable of fixing nitrogen. Recently, this bacterium has been used as a biofertilizer, especially in areas facing nitrogen deficiency. By fixing atmospheric nitrogen and converting it into a usable form, Azospirillum brasilense improves nitrogen efficiency and reduces demand for chemical fertilizers. The current study aimed to investigate the physiological responses of rice seedlings to different nitrogen concentrations when inoculated with Azospirillum brasilense. Methods and Materials In this experiment, Azospirillum brasilense was prepared from the Soil Biology Laboratory of the Agricultural Research and Education Center of Golestan province. Then, the bacteria were cultured and identified on a specific RC medium. After selecting a single colony of the desired bacteria, the bacteria were cultured on NFB liquid medium at 30 °C for 48 hours. Rice seeds of the Hashemi cultivar were obtained from the National Rice Research Institute (AML). After surface sterilization, the seeds were placed on moist filter paper at 29 °C for germination. Then, the 7-day-old seedlings were transferred to Yoshida culture medium under a photoperiod of 18 h day (33 °C) and 6 h night (22 °C), with a relative humidity of 71%. In the next step, 21-day-old rice seedlings were treated with Yoshida medium containing nitrogen levels: complete nitrogen (N), half nitrogen (N/2), quarter nitrogen (N/4), and zero nitrogen (control) under two inoculation conditions: inoculated (AB) and non-inoculated (NI). After 4 weeks of treatment, the roots of rice seedlings were collected, and various growth parameters and several biochemical factors were examined. The experiment was conducted in a completely randomized design with four replications. Results and Discussion Microscopic observations revealed that the bacteria first settled on the root epidermis and then entered the root cells. After passing through the epidermis, the bacteria penetrated the parenchyma layer and formed colonies. The results showed that small and large colonies, with single or scattered bacteria, were observed in the root cells. On the other hand, the current data showed that nitrogen concentration significantly affects all measured parameters in both AB and NI plants. The highest amount of root and shoot length, root fresh and dry weight, shoot dry weight, total dry and fresh weight, relative growth, soluble and total shoot sugar, chlorophyll b, and total chlorophyll were observed in the treatment with both full nitrogen concentration and inoculation with A. brasilense. Also, the present data revealed that seedlings inoculated with Azospirillum performed better than the non-inoculated samples.Meanwhile, current data showed that catalase enzyme activity increased with increased nitrogen concentration (in both inoculated and non-inoculated conditions). However, catalase enzyme activity was higher in inoculated seedlings at all nitrogen levels, compared to non-inoculated seedlings. These data indicate that inoculation with A. brasilense increases oxidative stress tolerance in rice seedlings. This evidence suggests that this bacterium improves plant resilience to treatment with different nitrogen concentrations and enhances their ability to scavenge reactive oxygen species (ROS) produced during nitrogen metabolism. The present data showed that peroxidase enzyme activity increased with higher nitrogen concentrations, in both inoculated and non-inoculated conditions. Conclusion The present results showed that inoculating rice seedlings with A. brasilense can significantly enhance their physiological responses. At all nitrogen concentrations, treatment improved the growth of inoculated plants, which showed higher protein content and enzyme activity compared to non-inoculated seedlings. These findings highlight the potential of A. brasilense to enhance nitrogen efficiency in crops. Meanwhile, our data showed that inoculation with A. brasilense can improve rice seedling productivity, especially under optimal nitrogen conditions. Azospirillum is one of the most important plant growth-promoting bacteria, with the ability to fix nitrogen. The current study aimed to investigate the physiological responses of rice plants with different nitrogen concentrations when inoculated with Azospirillum brasilense. In this experiment, the growth and physiological responses of 21-day-old rice seedlings (Oryza sativa L., cv. Hashemi) were investigated after 4 weeks of treatment with different nitrogen levels, including complete nitrogen (N), half (N/2), fourth (N/4), and zero nitrogen (control) concentrations under two inoculated (AB) and non-inoculated (NI) conditions. The experiment was conducted in a completely randomized design with 4 replications. The current data showed that nitrogen concentration significantly affects all measured parameters in both AB and NI plants. The highest values of growth traits, biomass accumulation, photosynthetic pigments, soluble sugars, and antioxidant enzyme activities (catalase and peroxidase) were observed in plants treated with full nitrogen and inoculated with A. brasilense. These findings highlight the potential of A. brasilense to enhance nitrogen efficiency in agricultural crops. Meanwhile, our data showed that inoculation with A. brasilense can improve rice plant productivity, especially under optimal nitrogen conditions. Introduction Rice (Oryza sativa) is a monocotyledonous plant that belongs to the Poaceae family. Rice is a strategic crop and the primary food source for more than half of the world’s population. The increase in rice cultivation areas in our country and the widespread use of chemical fertilizers have led to the emergence of various diseases. Today, the use of Plant Growth Promoting Rhizobacteria (PGPR) is considered an alternative to chemical fertilizers. These bacteria are present in the rhizosphere of plants and enhance plant growth in various ways, including increasing nutrient absorption, producing plant hormones, and protecting plants against pathogens. These bacteria enhance mineral nutrient uptake by stimulating root system development. A group of PGPRs is called endophytes. Endophytes are non-pathogenic bacteria that spend part of their life cycle within plant tissues. So far, various types of nitrogen-fixing endophytes have been identified and reported in the stems, roots, and seeds of rice. Azospirillum is one of the most important plant growth-promoting bacteria, capable of fixing nitrogen. Recently, this bacterium has been used as a biofertilizer, especially in areas facing nitrogen deficiency. By fixing atmospheric nitrogen and converting it into a usable form, Azospirillum brasilense improves nitrogen efficiency and reduces demand for chemical fertilizers. The current study aimed to investigate the physiological responses of rice seedlings to different nitrogen concentrations when inoculated with Azospirillum brasilense. Methods and Materials In this experiment, Azospirillum brasilense was prepared from the Soil Biology Laboratory of the Agricultural Research and Education Center of Golestan province. Then, the bacteria were cultured and identified on a specific RC medium. After selecting a single colony of the desired bacteria, the bacteria were cultured on NFB liquid medium at 30 °C for 48 hours. Rice seeds of the Hashemi cultivar were obtained from the National Rice Research Institute (AML). After surface sterilization, the seeds were placed on moist filter paper at 29 °C for germination. Then, the 7-day-old seedlings were transferred to Yoshida culture medium under a photoperiod of 18 h day (33 °C) and 6 h night (22 °C), with a relative humidity of 71%. In the next step, 21-day-old rice seedlings were treated with Yoshida medium containing nitrogen levels: complete nitrogen (N), half nitrogen (N/2), quarter nitrogen (N/4), and zero nitrogen (control) under two inoculation conditions: inoculated (AB) and non-inoculated (NI). After 4 weeks of treatment, the roots of rice seedlings were collected, and various growth parameters and several biochemical factors were examined. The experiment was conducted in a completely randomized design with four replications. Results and Discussion Microscopic observations revealed that the bacteria first settled on the root epidermis and then entered the root cells. After passing through the epidermis, the bacteria penetrated the parenchyma layer and formed colonies. The results showed that small and large colonies, with single or scattered bacteria, were observed in the root cells. On the other hand, the current data showed that nitrogen concentration significantly affects all measured parameters in both AB and NI plants. The highest amount of root and shoot length, root fresh and dry weight, shoot dry weight, total dry and fresh weight, relative growth, soluble and total shoot sugar, chlorophyll b, and total chlorophyll were observed in the treatment with both full nitrogen concentration and inoculation with A. brasilense. Also, the present data revealed that seedlings inoculated with Azospirillum performed better than the non-inoculated samples.Meanwhile, current data showed that catalase enzyme activity increased with increased nitrogen concentration (in both inoculated and non-inoculated conditions). However, catalase enzyme activity was higher in inoculated seedlings at all nitrogen levels, compared to non-inoculated seedlings. These data indicate that inoculation with A. brasilense increases oxidative stress tolerance in rice seedlings. This evidence suggests that this bacterium improves plant resilience to treatment with different nitrogen concentrations and enhances their ability to scavenge reactive oxygen species (ROS) produced during nitrogen metabolism. The present data showed that peroxidase enzyme activity increased with higher nitrogen concentrations, in both inoculated and non-inoculated conditions. Conclusion The present results showed that inoculating rice seedlings with A. brasilense can significantly enhance their physiological responses. At all nitrogen concentrations, treatment improved the growth of inoculated plants, which showed higher protein content and enzyme activity compared to non-inoculated seedlings. These findings highlight the potential of A. brasilense to enhance nitrogen efficiency in crops. Meanwhile, our data showed that inoculation with A. brasilense can improve rice seedling productivity, especially under optimal nitrogen conditions.

https://ijpb.ui.ac.ir/article_30080_ab4eb2901709d8feca9690e177663c40.pdf