The effect of the application of coal ash in soil on the symbiotic indices of white pea inoculated with Mesorhizobium and mycorrhizal fungi

Background and Objectives: Iranian chickpea (Cicer arietinum L.) ranks third globally in edible legumes, but leads in the Mediterranean and South Asia. It enhances soil nitrogen through symbiosis with Rhizobium bacteria, contributing to sustainable agriculture. Coal, a major energy source, produces ash that can pollute if misused but can improve soil properties when applied properly. Coal ash enhances plant growth and nutrient uptake but higher concentrations may reduce beneficial microbes. Mycorrhizal fungi and Rhizobium bacteria form valuable symbiotic relationships with legumes. The study aimed to investigate the effects of bituminous coal ash on the tripartite symbiosis between chickpea plants, mycorrhizal fungi, and rhizobial bacteria.

Materials and Methods: The study was conducted in a low-phosphorus soil with a loam texture, collected from Azandarian, Hamadan Province, Iran. Bituminous coal ash was obtained from the Shek-Maidan mine, Kermanshah Province. Physical and chemical properties of the soil and coal ash were analyzed using standard methods. A factorial experiment in a completely randomized design with three replications was conducted on sterilized soil. The factors included coal ash at four levels (0%, 2.5%, 5%, and 10%), mycorrhizal fungi inoculation (Funneliformis mosseae and Rhizophagus irregularis, or no inoculation), and Mesorhizobium ciceri inoculation (with or without). Chickpea seeds were surface-sterilized and inoculated with Mesorhizobium before sowing. Mycorrhizal inoculum and chickpea seeds were obtained from certified sources. Soil and coal ash mixtures were sterilized in an autoclave, and 3 kg of the mixture was placed in each pot. Mycorrhizal inoculum (100 g) was applied below the seeds. Six chickpea seeds were planted per pot and thinned to two after germination. The plants were grown under greenhouse conditions without any additional fertilizers, and pots were watered regularly. At flowering, plants were harvested, and roots were separated from the soil. Nodules were counted, and root colonization by mycorrhizal fungi was assessed using staining techniques. Glomalin-related soil proteins were extracted and quantified, and fungal spores in the soil were counted using centrifugation in a sugar solution. Data were analyzed using Excel and SAS software.

Results: The results indicated that the application of coal ash at the 2.5% level significantly improved the symbiotic interactions between chickpea plants and both mycorrhizal fungi and rhizobial bacteria compared to the control (no coal ash). Specifically, this treatment increased the number of nodules formed by Mesorhizobium and enhanced the establishment of mycorrhizal symbiosis. In contrast, applying higher concentrations of coal ash (5% and 10%) resulted in a notable decline in these beneficial symbiotic relationships, as evidenced by a decrease in both mycorrhizal colonization and rhizobial nodule formation.

The highest mean number of rhizobial nodules per pot (41.66) was found in treatments where both Mesorhizobium inoculation and 2.5% coal ash application were combined, along with inoculation of Funneliformis mosseae. This combination produced the most favorable environment for both the mycorrhizal fungi and rhizobia. On the other hand, the lowest number of rhizobial nodules (3.66) was recorded in the 10% coal ash treatment where no mycorrhizal inoculation was applied, suggesting that excessive coal ash inhibits the formation of beneficial microbial interactions.

In terms of fungal performance, both Funneliformis mosseae and Rhizophagus irregularis improved the symbiosis between the chickpea plants and the rhizobial bacteria. However, the beneficial effects were not as significant in the 10% coal ash treatment, where the overall soil conditions seemed less conducive to the establishment of both mycorrhizal and rhizobial symbioses. Interestingly, among the two mycorrhizal fungi, Funneliformis mosseae showed slightly greater tolerance to the coal ash, maintaining better performance compared to Rhizophagus irregularis, especially at higher ash concentrations.

Conclusion: This study demonstrate that the application of bituminous coal ash in small amounts significantly enhances biological parameters, such as the abundance and weight of Mesorhizobium nodules on the roots of white chickpea plants, the percentage of mycorrhizal colonization of the roots, the number of Glomale spores in the soil, and soil glomalin or glycopeptides. Applying coal ash in low amounts (2.5%) could be beneficial for chickpea growth by improving symbiotic relationships with both mycorrhizal fungi and rhizobia, thus enhancing plant growth and function. However, increasing the coal ash concentration to higher levels (5% and 10%) can deteriorate the soil's ability to support these beneficial symbiotic interactions, leading to reduced plant growth. Although the response of the two fungi species was not significantly different, Funneliformis mosseae demonstrated greater tolerance and better performance in response to the harmful effects of ash. Inoculation with Mesorhizobium increased symbiotic associations with fungi, particularly Rhizophagus irregularis, in the plant, while the inoculation and application of mycorrhizal fungi promoted nodule formation and symbiosis between Mesorhizobium and the chickpea plant. Therefore, the application of 2.5% bituminous coal ash in the soil is beneficial for chickpea growth and its symbiotic relationship with microorganisms, whereas higher doses may have adverse effects on plant growth and overall soil health. This highlights the potential risks associated with high concentrations of coal ash in agricultural systems.