Current indices of saline-alkaline (SA) tolerance are mainly based on the traditional growth and physiological indices for salinity tolerance and likely affect the accuracy of alfalfa tolerance predictions. We determined whether the inclusion of soil alkalinity-affected indices, particularly Ca²⁺, Mg²⁺, and their ratios to Na⁺ in plants, based on the traditional method could improve the prediction accuracy of SA tolerance in alfalfa, determine important indices for SA tolerance, and identify suitable alfalfa cultivars in alkaline salt-affected soils. Fifty alfalfa cultivars were evaluated for their SA tolerance under SA and non-SA field conditions. The SA-tolerance coefficient (SATC) for each investigated index of the alfalfa shoot was calculated as the ratio of SA to non-SA field conditions, and the contribution of SATC under different growth and physiological indices to SA tolerance was quantified based on the inclusion/exclusion of special alkalinity-affected indices. The traditional method, excluding the special alkalinity-affected indices, explained nearly all of the variation in alfalfa SA tolerance, and the most important predictor was the SATC of stem length. The new method, which included these special alkalinity-affected indices, had similar explanatory power but instead identified the SATC of shoot Ca²⁺/Na⁺ ratio, followed by that of stem length, as key markers for the field evaluation of SA tolerance. Ca²⁺, Mg²⁺, and their ratios to Na⁺ hold promise for enhancing the robustness of SA-tolerance predictions in alfalfa. These results encourage further investigation into the involvement of Ca²⁺ in such predictions in other plant species and soil types under more alkaline salt-affected conditions.

Soil salinization is considered a major global environmental problem due to its adverse effects on agricultural sustainability and production. Compost is an environmentally friendly and sustainable measure used for reclaiming saline–sodic soil. However, the responses of the physiological characteristics of alfalfa and the structure and function of rhizosphere fungal communities after compost application in saline–sodic soil remain elusive. Here, a pot experiment was conducted to explore the effect of different compost application rates on soil properties, plant physiological traits, and rhizosphere fungal community characteristics. The results showed that compost significantly increased soil nutrients and corresponding soil enzyme activities, enhanced leaf photosynthesis traits, and ion homeostasis compared with the control treatment. We further found that the rhizosphere fungal communities were dominated by Sodiomyces at the genus level, and the relative abundance of pathogenic fungi, such as Botryotrichum, Plectosphaerella, Pseudogymnoascus, and Fusarium, declined after compost application. Moreover, the α-diversity indexes of the fungal community under compost application rates of 15% and 25% significantly decreased in comparison to the control treatment. The soil SOC, pH, TP, and TN were the main environmental factors affecting fungal community composition. The leaf photosynthetic traits and metal ion contents showed significantly positive correlations with Sodiomyces and Aspergillus. The fungal trophic mode was dominated by Pathotroph–Saprotroph–Symbiotroph and Saprotroph. Overall, our findings provide an important basis for the future application of microbial-based strategies to improve plant tolerance to saline-alkali stress.

Key message Candidate pathways for alkaline tolerance in alfalfa seedlings were identified; these included those for homeostasis of ions and redox status, biosynthesis of phenylpropanoids, flavonoids, and amino acids, and MAPK signaling.Abstract Soil alkalization severely limits plant growth and development; however, the mechanisms of alkaline response remain largely unknown. In this study, we performed physiological and transcriptomic analyses using two alfalfa cultivars (Medicago sativa L.) with different sensitivities to alkaline conditions. The chlorophyll content and shoot fresh weight drastically declined in the alkaline-sensitive cultivar Algonquin (AG) following alkaline treatment (0-25 mM Na₂CO₃ solution), while the alkaline-tolerant cultivar Gongnong NO.1 (GN) maintained relatively stable growth and chlorophyll content. Physiological analysis revealed that compared with AG, GN had higher contents of Ca²⁺ and Mg²⁺; the ratios of Ca²⁺ and Mg²⁺ to Na⁺, proline and soluble sugar, and enzyme activities of peroxidase (POD) and catalase (CAT) decreased under the alkaline conditions. Further, transcriptomic analysis identified three categories of alkaline-responsive differentially expressed genes (DEGs) between the two cultivars: 48 genes commonly induced in both the cultivars (CAR), 574 genes from the tolerant cultivar (TAR), and 493 genes from the sensitive cultivar (SAR). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that CAR genes were mostly involved in phenylpropanoid biosynthesis, lipid metabolism, and DNA replication and repair; TAR genes were significantly enriched in metabolic pathways, biosynthesis of secondary metabolites, MAPK signaling pathway, and flavonoid and amino acid biosynthesis; the SAR genes were specifically enriched in vitamin B6 metabolism. Taken together, the results identified candidate pathways associated with genetic variation in response to alkaline stress, providing novel insights into the mechanisms underlying alkaline tolerance in alfalfa.