Physiological responses of black walnut to saline-alkali stress and comprehensive evaluation of saline alkali tolerance

【Objective】Soil salinization has become a global ecological and agricultural challenge, with its area continuing to expand. China has vast areas of potential saline-alkali soil, and improper management may exacerbate land degradation. Screening and utilizing salt-tolerant plants is an effective approach for the biological improvement of saline-alkali land, which holds significant importance for its remediation and sustainable development. Xinjiang, located in an arid region, experiences intense evaporation, and its saline-alkali land area accounts for approximately one-third of the national total, severely constraining local ecological and economic development. Black walnut (Juglans nigra), native to North America, is valued for its high-quality timber, edible nuts, and strong adaptability, making it an important tree species with both economic and ecological value. Currently, research on the saline-alkali tolerance of black walnut has primarily focused on single-salt stress or varietal comparisons, often cen- tered on specific cultivars, leaving a gap in the comprehensive evaluation and mechanistic understanding of saline-alkali tolerance at the family level. Given the complex saline-alkali environment in Xinjiang, studies based solely on salt stress have difficulties in fully reflecting actual conditions. Therefore, this study aims to investigate the growth and physiological responses of seedlings from different black walnut families to saline-alkali stress, thus comprehensively evaluate their saline-alkali tolerance, and identify families with strong tolerance. This research seeks to provide a theoretical basis and practical reference for the selection of saline- alkali-tolerant germplasm, the rational utilization of saline- alkali land resources, and the enhancement of ecological security and economic forest development in Xinjiang's oasis regions.【Methods】Seedlings of black walnut families BH01, BH02, BH03, and BH04 were cultivated from seeds. When the radicles emerged, the seedlings were transplanted into pots with 14 cm in diameter and 25 cm in height. The substrate consisted of a mixture of peat soil, vermiculite, and perlite in a volume ratio of 3∶1∶1. In June, healthy, pest-free, and uniformly growing seedlings were selected for the saline-alkali stress experiment, which was conducted in a greenhouse at the National Longterm Scientific Research Base for Jiamu Pomology in Xinjiang. The experiment was carried out in July 2025. A mixed saline- alkali solution was prepared with a molar ratio of NaCl∶Na2SO4 ∶NaHCO3 ∶ Na2CO3 at 12∶9∶8∶1. Different concentration gradients were set as 0.0%, 0.2%, 0.4%, 0.6%, 0.8%, and 1.0%. Each treatment was replicated three times. During salt application, the solution was irrigated once daily at 250 mL per application for four consecutive days until the total volume was administered. Sampling was completed after 40 days of treatment, and growth indicators, biomass, and photosynthetic parameters were measured.【Results】As the saline-alkali concentration increased, the seedling height increment, ground diameter increment, and total biomass of black walnut seedlings generally showed a declining trend. In terms of leaf phenotypic characteristics, with prolonged treatment duration, higher saline-alkali concentrations led to earlier symptom appearance. At a 0.8% saline-alkali concentration, leaves of all black walnut families exhibited scorching at the edges and tips. Under 1.0% saline-alkali stress, seedlings of the BH04 family showed localized yellowing and scorching symptoms, but the overall leaf condition was the best among the families. The other families exhibited symptoms such as leaf abscission and extensive scorching across the entire plant. With increasing saline- alkali concentration and prolonged stress duration, the net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), and chlorophyll relative content of black walnut family seedlings generally decreased, while the intercellular CO2 concentration (Ci) showed an overall trend of initial increase, followed by a decrease, and then another increase. At the 1.0 % saline-alkali concentration, the BH04 family maintained relatively higher levels of Pn, Gs, Tr, and relative chlorophyll content with a smaller decline, while its Ci value remained at a lower level with a smaller increase. This indicates that BH04 possesses a stronger capacity for maintaining photosynthesis and chlorophyll homeostasis, with both stomatal and non-stomatal limitations working together to mitigate damage from saline-alkali stress. As saline-alkali concentration increased and stress duration extended, the relative electrical conductivity (REC), malondialdehyde (MDA) content, soluble sugar (SS) content, and free proline (Pro) content of black walnut family seedlings generally showed an upward trend. In contrast, soluble protein (SP) content, superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities generally exhibited an initial increase followed by a decline. At the 1.0 % saline-alkali concentration, the BH03 family displayed lower REC and MDA values with the smallest increases, indicating relatively less damage to its cell membranes. The BH01 family accumulated substantial amounts of soluble sugars, soluble proteins, and free proline, with greater increases compared to other families, suggesting a stronger osmotic adjustment capacity to enhance overall stress resistance. Across various saline-alkali concentrations, the BH04 family exhibited higher SOD and POD activities with greater increases compared to other families. Its CAT activity remained the highest at 0.2%-0.4% saline-alkali concentrations, indicating a stronger and more persistent reactive oxygen species (ROS) scavenging capacity and oxidative stress tolerance.【Conclusion】 As saline-alkali stress concentration increased, growth and photosynthetic parameters (such as biomass, stomatal conductance, transpiration rate, and net photosynthetic rate) of black walnut family seedlings were generally inhibited, while intercellular CO2 concentration showed an upward trend. Concurrently, cell membrane permeability and membrane lipid peroxidation product (MDA) accumulated continuously. Antioxidant enzyme (SOD, POD, CAT) activities exhibited an initial increase followed by a decline, while the contents of osmotic adjustment substances (SS, SP, Pro) increased significantly. This reflects the dynamic process of self- regulation and damage accumulation in black walnut under stress conditions. Through principal component analysis combined with the subordinate function method for comprehensive evaluation of multiple growth and physiological indicators, the saline-alkali tolerance of the four families was ranked from strongest to weakest as: BH04＞BH01＞BH02＞BH03. The BH04 family (Thatcher), screened in this study, performed best in terms of growth maintenance, photosynthetic stability, and antioxidant capacity, demonstrating strong saline- alkali tolerance potential. This study provides excellent germplasm resources for afforestation in saline-alkali lands of Xinjiang and for breeding saline-alkali tolerant black walnut varieties.