Objective This study investigated the effects of different mycorrhizal fungi on the growth and photosynthetic characteristics of walnut (Juglans regia) seedlings under non-stress conditions, and clarified whether combined inoculation outperforms single inoculation, with the aim of providing a theoretical basis for mycorrhizal cultivation techniques in walnut production.

Method Through a greenhouse pot experiment, using ‘Yanyuanzao’ walnut seedlings as the test material, four treatments were established: non-inoculated control (CK), Funneliformis mosseae (F), Piriformospora indica (P), and combined inoculation of both fungi (FP). Chlorophyll content, gas exchange parameters, chlorophyll fluorescence parameters, stomatal parameters, and biomass of walnut were measured.Then comprehensively evaluated the effects of composite mycorrhizal fungal inoculation on walnut seedlings from the dimensions of growth and photosynthetic characteristics using one-way and two-way analysis of variance, correlation heatmaps, and principal component analysis.

Result (1) Single and combined inoculations with Funneliformis mosseae and Piriformospora indica both established symbiotic associations with walnut seedlings, significantly enhancing their photosynthetic performance and promoting their growth. (2) The combined inoculation treatment exhibited significantly higher values than the single-inoculation treatments in chlorophyll a content, total chlorophyll content, net photosynthetic rate, OJIP-test parameters, leaf specific activity parameters, and stomatal length-to-width ratio parameters. (3) Walnut seedlings inoculated with Funneliformis mosseae alone showed the highest stomatal density, stomatal conductance, and transpiration rate; Piriformospora indica alone significantly increased chlorophyll b content, the effective photochemical quantum yield of PSII (F_v/F_m), stomatal length, and stomatal width. (4) Principal component analysis and two-way analysis of variance indicated that mycorrhizal fungal inoculation promoted the photosynthetic capacity of walnut seedlings, and the combined inoculation produced synergistic effects on the photosynthetic physiological indices of walnut seedlings.

Conclusion After the formation of mycorrhizal symbiosis, various photosynthetic physiological parameters of walnut seedlings, including chlorophyll synthesis, net photosynthetic rate, and PSII light energy conversion efficiency, were significantly improved, and stomatal characteristics were markedly altered, ultimately leading to a significant increase in biomass in the inoculated treatments. A comprehensive evaluation of growth, photosynthetic physiology, and stomatal traits indicated that the combined inoculation treatment outperformed single inoculation under the present experimental conditions and exerted a synergistic effect on walnut seedling growth. Therefore, combined inoculation represents a superior inoculation strategy for promoting walnut seedling growth and can provide a theoretical basis for mycorrhizal seedling cultivation of walnut.