Abstract：【Objective】Walnut bacterial blight poses a serious threat to the yield and quality of Juglans regia L. In the process of disease resistance, the plant's own metabolic system plays a crucial role. 【Methods】This study used Juglans regia L. Qingxiang walnut as the experimental object to investigate the effects of black spot disease on osmoregulatory substances, oxidative substances, key enzymes and secondary metabolites in phenylpropane metabolism pathways during different stages of walnut fruit development. 【Results】The results showed that as the disease progressed, the accumulation of soluble sugar (SS), soluble protein (SP), proline (Pro), and malondialdehyde (MDA) in walnut fruits increased with the increase of black spot stress after infection; The activities of PAL and 4CL, as well as the contents of lignin and TP, increased significantly (p<0.01), while the activities of C4H and the content of flavonoids increased significantly (p<0.05) during the course of the disease, reflecting the role of the phenylpropane metabolic pathway in the resistance of walnuts to black spot disease. Under normal conditions (CK) and black spot susceptibility stress conditions (Xaj), the results showed that: The contents of 6 indexes of (soluble sugar, SS), (soluble protein, SP), (proline, Pro), (malondialdehyde, MDA), were determined, and the dynamic changes of 4 indexes in three different periods were compared. The results showed as follows: the SS content of Xaj decreased by 24.36% compared with CK at day 30; SS content of Xaj increased by 30.48% compared with CK at day 60; SS content of Xaj decreased by 21.00% compared with CK at day 90; Compared with CK, SP content of Xaj increased by 30.46% at day 30, SP content increased by only 2.23% at day 60, and SP content decreased by 77.74% at day 90. The Xaj content was 60.43% lower than CK at 30 d, 185.15% higher than CK at 60 d, and 102.60% higher than CK at 90 d. The MDA content of Xaj was 200.98% higher than that of CK at 30 days, 2.34% higher than that of CK at 60 days, and 16.33% lower than that of CK at 90 days. The research results indicate that black spot disease has a significant periodic effect on the activities of PAL, C4H, and 4CL key enzymes in the phenylpropanoid metabolism pathway of walnut fruit, as well as on the content of lignin, flavonoids, and TP in secondary metabolites. The activity of PAL and 4CL, as well as the content of lignin and TP, showed a significant increase (p<0.01) in the disease group after infection, reflecting the key role of these enzymes in resisting black spot disease, indicating that walnut fruit attempts to resist pathogen invasion by activating defense mechanisms. The activity of C4H enzyme and the content of flavonoids also significantly increased in the subsequent course of the disease (p<0.05), indicating that they play an important role in the walnut fruit's response to pathogen infection. Over time, there will be a decrease in enzyme activity in walnut fruits, indicating that after walnut infection, a large amount of energy and substances will be consumed to improve the efficiency of phenylpropane metabolism, which may lead to gradual depletion of resources. At this point, the walnut fruit may need to be restored to a more energy-efficient state to maintain basic life activities, so the activity of PAL, C4H, and 4CL enzymes, as well as the content of lignin, flavonoids, and TP, may decrease to conserve resources and avoid self injury caused by excessive defense. The phenylpropane metabolic pathway is a complex biochemical process, starting from phenylalanine and undergoing a series of enzymatic reactions to produce various secondary metabolites including lignin, flavonoids, TP, etc. There is a close relationship between key enzymes in the phenylpropane metabolic pathway and changes in the content of secondary metabolites, which jointly participate in the response and adaptation process of plants to biological stress. The upregulation of the phenylpropane metabolic pathway not only increases the content of lignin, but also enhances the physical defense of plants. Meanwhile, the synthesis of flavonoids has been increased through branching pathways, providing the ability for chemical defense and adaptation to environmental changes. 【Conclusion】This comprehensive effect is a synergistic strategy for plants to cope with adversity, ensuring their survival and reproduction. Therefore, selecting and cultivating varieties with high PAL, C4H, and 4CL enzyme activity backgrounds is expected to enhance the resistance of walnuts to black spot disease. In addition, understanding the dynamic changes of these key enzymes and secondary metabolites during the disease course can help develop targeted disease prevention and control strategies, such as applying biostimulants or chemical regulators in advance during the disease prone period to activate or maintain the activity of these key enzymes and improve the plant's disease resistance.
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