Citrus canker is a bacterial disease caused by Xanthomonas citri subsp. citri (Xcc), which can infect branches, leaves and fruits, and affects almost all major citrus varieties. The results of a previous survey of 39 citrus orchards in Hunan Province showed that soil acidification and exchange calcium deficiency in citrus orchards were serious and calcium deficiency existed in all leaves. Calcium is one of the elements required by plants in large quantities, and calcium deficiency causes nutritional imbalance, reduced growth potential and compromised plant immunity level. However, the effect of calcium element on the process of citrus infection with canker is not clear.

The objective of this study is to analyze the pathogenic differences after inoculation with Xcc in Poncirus trifoliata (sensitive to citrus canker) leaves under different calcium concentrations, and to explore the role of calcium in Xcc infection of P. trifoliata leaves.

The seedlings of P. trifoliata were sand cultured with calcium concentrations of 0, 0.75, 3, and 30 mmol·L^-1. During the growth period of P. trifoliata, the biomass, chlorophyll a and b concentrations, and calcium content in roots and leaves were determined, as well as the formation of reactive oxygen species (ROS) in roots and callose deposition. The effects of Xcc inoculation on cell wall synthesis-related genes and immune-related genes in P. trifoliata leaves were investigated.

Compared with 3 mmol·L^-1 calcium treatment, 0, 0.75 and 30 mmol·L^-1 calcium treatments inhibited the growth and development of aboveground and underground parts of P. trifoliate, and chlorophyll a and b concentrations decreased. The calcium content in roots and leaves was proportional to the amount of exogenous calcium. ROS and callose deposition were generated in roots after treatment with different calcium concentrations, reaching the maximum at 3 mmol·L^-1. After inoculation with Xcc, the leaf symptoms gradually decreased with the increase of calcium concentration, but the growth of Xcc had no significant difference. Compared with 3 mmol·L^-1 treatment, PtCESA4, a gene involved in cell wall synthesis, was up-regulated and then down-regulated by Xcc under 0 mmol·L^-1 treatment, and up-regulated by Xcc under 30 mmol·L^-1 treatment. PtPME and PtFLA were down-regulated by Xcc under 0 mmol·L^-1 treatment, and up-regulated by Xcc under 30 mmol·L^-1 treatment. The expression levels of immune pathway related genes PtGSL, PtGST1 and PtWRKY22 induced by Xcc at 30 mmol·L^-1 were higher than those at 0 and 3 mmol·L^-1 after inoculation with Xcc at 0, 2, 4 and 6 dpi.

The growth and development of P. trifoliate is affected by calcium deficiency and excess, resulting in leaf chlorosis, and ROS production and callose deposition in roots decreased. The sensitive symptoms on the leaf surface caused by Xcc were greatly attenuated after calcium application, but the bacterial content was not significantly different from that of the control. Calcium may promote cell wall thickening by regulating genes related to cell wall synthesis, thereby inhibiting Xcc from breaking through leaf epidermis and forming typical symptoms.