Anatomical observation of the characteristics of the abscission zone during leaf abscission induced by citrus canker in Bingtang orange

Abstract: 【Objective】Citrus canker is a worldwide quarantine disease caused by Xanthomonas citri subsp. citri (Xcc), which can infect various organs of citrus plants, causing severe damage and leading to defoliation and fruit drop. The mechanism of leaf abscission induced by citrus canker is still unclear, and there is limited research on the mechanisms of the leaf abscission caused by other diseases. To explore the characteristics and patterns of leaf abscission induced by Xcc in the leaves of Bingtang sweet orange, we tried to bserve the dynamic changes in the structure of separated tissue during the process of leaf abscission in Bingtang sweet orange induced by Xcc, and identify the features and rules of leaf abscission in Bingtang sweet orange induced by Xcc, to provide new insights for the study of leaf and fruit drop in citrus caused by Xcc.【Methods】Using the leaves of Bingtang sweet orange susceptible to citrus canker disease as materials, the different concentrations of wild- type Xcc were injected into the blades to observe the different symptoms and the time of abscission of the leaves induced by Xcc for determining the concentration of Xcc that could significantly promote leaf shedding. The different patho-genic bacteria strains were injected into the blades, and the different symptoms on the back of blades and the shedding time of the blades were observed. Using different live inoculation methods of Xcc, the suitable inoculation methods to promote leaf shedding were selected. Finally, the selected concentrations of Xcc and the method of inoculation in vivo were used to inoculate the leaves, and then the tissues of abscission zone were fixed, embedded, and resin sectioned at different time points after inoculation. The dynamic changes in the structure of the tissues in abscission zone were observed using microscope during the shedding process, and the changes in the number and the average area of the cells in unit area of the abscission zone were analyzed.【Results】After injection of 108 CFU·mL-1 Xcc into the leaves of Bingtang sweet orange, the back of the blades showed watery protuberant lesions at 3 dpi (day post inoculation), the blades began to fall off at 4 dpi, all blades fell off at 6 dpi, and the watery lesions completely covered the back of the blades, without forming the typical volcanic crater-like protuberant lesions of Xcc. As the concentration of Xcc decreased, the watery lesions decreased, and the time of the starting and completing of leaf shedding was correspondingly delayed. There were no obvious symptoms on the surface and no shedding of the wing leaves occurred. After injection of 108 CFU·mL-1 Xcc 049A (Xcc with the main pathogenic effector pthA4 missing) into the leaves of Bingtang sweet orange, the veins on the back of the blades showed water stain at 3 dpi, and there were no watery protuberant lesions on the blades, the blades began to fall off at the same time as the leaves injected with the same concentration of Xcc, all blades fell off at 9 dpi, 2 days after the blades were injected with the same concentration of Xcc, there were no obvious symptoms on the surface and no shedding of the wing leaves occurred. After the injection of 108 CFU·mL-1 Xoo (the same genus of Xcc but non-host bacterial strain) into the leaves of Bingtang sweet orange, no obvious symptoms were observed and no shedding of leaves occurred. After soaking inoculation of 108 , 109 CFU·mL-1 Xcc, callus-like protuberant lesions appeared on the back of the blades at 8 dpi and 3 dpi, and at 14 dpi and 8 dpi, the lesions completely covered the back of the blades respectively. The blades were curled, and no shedding of the baldes or wing leaves was observed at 15 dpi; After the injection of 108 CFU·mL-1 wild-type Xcc, there was no significant difference in the tissue structure of the abscission zone compared with the control. At 4 dpi, the cells differentiated into an isolation layer and a protective layer, and the cells expanded and the average area of individual cells decreased in the abscission zone. At the junction of the separation layer and protective layer, two layers gradually separated from the outer circumference to the central axis.【Conclusion】The injection of 108 CFU·mL-1 of wild-type Xcc could significantly promote leaf abscission, and the injection was more effective than the soaking. Xcc with the main virulence effector pthA4 deficiency could still induce leaf abscission, but the time of abscission was significantly delayed. The leaf cells responded to the signal of abscission; the cells in the abscission zone enlarged and separated, resulting in leaf abscission.