冰糖橙叶片受柑橘溃疡病菌诱导脱落的离区解剖特性分析

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

【目的】观察冰糖橙叶片受柑橘溃疡病菌(Xanthomonas citri subsp.citri,Xcc)诱导脱落过程中离区组织结构的动态变化特征,探明冰糖橙叶片受Xcc诱导脱落的特征和规律。【方法】通过不同的活体接种方式和接种不同浓度的病原菌,筛选加速叶片脱落的Xcc接种方式和接种浓度,并采用树脂切片观察叶片脱落过程中离区组织结构变化。【结果】与注射接种10^(6)或10^(7)CFU·mL^(-1)Xcc、10^(8)CFU·mL^(-1)主要致病效应子pthA4缺失的菌系Xcc 049A,以及浸泡接种10^(8)或10^(9)CFU·mL^(-1)Xcc相比,注射接种10^(8)CFU·mL^(-1)Xcc显著促进本叶脱落,翼叶未脱落,4 dpi(day post inoculation)离区细胞分化形成离层和保护层,离区细胞膨大且细胞平均面积减少。【结论】冰糖橙叶片注射接种10^(8)CFU·mL^(-1)Xcc后,在本叶和翼叶衔接处从外周向中轴逐渐分离,分化形成成熟的离层和保护层,最终导致本叶脱落。

【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 10^(8)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 10^(8) 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 10^(8)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 10^(8),10^(9) 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 10^(8)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 10^(8) 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.