Preliminary Study On The Distribution Regularity Of "Candidatus Liberibacter Asiaticus" In Sweet Orange Tree And The Reasons For Inducing Leaf Structure Change

Citrus Huanglongbing(HLB)is caused by the infection of the gram-negative bacteria(Candidatus Liberibacter asiaticus,C.Las).It is a devastating bacterial disease with extremely high infectiveness and currently there is no efficient eradication method.Its existence seriously endangers the sustainable development of Citrus industry and rural economy in China.In this paper,C.Las in the orange tree on space-time distribution system analysis,and the relationship between lignin content and the expression level of related genes and the content of HLB was studied from the tissue structure,material content and gene expression level of leaf cells.The causes of lignification of leaf structure infected with HLB were preliminarily explored.The results further understand the damage characteristics and pathogenesis of HLB to trees,and then provided a reference basis for the prevention and control of HLB.The research results of this paper are as follows:1.The distribution of HLB in the sweet orange tree has a seasonal dynamic law.The distribution of C.Las in different parts of the sweet orange tree in different seasons was detected by real-time fluorescence quantitative PCR,and it was found that there was no significant difference in the positive rate of branches and samples between different parts in summer and winter,but the lower leaf samples had no significant difference.The positive rate was slightly lower than that of the middle and upper samples;there were extremely significant differences between seasons.The positive rate of branches and leaf samples in winter was higher than that of leaf samples in summer.The Cq values ? ? of the positive samples(detected with HLB bacteria)were compared and analyzed in the upper,middle and lower parts of the tree.The bacterial contents in summer and winter showed that the content of bacteria in the lower branches > the content of bacteria in the middle branches > the bacteria in the upper branches However,there was no significant difference in the Cq value of the susceptible samples from different branches of the tree in summer,but there were significant differences in the bacterial content of the three parts in winter.In addition,the positive leaf samples were further analyzed according to their positions on the tree body and branches,and their Cq mean values.In spring and summer,the bacterial content of HLB in the upper leaves of the lower branches of the tree was the highest,and the bacterial content of the lower leaves of the upper branches of the tree was the highest.the lowest,but there is no significant difference.In autumn and winter,the bacterial content of HLB in the middle leaves of the lower branches of the tree was the highest,and the bacterial content of the middle and lower leaves of the upper branches of the tree was lower,and there was a significant difference with the middle leaves of the lower branches.2.Difference analysis of tissue and cell structure in leaves of HLB.Three types of leaf samples with the same leaf age but different bacterial contents detected by real-time quantitative PCR were observed by paraffin section(H-CLas,Cq value was23-25;L-CLas,Cq value was 32-34;healthy,Cq value was 32-34;N/A),statistical analysis of the differences in leaf tissue and cell structure showed that the palisade tissue of mesophyll cells was loosely arranged after the invasion of host cells by HLB bacteria,with large gaps and relatively few layers.Sponge tissue cells were loosely arranged,with large gaps and thickened thickness.The thickness of sponge/palisade tissue of infected samples became larger,and was significantly different from CK.The higher the bacterial content,the more serious the destruction of mesophyll cells.Observation and statistical analysis of leaf midrib tissue of the three samples showed that a series of changes occurred in the phloem,xylem and pith after the invasion of the host cells by HLB bacteria.Compared with healthy leaves,the thickness of phloem and pith of infected HLB veins decreased,and the thickness of xylem increased,and there were significant differences between susceptible samples and CK.The areas of vein phloem,xylem and pith of leaves infected with HLB were significantly different from those of healthy leaves.The samples with lower bacterial content had larger areas of phloem and xylem,while the area of ? ? pith was the opposite.the larger the area.3.Quantitative differential expression and analysis of lignin content and related genes in leaves of HLB.The quantitative differential expression and analysis of lignin content and related genes in leaves infected with HLB showed that there were significant differences in lignin content between leaf samples with different bacterial concentrations at the same leaf age.The higher the bacterial content,the higher the lignin content.Real-time quantitative PCR(q-PCR)was used to verify the expression characteristics of five lignin synthesis-related genes in the veins of different samples.And there were significant differences between them and the control;Cs2g28060,Cs7g30160 were down-regulated in the veins of H-CLas and L-CLas,but there was no significant difference in the expression of H-CLas and L-CLas veins.