Establishement Of Magnetic Separation In Detection Of Two Important Bacterial Pathogens On Citrus And Transcriptome Research Of Citrus Infected With HLB

Xanthomonas citri subsp.citri and Candidatus Liberibacter are two major bacterial pathogens of citurs that seriously affect the citrus cultivation,production and trade.Since there are no effective prevention and cure measures,it would cause big economic loss in citrus industry if the disease is not properly controlled.Most of the countries in the world have established quarantine and inspection regulations to prevent spreading and propagation of these two bacterial citrus diseases.Because of differences in control measures,agronomy,the influence of climate and enviroment as well as the variable latent period of the disease,it always fails to trace the disease before its further development.Effective sample preparation and separation as well as fast and accurate detection method are the basis of disease screening and prevention.This study systematically established the bacteria and citrus genomic DNA extraction method,non-specific bacteria separation method and targeted separation method of Xanthomonas citri subsp.citri based on magnetic separation.Duplex real-time PCR of Xanthomonas citri subsp.citri and Candidatus Liberibacter asiaticus and Immunomagnetic particles–sandwich ELISA of Xanthomonas citri subsp.citri were also established.This study also explored the transcriptome of field-grow citrus infected with Candidatus Liberibacter asiaticus based on RNA-seq technology.The magnetic separation-based bacteria and citrus genomic DNA extraction method had a yield of 192.8±11.7μg DNA/3.3×10~8 CFU Xanthomonas citri subsp.citri and 89.7±5.2μg DNA/100 mg citrus material.Commecial column-based DNA extraction method had a yield of 176.5±3.3μg DNA/3.3×10~8 CFU bacteria and 118.5±2.2μg DNA/100 mg citrus material.It proved that the magnetic separation-based DNA extration method had a equal performance with the commecial kits in DNA yielding.Magnetic separation is more simple and efficient compared with the column-based separation method.The average recovery rate of non-specific magnetic separation of Xanthomonas citri subsp.citri was 73%.The viability of Xanthomonas citri subsp.citri was not affected by magnetic particles.The conjugation between magnetic particles and Xanthomonas citri subsp.citri was affected by the buffer solution.The highest separation rate was acquired with pH 8.0 Tris buffer.Zeta potential of magnetic particles in different buffer solution demonstrated that electrostatic interaction had significant impact with the separation rate besides embedding effect.Immunomagnetic particles–sandwich ELISA(IMP-sELISA)established in this study was very specific to Xanthomonas citri subsp.citri which could distinguished it from Xanthomonas oryzae pv.oryzicola RS105,Xanthomonas campestris pv.campestris,Burkholderia gladioli pv.alliicola,Clavibacter michiganensis subsp.nebraskensis ATCC 27794,Acidovorax avenae subsp.citrulli XY-1 and Pantoea stewartii subsp.stewartii ATCC 29231.The sensitivity of IMP-sELISA was 3.3×10~5CFU/m L.Using duplex real-time PCR instead of IMP-sELISA as the detection method,the sensitivity could achieve 3.3×10~2 CFU/mL which proved the high separation efficiency of the targeted magnetic separtion.RNA-seq results of transcriptome analysis of citrus infected with Candidatus Liberibacter asiaticus showed that 88 genes were differentilly expressed at the gene level of infected citrus with 57 up-regulated and 31 down-regulated.It also showed that117 genes were differentilly expressed at the transcription level with 75 up-regulated and 42 down-regulated.The differentially expressed genes associated with disease resistance included Endochitinase、Glucan endo-1,3-beta-glucosidase 5、Linoleate 13S-lipoxygenase 2-1、Non-specific lipid-transfer protein、Transcription factor MYB82、Transcription factor MYB1R1、Thaumatin-like protein 1、Systemin receptor SR160、putative disease resistance gene protein(pY14)and plant defensin 2.2(PDF2.2).The differentially expressed genes associated with starch metabolism included Isoamylase 2、Beta-amylase and Glucose-6-phosphate/phosphate translocator 2.Other differentially expressed gene included Callose synthase 2、Chlorophyllase-1 and Cathepsin.The study revealed the physiology and pathology differences between filed-grown citrus and greenhouse-grown citrus within different infection period.