The Sequence Analysed Of 'Candidatus Liberibacter Spp' And Their Distribution In Citrus Fresh Shoots In Ganzhou

Citrus Huanglong(HLB)is the most destructive disease of citrus worldwide.Trees will decline and fruit yield will significant reduced after infected.Due to lack of feasible method to cure HLB,control is largely based on removal of infected trees and spray pesticide to against psyllid.HLB is caused by a kind of Gram-negative bacterium,still unsuccessfully cultured in vitro,Koch's postulates have not been completed,so the classification status doesn't determine.Currently,the HLB bacterium belongs to the genus 'Candidatus Liberibacter',which has three species,including 'Candidatus Liberibacter asiaticus'(CLas),'Candidatus Liberibacter africanus'(CLaf),and 'Candidatus Liberibacter americanus'(CLam).All species and cultivars of citrus can be infected.This disease has become a major risk in citrus industry.HLB short-distance spread occurs by grafting with infected budwood or citrus psyllid insect vectors.In the past few years,due to global warming leads to citrus psyllid break out,and in addition to citrus seeding no specification inspected and quarantined,HLB becomes more and more serious and expanding their normal survival range from south to north.The most efficiently measures to control HLB are applying pesticide during flushing,and planting approved HLB-free seedings as well as removal of HLB-infected trees.In this study,the following two aspects have been focused: 1)Quantitative detection of HLB pathogen in fresh shoots form HLB-infected trees.2)Four different CLas DNA frangments were evaluated forgenetic diversity analysis,including 16 S rDNA sequences,16S/23 S rDNA intergenic spacer regions,ribosomal protein genes and outer membrane protein genes in Ganzhou region.The main results acquired are as follows.1.Distribution of HLB pathogen in fresh shoots from HLB-infected trees.Three different varieties were detected HLB in fresh shoots from HLB-infected trees by quantitative real-time PCR,including ‘Newhall',‘Key Lime' and ‘Hamlin'.The results suggested that the fresh shoots from HLB-infected trees could be detected HLB pathogen,and it shows HLB pathogen is uneven distribution.Six leaves from fresh shoots of ‘Newhall' all could be detected HLB pathogen.Six leaves from shoots from fresh shoots of ‘Key Lime' and ‘Hamlin',the top of the morphology could not be detected.2.HLB samples collection and CLas decetion in Ganzhou region.The CLas PCR identification primer sets OI1(OA1)/OI2 c and GB1/GB3 were used for detecting 118 samples from Ganzhou region.The primers of GB1/GB3 shows no detectable bands,indicating that there are no CLam in Ganzhou region.What is more,PCR-RFLP of Xba?restriction enzymes shows that HLB bacterium in Ganzhou region are all CLas,which have no significant differences of the restriction endonuclease digestion bands between 118 isolates.The primer set of OMP-F/OMP-R designed according to out membrane protein genes by Oligo 7software.32 samples were selected through primer sets IR1/IR2,?1/?2 and OMP-F/OMP-R for further DNA analysis.3.The PCR-RFLP(Polymerase chain reaction with restriction fragment length polymorphism)technique for analysing 32 isolates in Ganzhou region.Two restriction enzymes Msp? and Hae? were used for analysing 16 S rDNA sequence by PCR-RFLP method,16S/23 S rDNA intergenic spacer regions digested by Rsa? and Alu?,ribosomal protein genes digested by Alu?,Hinf? and Taq?,outer membrane protein genes digested by Taq? and Rsa?.The results suggested that 32 isolates were identical and showed no polymorphism.4.Four DNA fragments sequence analysed for 32 CLas isolates from Ganzhou.There are four DNA locus from 32 isolates were analysed by MEGA 5.05,including the bases content,transition and transversion,homology,nucleotide genetic distance and system evolution characteristics.The results suggested that 32 isolates of homology level were around98% to 100%.The sequences of bases with transition/transversion of 16 S rDNA sequence were the lowest,but the GC content were the highest.They are shared high homology and closer nucleotide genetic distance between 32 isolates and CLas sequences from NCBI database.Phylogenetic analysis showed that the HLB bacterium from Genzhou region belongs to CLas.