Establishment Of RT-LAMP Assay For Detection And Genome Sequence Analysis Of Citrus Psorosis Virus

III Citrus is one of the most widely grown fruit crops in the world,of which China is a major citrus producer with the largest area and production.Citrus psorosis virus(CPsV)is a globally devastating disease of citrus.CPsV is a graft-transmissible viral disease on citrus.CPsV,the type species of the genus Ophiovirus,have single stranded negative RNA genome(11.3-12.5 kb size)containing 3 parts.There are two types of psorosis,psorosis A(PsA),the more common disease,characterized by the presence of bark-scaling in the trunk and limbs of infected field trees and staining of interior wood,Chlorotic flecks in young leaves.The more aggressive psorosis B(PsB),causes rampant bark scaling even in fine twigs in field trees,chlorotic blotching in old leaves with brownish gummy pustules in the leaf underside,and sometimes oak-leaf symptom in young leaves.Plants infected with PsA were protected against challenge inoculation with PsB.only one of genomic sequences of CPsV,the P-121 strain of Spain,has been published.Four sets of specific primer pairs were designed for one step RT-LAMP detection system based on the conserved region of the CP(Coat protein)gene of CPsV from NCBI database.One feasible pair of primers was screened to be suitable for this system.Template RNA extracted from CPsV infected citrus leaves was used for One-step RT-LAMP and isothermal condition was optimized to be reaction temperature of 60 ? for 60 minutes.The agarose gel electrophoresis analysis of RT-LAMP products indicated that the established RT-LAMP system was specific and sensitive in detecting CPsV and 10-fold higher than RT-PCR method in sensitivity.Of 258 samples detected by this RT-LAMP system,5 were found to be infected by CPsV,which was consistent with RT-PCR detection method.Hence this RT-LAMP system is applicable for rapid and accurate detection and identification of CPsV.CPsV is a destructive viral disease on citrus,while viral genome information associated with CPsV is still limited to date.In this study,we obtained the complete genome sequences of five CPsV isolates CHN-1,CHN-2,CHN-3,CHN-HN and CHN-UN21 by using transcriptome sequencing(RNA-seq),RT-PCR,RACE,cloning and Sanger sequencing approaches.Genome sequence analysis showed that the full length sequences of CHN-1,CHN-2,CHN-3,CHN-HN and CHN-UN21 were 11282 nucleotide(nt),11279 nt,11278 nt,11283 nt and 11305 nt,respectively,and comprised of four open reading frames(ORFs)each.Nt sequence similarity between MP genes of the five CPsV isolates ranged from 80.78% to 99.37%,whereas their amino acid(aa)sequence similarity was from 90.55% to 99.16%.Comparing with MP genes of other CPsV-isolates reported,the nt sequence similarity was from 80.78% to 99.79% while aa sequence similarity from 89.71% to 99.58%.Nt sequence similarity between CP genes of the five CPsV isolates ranged from 81.44% to 99.02%,whereas their aa sequence similarity was from 88.38% to 99.32%.Comparing with CP genes of other CPsV-isolates reported,the nt sequence similarities were from 81.29% to 100.00% while aa sequence similarities from 88.38% to 100.00%.Phylogenetic analysis indicated that the isolates CHN-1,CHN-2,CHN-3 and CHN-HN clustered together into a branch of evolutionary tree,while CHN-UN21 on a branch alone.Sequence analysis revealed that there were large differences in the sequences of CHN-3 and CHN-UN21.Since no citrus seedlings infected by CHN-3 and CHN-UN21 alone were obtained,in order to verify whether the two were also different in symptoms,the 24 k gene of CHN-3 and CHN-UN21 MP gene was cloned and inserted into the expression vector TRV to observe differences in symptoms on Nicotiana benthamiana.As a result,their symptoms were similar and showed rolling of the leaf blades into mild cup-shaped form and blistered leaf epidermis.