| Citrus leaf blotch virus(CLBV)is a type species of the genus Citrivirus,family Betaflexiviridae,which can infect citrus,kiwi,cherry and other fruit trees and spread through grafting and mechanical friction inoculation.The scale and area of kiwi planting in Shaanxi is growing,and it has become an important pillar industry.In recent years,the disease of kiwi has received widespread attention,with the exception of canker,anthracnose and root rot of kiwi tree,growing number of viruses were found to infect kiwi fruit in Shaanxi,the hometown of Chinese kiwi fruit.Some viruses can cause leaf mosaics,chlorotic ring spots,or plant decline.However,there have been few studies on the species,distribution,genetic diversity and pathogenicity of kiwi tree infecting virus in Shaanxi Province.In this study,the viral diseases in Shaanxi kiwi fruit was identified by small RNA sequencing analysis.CLBV was the major virus in kiwi cultivation.Furthermore,and CLBV distribution,population genetic structure and pathogenic genes were studied.The results were described as follows:1.The real-time fluorescent quantitative polymerase chain reaction(RT-q PCR),reverse transcription loop-mediated isothermal amplification(RT-LAMP)and ELISA detection methods of CLBV was established,which is suitable for the actual needs of CLBV detection by scientific research and agricultural technology departments.The RT-q PCR method for CLBV detection based on SYBR Green I fluorescent dye method was developed.The results showed that the method was specific,with the slope of the standard curve was-3.378,the determination coefficient was 0.9979 and the amplification efficiency was 97.7%.The sensitivity was 100 times higher than that of conventional RT-PCR.It could be used for low abundance virus samples(such as dormant branches of kiwi fruit).A CLBV detection method based on RT-LAMP was established,which is 100 times more sensitive than the previously reported RT-PCR assay.In addition,polyclonal antibodies for detection of CLBV were prepared at a concentration of 2.91 mg/m L,and a 1:1000-fold dilution could detect 500 pg of antigen.The CLBV in kiwi tree can be detected by Dot-Elisa,which can satisfy the large-scale detection of field samples,and can also be used for study of the pathogenesis of CLBV.2.The incidence and genetic diversity of kiwi tree-infecting CLBV in Shaanxi were investigated.Using RT-LAMP,RT-PCR and Dot-Elisa methods,361 leaf samples from the major regions of the kiwi growing areas in Shaanxi Province were tested positive for CLBV infection with an average incidence of 28.5%.The complete sequence of CP and MP genes were obtained from 60 positive samples from different regions.The results showed that the CP and MP genes were conservative,and the pairwise identities among the 60 CP nucleotide sequences were 89%～100%.The nucleotide differences between different host isolates were only 78%～86%.The complete sequence of the CP gene of all CLBV isolates from the same host were clustered together.The pairwise identities among the 60 MP nucleotide sequences were 90%～100%,and the identities between different host isolates were 77%～79%.Compared with the CP gene,MP gene showed more obvious host specificity.However,preliminary judgments are not related to the geographical locations.3.The CLBV genomic sequence was determined and infectious c DNA clones of high virulence strain and attenuated strain of CLBV were constructed.The full-length genome sequences of CLBV isolates from Zhouzhi(CLBV-ZZ;8792 bp)and Meixian(CLBV-MX;8791 bp)were obtained by PCR and RACE methods,and the vectors of CLBV infectious clone p Cass-CLBV-ZZ and p Cass-CLBV-MX were constructed.The infectious clones were inoculated separately with N.benthamiana,and 14 days later,the new leaves inoculated with p Cass-CLBV-ZZ showed severe yellowing and rolling leaf symptoms.The new leaves inoculated with p Cass-CLBV-MX showed no obvious symptoms,but slight vein clearing could be seen in the leaves through light.Both infectious clones were confirmed to be infectious by RT-PCR,ELISA and transmission electron microscopy.In view of the different virulence of p Cass-CLBV-ZZ and p Cass-CLBV-MX,they were named as high virulence and attenuated strains.4.Pol gene was identified as the pathogenic factor of CLBV.The ORFs of infectious clones of virulent and attenuated strains were recombined.The results showed that the symptoms of the two CLBV infectious clones in N.benthamiana were not changed after replacing the MP or CP gene.After replacing the Pol replicase gene with each other,the symptoms caused by the virulent and attenuated strains of CLBV in N.benthamiana were also interchanged,which confirmed that the Pol gene determines the pathogenicity of CLBV.The amino acid sequence derived from the Pol gene of the two CLBV strains was found to have 114 differential sites.Finally,it was confirmed that the 10 amino acid differences in the1P-2 region of the Pol gene determine the pathogenicity of CLBV.5.A novel Actinidia virus was identified,which was temporarily named as “Actinidia leaf blotch virus Ac”(Ac LBV),and its genomic characteristics were analyzed.The Ac LBV genome contains three ORFs with a full length of 8678 bp and a Poly(A)sequence at the 3’end.Genome characterization showed that it is a new member of the genus Citrivirus,family Betaflexiviridae.The amino acid sequences deduced from the three ORFs were analyzed by phylogeny,and Ac LBV was clustered with members of the genus Citrivirus.The identities of the Pol,MP and CP amino acid sequences with other members of the family Betaflexiviridae were 23.64% to 52.37%,9.67% to 95.58% and 7.10% to 93.94%respectively.The amino acid sequence deduced from the three ORFs had the highest identities with that of CLBV,which were 52.37%,95.58% and 93.94% respectively.Moreover,its whole genome sequence is the most similar to CLBV,with 65.32% nucleotide sequence identity.These sequence similarities are much lower than ICTV’s criteria for new species classification. |
