| Citrus yellow vein clearing virus?CYVCV?is a member in genus Mandarivirus,family Alphaflexiviridae.The virus mainly infects some common citrus hosts like lemon,orange,and lime and grapefruit.Since the virus was first reported in China in2014,citrus yellowing vein disease caused by CYVCV has been plague in many areas with an increasing trend.To clarify the genetic variation of CYVCV in China could provide a better basis for understanding the pathogenicity and occurrence of CYVCV,and it is of great significance the management of CYVCV in China.In this study,86 samples with yellow vein symptom were collected from Sichuan,Hubei,Guangxi and Yunnan Provinces,and detected by RT-PCR using CYVCV-specific primers.The results showed that CYVCV was detected in 56 out of86 samples,and the total detection rate was 65.12%.By counting the detection rate of CYVCV in different hosts,it was found that the detection rate of CYVCV was 83.33%in lemon and 16.67%in citron.Detection of samples from different hosts and geographical sources was performed.The results showed that there were significant differences in the detection rate of CYVCV among samples from various regions,among which the detection rate of Hubei samples was the highest,100%,and the detection rate of Yunnan samples was the lowest,43.48%.In addition,the symptoms in the Guangxi region were more complicated than those in other regions.Some CYVCV-positive samples did not show typical symptoms of chlorotic yellow vein disease.It is suggested that upon CYVCV infection,different citrus cultivars may have different symptoms.In this study,citrus-infecting virus like Citrus tristeza virus?CTV?,Citrus tatter leaf virus?CTLV?,and Apple stem grooving virus?ASGV?were investigated in Guangxi samples.The detection rate of CTV was the highest,82.14%.Moreover,more than 50%of the samples were complex-infected by virus,indicating that the compound infection on citrus was serious.In order to clarify the genetic diversity of CYVCV isolates in different regions,the entire sequences of 5 isolates?YN-RL?SC-AY?HB-WH?GX-NNX and GX-NNZ?were used in this study.Genome structure analysis was performed and results showed that all isolates shared similar genomic structure with CYVCV,and the full length ranged from 7529 bp to 7531 bp.The phylogenetic analysis with other 40 CYVCV isolates collected from different regions of the world showed that The intraspecific geneticdiversity in the same virus phylogenetic analysis suggested geographical selection force played amore significant role in virus evolution than host.And the D value of the population neutral detection parameter reached a significant negative value,indicating that the CYVCV population showed a clear trend of expansion.Recombination analysis showed that there were one recombination event and four potential recombination events in 40 isolates.Genetic diversity analysis revealed that the CYVCV population showed lower nucleotide diversity in each segment.The haplotype diversity?Hd?and nucleotide diversity?Pi?of the CYVCV population were1.000±0.006 and 0.01629±0.00192,respectively,indicating that the CYVCV population has abundant genetic diversity.The most significant variation was in the TGBp3 region,with more site mutation in the TGBp1?RdRp and TGBp2 regions,and fewer site mutation in the P23 region.The Hd and Pi values in the IR2 region were0.192±0.083 and 0.01190±0.00572,respectively,which were smaller than the Pi values in the RdRp?TGBp1?TGBp2?TGBp3and CP regions and greater than the Pi values in the P23 regions.This shows that the genetic variation of the TGBp3 region in the CYVCV population is the largest,and the selection pressure is weak;the coding region P23 is more conservative and the selection pressure is strong.In this study,Sichuan lemon,Hubei lemon,Guangxi diseased fragrant citrus and Guangxi disease-free fragrant citrus were selected as materials,15 populations of CYVCV was constructed based on TGB?CP and P23 genes:SC-AY-TGB?YN-RL-TGB?HB-WH-TGB?GX-NNX-TGB?GX-NNZ-TGB?SC-AY-CP?YN-RL-CP?HB-WH-CP?GX-NNX-CP?GX-NNZ-CP,SC-AY-P23?YN-RL-P23?HB-WH-P23?GX-NNX-P23?GX-NNZ-P23?The genetic structure,variation characteristics,and types of base mutations in each population were analyzed.The results show that CYVCV has a"quasi-species"structure,and no identical clones were found between random two regions.Among the clones of 15 populations,the main sequence of each group did not occupy the absolute advantage,and the proportion of mutation sequences was large.The percentage of CYVCV mutant clones exceeded70.00%and the mutation frequency was 10-3.In the four regions,the natural population variation types of CYVCV TGB?CYVCV CP and CYVCV P23 were mutations,with mutation frequency of 10-3,but the mutation frequency varies among populations in different regions.Among them,the highest frequency of mutation was in Guangxi's disease-free fragrant citrus population?GX-NNZ-P23?,which was 3.17×10-3.The lowest frequency of mutations was in Sichuan lemon population?SC-AY-TGB?,which was 1.27×10-3.The base mutation types of two population CYVCV TGB and CYVCV CP have a pronounced tendency of T?C and A?G,respectively,However,The base mutation type of two population CYVCV P23 have a significant T?C and A?G base mutation tendency.The base mutation of the CYVCV TGB population was mainly distributed in the 5'end of the TGBp1 region and the TGBp2 region.There was fewer base mutations in the TGBp3 regione.It shows that TGBp1 and TGBp2 have a higher degree of variation and TGBp3 is more conservative within the population.However,Base mutations were evenly distributed in the CP gene.Base mutations were mainly distributed in the 5'end and 3'end of the P23 region.Fewer base mutations distributed in the central region of CYVCV P23 population. |
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