| Citrus chlorotic dwarf disease(CCDD)is an important viral disease,caused by Citrus chlorotic dwarf-associated virus(CCDaV).The disease is spread through grafting and seedlings,which can infect most citrus cultivars.CCDaV was first reported in Turkey’s Eastern Mediterranean Region in the late 1980 s.Currently,it was reported in Thailand.Previous studies reported that the occurrence of CCDaV was restricted to few Eureka lemon(Citrus limon)orchards in Ruili of Yunnan province in China.Recently however,some citrus viral diseases have spread quickly due to frequent domestic exchange,Brings a potential threat to the healthy development of citrus industry in China.Identification of citrus virus plays an important role in its prevention and control.The methods used for virus identification include indicated plant identification,serology,PCR and so on,but the application of traditional detection methods should be based on known information of the virus.Next generation sequencing(NGS)can overcome the drawbacks of traditional detection,and it is not limited by the type of viral genome,nor dependent on any known sequence information,so it can detect and identify known viruses and new viruses quickly and sensitively.In the field survey,virus-like symptoms were observed on some Ruby Green pomelo(C.grandis)orchards in Nanning,Guangxi,with symptoms of severe leaf curling,distortion and chlorosis on young flushes.The infected Ruby Green pomelo expressed dwarfing,stunted development and fruit small,which result in huge economic losses in the local pomelo industry.In order to provide theoretical guidance for the control of this disease,NGS and biological identification were used in this study to identify the virus species causing the disease of Ruby Green pomelo,and then the occurrence,distribution and molecular characteristics of the virus in China were investigated.The geographical distribution and genetic evolution of the pathogen in citrus growing provinces of China were studied.Meanwhile,the production of monoclonal antibody was prepared to the foundation for the rapid detection method in the field.The main results are as follows:1)The pathogen of Ruby Green pomelo was identified.All the Ruby Green pomelo samples were tested negative for Citrus leaf blotch virus(CLBV),Citrus psorosis virus(CPs V),Citrus tatter leaf virus(CTLV),Citrus tristeza virus(CTV),Citrus yellow vein clearing virus(CYVCV),Citrus exocortis viroid(CEVd)and Satsuma dwarf virus(SDV)by RT-PCR.The total RNA was extracted from the infected Ruby Green pomelo for RNA-seq analysis.PCR result showed that the isolate is CCDaV,which is consistent with the genome structure of 15 previously reported CCDaV isolates,with sequence identity of the 15 CCDaV isolates was 99.5% to 99.6%.The results showed that only CCDaV was identified in the transcriptome database.The bud woods from Ruby Green pomelo plants were grafted onto healthy Sanhongmiyou pomelo(C.grandis)plants and covered by insect-proof net.Three months later,the young flushes of these graft-inoculated plants displayed typical symptoms of severe leaf curling,distortion and chlorosis on young flushes.2)The distribution in China and molecular characteristics of CCDaV were investigated.The field survey and sampling were conducted between 2017 and 2019.A total of1,772 citrus samples were collected from 145 orchards in 11 major citrus production provinces of.During sampling,the symptoms on leaves were carefully observed.CCDaV was detected by PCR in 134 out of 1,772 collected samples.CCDaV-positive samples originated from Yunnan(73/704),Guangxi(60/195),and Guangdong(1/136)provinces.CCDaV was not found in Sichuan(0/196),Hunan(0/140),Jiangxi(0/146),Chongqing(0/127),Fujian(0/54),Zhejiang(0 /40),Hubei(0/18),and Guizhou(0/16)provinces.Among the surveyed citrus cultivars,Ruby Green pomelo was the most frequently infected cultivar,with an infection rate of 50.8%(61/120).Other CCDaV-infected cultivars were Thailand Green pomelo(C.grandis,6/25),Eureka lemon(57/274),Mexican lime(C.aurantifolia,5/25),Tahiti lime(C.aurantifolia,4/22)and Sanhongyou pomelo(C.grandis,1/31).CCDaV was not detected on sweet orange(0/120),Sour orange(0/1),Blood orange(0/12),Navel Orange(0/136),tangor(0/266),mandarin(0/59),tangerine(0/110),trifoliate(0/2)or kumquat(0/19).Typical leaf symptoms such as V-shaped notch,chlorosis,curling and distortion were observed on CCDaV-infected Eureka lemon,Ruby Green pomelo,Thailand Green pomelo and Sanhongyou pomelo.Additionally,the three pomelo cultivars Ruby Green pomelo,Thailand Green pomelo and Sanhongyou pomelo plants developed more severe symptoms than Eureka lemon,and young flushes of the pomelo cultivars exhibited vein clearing.Multiple nucleotide sequence alignments of the complete genome of the 17 CCDaV-positive samples were randomly selected from different citrus cultivars and geographical origins from this study,and 15 CCDaV isolates available in Gen Bank.Sequence alignments showed that the complete sequence identity of the 32 CCDaV isolates was 98.98% to 99.97% similar.The phylogenetic tree showed that the 32 CCDaV isolates,including 15 sequences from Gen Bank,were clustered into four groups.CCDaV isolates from China were phylogenetically distinct from the isolates from Turkey.CCDaV isolates from Turkey and isolate Tha30 from Thailand belonged to group 1.CCDaV isolates from‘Ruby Green’ pomelo,‘Thailand Green’ pomelo and ‘Sanhongyou’ pomelo were grouped together with isolates Tha1-17 and Tha1-19 from Thailand and belonged to group 2.Isolates YN-EL-9 and YN-EL-19 from Ruili,Yunnan province,belonged to group 3.The remaining CCDaV isolates from Ruili,Yunnan province,were clustered into group 4.3)The production of monoclonal antibody of CCDaV.In order to fill the gap in the serology of CCDaV and lay the foundation for the rapid detection in the field,the monoclonal antibody were produced by targeting the conserved domain of CCDaV coat protein in this study.The titer and specificity of the antibody was verified respectively by ELISA and Western Blot.The results showed that CCDaV monoclonal antibody are sensitive and specific highly,which could be used in the detection of CCDaV positive plants. |
