Detection Of Apple Chlorotic Leaf Spot Virus In Hawthorn And Comparison Of Virus Elimination Methods

Apple chlorotic leaf spot virus(ACLSV)is one of the important latent recessive viruses in fruit trees.Duo to lack of obvious symptom in early time,this virus is difficult to detect.With the growth of the tree,the virus will be accumulated year by year,causing orchard production,and even bring devastating disasters in fruit yield.ACLSV infection of many plant species,different isolates have great genetic diversity and molecular variation of ACLSV.So based on the transcriptome sequencing data using Illumina technology for the young fruits of hawthorn(Crataegus pinnatifida),the detection primers of ACLSV was designed,establishing an optimized system for detection ACLSV in hawthorns.At the same time,we studied the technology for ACLSV elimination from hawthorn with tissue culture.The main research is as follows:1.Primers were designed based on the genomic sequence of ACLSV hawthorn isolates.The RT-PCR detection system for ACLSV was optimized by comparing the cDNA concentration,the kinds of Taq polymerase and the kinds of organs.By comparison,we established an optimized RT-PCR detection system for detection of ACLSV in hawthorn plants.The optimal primer pair F3/R3 was selected.An optimized PCR system for detection ACLSV in hawthorns was as below:1 ?L of undiluted cDNA was used for a 20 ?L PCR volume;Taq DNA polymerase of Promega was screened out;and young leaves were used as the material.2.TaqMan primers and probes used in our research were designed to fit the most conservative CP genome sequence.Standard curve from a plasmid that contained a CP gene sequence of ACLSV used to obtain an absolute quantitation of ACLSV.The sensitivities and reproducibility of this method were investigated.The standard curve was generated using different template concentrations ranging from 6.9 × 108 to 6.9 × 102 copies·?L-1 obtained by 10-fold serial dilutions.This curve between log of DNA concentration versus Ct value generated a linear fit with a slope of-3.18 and linear regression coefficient(R2)of 0.99 and the PCR efficiency was more than 90%.The sensitivity of the qRT-PCR is 100 times higher than that of conventional RT-PCR,the coefficients of variation(CV)values of both intra-and inter-method were less than 0.91%.3.An optimized virus detection method established for hawthorn ACLSV was applied to eighty hawthorns samples detection.Eighty hawthorn varieties were analyzed,and ACLSV was found in 18 varieties.We found the incidence of the viral infections counts for 22.5%in eighty hawthorn plants.4.To study the technology for ACLSV elimination from hawthorn with tissue culture,we used 'Qiuhong' and 'Xinbinruanzi' hawthorn as materials to establish the tissue culture plantlets of hawthorn.The annual branch shoots as explants,by initiation culture and suitable subculture,we obtained some robust tissue culture plantlets.Then,thermotherapy(37?,30 d)and chemistry method(supplemented with 20 mg·L-1 ribavirin in proliferation medium,40 d)were used to eliminate ACLSV from tissue culture plantlets of hawthorn and the efficiencies of virus elimination were compared between thermotherapy and chemistry method.The result showed that:the rate of virus-free plantlets was 100%after thermotherapy or the treatment of thermotherapy combined with chemotherapy,while the rates of virus-free plantlets rate were 88.9%for 'Xinbinruanzi' and 57.1%for 'Qiuhong' after chemotherapy.Thermotherapy was the most effective method to eliminate ACLSV form hawthorn tissue culture plantlets.