| Citrus（Citrus reticulata Blanco）belongs to the family Rutaceae and the genus Citrus.It is the largest fruit in the world,with the largest planting area and yield in the world.However,citrus virus and viroid diseases prevent the normal growth of citrus plants,resulting in reduced fruit quality and even death of the plants.It has brought serious economic losses to the citrus industry.In this study,to diagnose and identify the citrus virus and viriod pathogens,multiplex RT-PCR detection system of citrus tristeza virus（CTV）,citrus yellow vein clearing virus（CYVCV）and hop stunt viroid（HSVd）were established.Meanwhile,recombinase polymerase amplification（RPA）detection system of CTV was also established.At the same time,two sets of primers designed by Hilf and Roy were used to identify the CTV genotypes of the susceptible plants and analyze their infection status.Main results were as follows:（1）Using citrus samples co-infected by CTV,CYVCV and HSVd,the multiplex RT-PCR reaction system was established by optimizing primer concentration,annealing temperature and sensitivity.The optimal concentration ratio three pairs of primers of CYVCV,CTV and HSVd was1:1:2,and the optimal annealing temperature was 52.9℃.The sensitivity test showed that the system could detect positive samples when the template was diluted to 10-2.When the system was used to detect 157 citrus samples collected from some areas of Fujian province,the results showed that numbers of positive samples of CTV,CYVCV and HSVd was 89,74and 36,respectively,with the detection rate of 56.7%,47.1%and 22.9%,respectively.（2）The genotypes of CTV were identified by Hilf and Roy primers.The results showed that single genotype infection only occurred in the Hilf primer,while the results of Roy primer identification were all complex infection.Among them,the Hilf primer did not identify the corresponding genotype in one positive sample,while the Roy primer could identify the genotypes of all positive samples,and the genotype results identified by the two types of primers were not completely consistent.The results of Hilf primers showed that there were 16 VT genotypes,17 T30 genotypes,3 T3genotypes and 9 T36 genotypes.The co-infection of strong and weak strains accounted for 93.7%of the total number of co-infection,and the co-infection of strong strains accounted for only 6.3%.The results of Roy primers showed that there were 13 VT genotypes,20 T30 genotypes,15T3 genotypes,18 T36 genotypes,and 12 B165 genotypes.The co-infection of strong and weak strains accounted for 83.3%of the total number of co-infection,and the co-infection of strong strains accounted for 16.7%.（3）Four pairs of primers were designed according to the conserved region of the coat protein gene sequence of CTV.After screening and optimization experiments,the RPA detection system of CTV was established.The CTV-RPA2-F/R with a length of 195 bp was selected as the detection primer for the RPA reaction.After optimization,the optimal reaction time and temperature were determined to be 30 min and 39℃,respectively.This system can specifically detect CTV,and the detection sensitivity is higher than that of single RT-PCR.When the system was applied to detect CTV in 24 randomly selected citrus samples,the results showed that 17 positive samples were detected,which was consistent with the results of single RT-PCR.In this study,the multiplex RT-PCR detection system for CTV,CYVCV and HSVd and the RPA detection system for CTV were successfully established,which provided technical support for the diagnosis and control of citrus virus and viroid diseases. |
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