Dissertation > Agricultural Sciences > Plant Protection > Pest and Disease Control > Horticultural Crops Pest and Disease Control > Fruit tree pests and diseases > Citrus pests and diseases

Development and Application of the Detection Technique of Real-Time Fluorescence RT-qPCR on Citrus Tristeza Virus

Author YuQingTao
Tutor ZhouChangYong;LiZhongAn
School Southwestern University
Course Plant Pathology
Keywords Citrus tristeza virus Real-time fluorescence RT-qPCR Detection Mild Strain Cross Protection (MSCP) Resistance effect
CLC S436.66
Type Master's thesis
Year 2008
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Tristeza caused by Citrus tristeza virus(CTV)is an economically important citrus disease in the world.It distributes widely in the citrus-producing areas in the world.China is a big citrus producer with the planting areas ranked the first and the output ranked the second in the world.With the readjustment of citrus industrial structure in China since the late 1980s,the cultivation proportion of Pomelo and sweet orange has been increased;the damage caused by stem pitting isolates of CTV has been becoming more and more serious.Based on overseas practice,Mild Strain Cross Protection (MSCP)is the most effective control method in areas where highly virulent CTV isolates and brown citrus aphids(main vector coexist).A method for quantitative,rapid and accurate detection of CTV is needed,which is not only requied to detect the occurrence and spread of CTV quantatively in individual plants,but also to identify the infection of different CTV isolates,and will further help study on the mechanism of MSCP.In this study,a real-time fluorescence RT-qPCR(Reverse Transcription and quantitative Polymerase Chain Reaction)system to detect CTV has been established with fluorescent dyes SYBR GreenⅠbased on a pair of primers designed and screened from the 3’ with a higher homology in CTV genome.This system was used to detect the different isolates collected from fields.A control effect detection system for MSCP was also established between the severe isolate CT14 and the mild isolate CT11 to examine the antagonism control effect of CT11 against CT14 challenged inoculation by 25 and 50 aphids prefeed on infected Jincheng sweet orange by CT14,respectively.The achieved results mainly are as follows: 1.A real-time fluorescence RT-qPCR system to detect CTV has been established firstly in china and it has many advantages such as low pollution(completely closed system reaction),rapid detection (the whole process only 2.5 h),easy operation and experiment design,and low cost.2.This system has good specificity(specific from CTLV、SDV and CPV),good linear range (8×10~2~8×10~8 copies/μL,r~2=1.000),good sensitivity(8×10~0 copies/μL,and it is higher than that of conventional PCR by 100 fold)and good reproducibility(intra-assay variation coefficients of 1.6% and inter-assay variation coefficients of 3.2%).It can provide an effective method for quantitative analysis of CTV.3.This system can detect different CTV isolates in the field stably,so it can be used for the examination of the MSCP at the early stage.4.Based on T3K17 of CTV genome,a pair of primers TQ1 and TQ2 were designed and screened to distinguish the severe isolate CT14 and the mild isolate CT11,and two pairs of primers ACT2 and 18S rRNA were screened as the reference genes for their quantitative analysis.Both general PCR and real-time fluorescence RT-qPCR systems to detect the three genes have been established.To compare with the former,the latter can detect the target gene quantitatively.5.The real-time fluorescence RT-qPCR established was used to monitor the distribution of the CTV severe isolate quantitatively in the immunized plants spatially and temporally firstly.As a result,the mild isolate CT11 can antagonist absolutely or delay the invasion or infection process of the severe isolate CT14 and hence it had some protection effect.The quantity of severe isolates in the same plant increased while the mild decreased and vice versa.The detection percentage of CT14 inoculated by 50 aphids was higher than 25 aphids.The detection results of CT14 varied from plant to plant within the same treatment and there existed the different detection percentages of CT14 from part to part within the same plant.The time order of detection of CT14 in the same plant was young bark→young leaves→old bark→young root→old leaves.

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