Development Of Diagnostic Detection And Stable Infectious Clones For Areca Palm Necrotic Ringspot Virus

Areca palm(Areca catechu L.)is an important tropical economic crop in Hainan.There are three kinds of areca palm viruses,areca palm necrotic ringspot virus(ANRSV),areca palm necrotic spindle spot virus(ANSSV)and areca palm velarivirus 1(APV1).Among them,ANRSV was found in the northern and eastern regions of Hainan.It can cause yellow and necrotic ringspot on areca leaf,which seriously affects the normal growth of areca plants and quantity of flowers and fruits.It seriously threatens the sustainable and healthy development of areca planting and industry.There are no effective physical,chemical and biological methods for ANRSV prevention and control.Therefore,the establishment of specific and sensitive detection technology is the key to prevent and control ANRSV.In view of the current situation,there is no other detection technology except RT-PCR and RT-LAMP.In this study,polyclonal antibodies to ANRSV were prepared from ANRSV CP protein and a double-antibody sandwich enzyme-linked immunosorbent assay(DAS-ELISA)was established.In addition,the recombinant enzyme polymerase nucleic acidamplification(RPA)technique was combined with lateral flow dipstick(LFD)detection method for ANRSV was established.These methods provide technical support for ANRSV diagnosis and monitoring,epidemic pre-warning and comprehensive scientific prevention and control system.The analysis of molecular characteristics and pathogenic mechanism of ANRSV is helpful for the development of virus strategy and the selection of antiviral varieties.The infectious cloning of plant virus is an effective tool for reverse genetics study of viral gene power and viral pathogenic mechanism.In this study,Agrobacterium-mediated cloning of ANRSV was successfully constructed using In-Fusion seamless cloning technology.It lays a foundation for the further study of gene function of ANRSV virus and molecular mechanism of virus-host interaction.The main findings are as follows1.Preparation of ANRSV polyclonal antibody and establishment of DAS-ELISA assayIn this study,the ANRSV-CP was expressed by PVX plant virus and pMAL-C5X prokaryotic expression system respectively.The recombinant expression vector of PVX-ANRSV-CP-Strep was successfully constructed.The Nicotiana benthamiana was infected by Agrobacterium injection.The results showed that PVX vector could mediate the effective expression of ANRSV-CP-Strep in Nicotiana benthamiana,and its expression was 22μg/g(fresh leaf quality).In addition,we construct the prokaryotic expression vector of pMAL-C5X-ANRSV-CP-His successfully.After the induction of BL21 strain and IPTG,SDS PAGE results showed that the soluble expression of ANRSV-CP-His was realized by fusion with MBP label.The recombinant protein of MBP-ANRSV-CP-His was obtained by His Ni 60 Superflow resin,and the yield reached 1.275mg(protein)/g(cell).The antiserum of ANRSV-CP was obtained by immunizing the rabbits with recombinant protein expressed in prokaryotic expression as antigen.The titer of the antiserum was more than 3 x 10~5by indirect ELISA.After purification,DAS-ELISA was established.The results showed that the best working concentration of the antibody was 0.5ug/m L and the best working concentration of enzyme standard was 10 ug/m L.ANSSV and APV1 were detected by the established assay of ANRSV DAS-ELISA.The results showed that APV1 were negative and positive with ANSSV,indicating that ANSSV and ANRSV had no serotype difference.However,The positive serum of SPFMV,Tu MV,APV1,PRSV and Cs CMV was negative,indicating that the method was specific.The sensitivity analysis showed that the sensitivity of DAS-ELISA was 1:20 times diluted(w/v,g/m L).2.A fast nucleic acid strip detection technology named ANRSV RT-RPA-LFD was establishedIn this study,a lateral flow dipstick(LFD)combined with the recombinant enzyme polymerase nucleic acidamplification(RPA)was developed for rapid,specific,and sensitive diagnosis of the areca palm necrotic ringspot disease caused by ANRSV.Four primer pairs specific to different regions of the ANRSV genome were designed and screened.The experimental results show that using the ANRSV disease-like cDNA template,the RPA constant temperature reaction can be completed at 34°C for 20 minutes,and the result can be determined within 3 minutes through the colored detection line on the test strip.The sensitivity test results showed that with the positive plasmid PVX-ANRSV-CP as the template,the detection limit of RT-RPA-LFD was 1×10~2copies/μL.This method has no cross-reactivity with 6 RNA viruses ANSSV,APV1,SPFMV,Tu MV,PRSV,and Cs CMV.Detection of latent infection of ANRSV in the areca palm by the developed RT-RPA-LFD was verified by PCR.The obtained results confirmed that RT-RPA-LFD has great potential for highly sensitive detection of latent infection.Therefore,the RT-RPA-LFD assay is simple,rapid,and cost-effective for diagnosing ANRSV in resource-limited settings or even on-site.3.Application of DAS-ELISA and RT-RPA-LFD detection methodsUsing established DAS-ELISA,RT-RPA-LFD and RT-PCR methods to detect 24suspected diseased leaves of ANRSV areca palm collected from Ding an,Qionghai and Wanning cities in Hainan.When using DAS-ELISA method,17 of 24 samples of ANRSV suspected leaves of areca palm showed positive reaction.The positive rate was 70.8%,which was consistent with the results of RT-PCR.lt indicated that the established DAS-ELISA method was effective.When detected by RT-RPA-LFD method,17 out of 24 ANRSV suspected diseased leaves of areca palm were positive.The positive rate was 70.8%,which was consistent with the results of RT-PCR.The results show that the ANRSV RT-RPA-LFD detection method is effective and feasible in field detection.The mixed infection of ANRSV and APV1 was found for the first time.4.Construction and analysis of ANRSV infectious clone mediated by Agrobacterium tumefaciensThe 3 fragments I,II and III including the full-length viral genomic sequence of ANRSV was obtained by PCR and then cloned between the 35S promoter and the poly(A)signal of T-DNA binary vector pGreenⅡ35S to generate pGreenⅡ35S-ANRSV using In-fusion seamless cloning strategy.To test the infectivity of pGreenⅡ35S-ANRSV,Nicotiana benthamiana were infiltrated with agrobacterium-carrying pGreenⅡ35S-ANRSV.After 10 days,it was found that the systemic leaves of the plants inoculated with pGreenⅡ35S-ANRSV infected clones showed obvious leaf curl and mosaic symptoms.The pGreenⅡ35S-ANRSV genomic RNA was detected in symptomatic Nicotiana benthamiana inoculated with pGreenⅡ35S-ANRSV but not in mock-inoculated control plants by RT-PCR.Six out of eight Nicotiana benthamiana were infected with pGreenⅡ35S-ANRSV,indicating that the infection efficiency in Nicotiana benthamiana could reach 75%.The same method was used to inoculate the seedlings of areca palm,and no systemic infection of pGreenⅡ35S-ANRSV was detected.