Development And Target Screening For A Small RNA-based Management That Can Prevent Citrus Tristeza And Citrus Huanglongbing

Citrus tristeza virus(CTV)and Citrus Huanglongbing(HLB)are the two most serious diseases of citrus.Citrus aphids and psyllids are important pests of the two viruses that spread the virus.Citrus is one of the important cash crops in our country,and the output ranks first in the world,so the prevention and treatment of CTV disease and HLB disease has great economic significance and research significance.At present,the control of citrus aphids and psyllids mainly relies on traditional control methods such as pesticides.Pesticide control pests not only cause harm to the environment and humans,but also cause resistance to aphids and psyllids.Therefore,it is necessary to find safer and more sustainable methods for prevention and control.RNA interference(RNAi)as a pest control method,has been applied in some agricultural protection fields,especially pest control and beneficial insect protection.Because of its simple structure design and high specificity,small RNA can be used as a new type of biopharmaceutical to provide a new strategy for the control of citrus diseases caused by citrus aphids and psyllids.This research takes citrus aphids as the research object,and attempts to develop an application system that uses RNAi technology to control citrus aphids by screening high-efficiency target genes and synthesizing ds RNA.Relying on this system,target genes with a higher lethality rate were screened out,and applied research was carried out in citrus orchards.The specific research content is as follows:1.Synthesis of ds RNA and establishment of target gene screening system for citrus aphidsThe candidate targets were selected based on the existing literature on the use of RNAi to study other insects,and 9 candidate target gene fragments were successfully amplified in Acyrthosiphon pisum.Using the target gene sequence as a template,ds RNA was synthesized through in vitro transcription and bacterial induced expression.We used Ap AK gene of aphid as template to synthesize double-stranded RNA and verified its optimal delivery mode of ds RNA in Acyrthosiphon pisum.In this study,three methods of delivering ds RNA were compared,and the results showed that the feeding and soaking method had the best effect.Using this method as a system,four genes,including Ap AK,Ap All-A,Apabd-A,and Ap Cth D,were screened among the candidate gene targets that have obvious control effects on Acyrthosiphon pisum.2.The control results of important genes of citrus aphids and the transmission of small RNA in plant systemsBased on the screened target gene Ap AK with a higher lethality rate,the silencing efficiency was tested.The high-efficiency lethal gene Ap AK selected in the previous chapter is used as a target gene.Acyrthosiphon pisum were treated with ds-Ap AK at a concentration of 400 ng/μL,and the silencing efficiency of Ap AK was tested.The results showed that at 24 h,48 h and 72 h,the silencing efficiency of ds-Ap AK was 24.3%,48.2%and 51.82%,respectively,which further confirmed that ds-Ap AK had inhibitory effect on the growth and development of Acyrthosiphon pisum.Using ds-Ap AK as the material,a small RNA agent application experiment was carried out in a citrus orchard.It was found that the 8-d lethality rates of small RNA agents with a concentration of 400 ng/μL and 800ng/μL were 56.88% and 69.38%,respectively.High concentrations of small RNA agents can increase the mortality of aphids,but the effect is not significant.The citrus shoots were soaked in the ds RNA solution,and the leaves and stems of the citrus shoots after absorption of ds RNA solution were intercepted for verification.The study proved that ds RNA can be transported through the plant transport system up to 8 cm after 24 h,and it can’t be degraded for 120 h under natural conditions.This is an optimal strategy for the control of aphids and other Hemiptera with stinger-sucking mouthparts.In summary,we synthesize ds RNA based to the target genes of citrus aphids and inhibit their expression,thereby affecting the growth and development of aphids and causing death,and achieving the purpose of cutting off the transmission route of CTV disease.It overcomes the various defects existence of chemical control and physical control.Establish a system for exploring the target gene screening and ds RNA delivery of citrus psyllids,and to lay the foundation for the prevention and treatment of citrus Huanglong disease.