Indoor And Field Evaluation Of Cross Protection Against Tobacco Virus Disease Based On Mild Vaccines From Cucumber Mosaic Virus

Virus diseases were one of the important plant pathogens and negatively affected the economic development of agriculture in China as well as throughout the world.With the development of genetic technology,cross protection from attenuated vaccines had become the effective strategy against plant viruses.Cucumber mosaic virus（CMV）has the characteristic of wide host range and multi-segments genome,which ensured it as an ideal basic vector for the creation of attenuated vaccines.In this study,new attenuated vaccines based on CMV_Fnywill be constructed.Different types of mild vaccines will be evaluated on the stability and cross-protection on target viruses in door and field.It will provide useful materials and data for management on plant virus diseases.Based on p CC_FR2-2b PTII containing CMV_Fny RNA2 with pre-termination of the 2b protein,two different regions of 100 bp fragments from PVY was respectivbely inserted downstream 2b ORF.The RNA2 mutant with the insertion of 100bp fragments from PVY were mixed with wild-type RNA1 and RNA3 of CMV_Fny.The mixture of RNA1/RNA2-mutant/RNA3 was inoculated into Nicotiana benthamiana.It is suggested that these two RNA2 mutant with 100 bp fragments from PVY presented genetic stability and effective cross-protection on CMV and PVY.Meanwhile,it is also suggested the suitable candidates of PVY fragments during construction of multivalent attenuated vaccines in future.Subsequently,six RNA2 mutants with the insertion of PVX fragments（R2-PX-150I,R2-PX-150II,R2-PX-150III,R2-PX-150IV,R2-PX-200I and R2-PX-200II）,four RNA2mutants with the insertion of TMV fragments（R2-TMV-200I,R2-TMV-200II,R2-TMV-200III and R2-TMV-200IV）and two RNA2 mutants with the insertion of TVBMV fragments（R2-TVB-200I and R2-TVB-200II）were also evaluated for gentic stability and the cross-protection on CMV and targets viruses through the inoculation of tobacco in the lab.It is suggested that six RNA2 mutants with PVX insertion,two RNA2 mutants with TMV insertion（R2-TMV-200I and R2-TMV-200II）and two RNA2 mutants with TVBMV insertion presented genetic stability and effective cross-protection on CMV and corresponding target viruses.These RNA2 mutants can be the suitable candidate vaccines for management on tobacco virus diseases in the field.Three RNA2 mutants（R2-PX-150I,R2-TMV-200II and R2-TVB-200I）were selected as bivalent attenuated vaccines against CMV/PVX,CMV/TMV and CMV/TVBMV respectively to deal with the tobacco virus diseases in the field.Field experiments were carried out in 6type of treatment（CM/PX single agent;CM/TM single agent;CM/TVB single agent;CM/PX+CM/TM mixture;CM/PX+CM/TVB mixture;CM/TM+CM/TVB mixture）in Gaomi and Changle,Weifang respectively.In order to objectively evaluate the effectiveness of cross-protection of these three types of attenuated vaccines,we firstly analyzed species of virus naturally infecting tobaccos in these two regions.In Gaomi county,CMV,TMV,TVBMV and PVY were detected,and PVY had the highest occurrence at the rate of 57%.In Changle county,CMV,TMV and PVY were detected,and TMV had the highest occurrence at the rate of 80%.Tobaccos in Gaomi and Changle counties of Weifang city were treated by these three types of attenuated vaccines in six different ways including single vaccines or mixture of two vaccines.The treatment of attenuated vaccines presented the following characteristic of cross-protection on tobacco virus diseases in the field.In general,the average effectiveness of cross-protection in Gaomi county at 47.04%was better than that in Changle county at 17.99%.Different treatment of attenuated vaccines presented different effectiveness of cross-protection.CM/TM single agent presented the hightest effectiveness compared with other types of treatment.In Gaomi county,the CM/TM single agent had 63.0%effectiveness of cross-protection.In Changle county,the CM/TM single agent had 22.2%effectiveness of cross-protection.However,CM/PX+CM/TM mixture presented the worst effectiveness compared with other types of treatment and was far below the average effectiveness.In Gaomi county,CM/PX+CM/TM mixture had 11.5%effectiveness.In Changle county,the CM/PX+CM/TM mixture had 0.04%effectiveness of cross-protection.The control effect of CM/PX single agent;CM/TVB single agent;CM/PX+CM/TVB mixture;CM/TM+CM/TVB mixture were better,and the relative control effect fluctuated.Taken together,this study provided the practical prodecure of application of attenuated vaccines,the suitable types of attenuated vaccines and the detailed data for cross protection on tobacco virus diseases in the field.