Molecular Strain Identification Of Potato Virus Y And Tobacco Mosaic Virus Isolates From Tobacco Plants In Heilongjiang And Screening Of Effective Fragments For Cross Protection

Tobacco is a major cash crop in China.Heilongjiang Province is one of the main producing areas of tobacco leaves in China.The tobacco leaves produced in Heilongjiang is famous for its good physical properties and low nicotine content.Potato virus Y(PVY)and Tobacco mosaic virus(TMV)are the prevalent viruses that threatening the production of tobacco leaves in Heilongjiang.PVY produces symptoms of mosaic and veinal necrosis in tobacco.TMV induces symptoms of mosaic and malformation in tobacco.So far,there has been no PVY-or TMV-resistant tobacco cultivar or effective antiviral agent available in practice.Cross protection is an effective method to control plant virus diseases.After identifying the molecular strains of PVY and TMV isolates from tobacco plant in Heilongjiang,we used an attenuated clone of Tobacco vein banding mosaic virus(TVBMV)to screen fragments that can effectively mediate resistance to PVY and TMV,and evaluate their efficiency in the field.The main results are listed as follows:1.The genomic sequences of two PVY isolates,Harbin and Mudanjiang,collected from tobacco plants in Heilongjiang Province were determined.Sequence identities,recombination,phylogenetic relationship and selection pressure were analyzed.The results showed that the full-length genome of isolates Harbin(Accession no.MH933741)and Mudanjiang(Accession no.MH933742)were both 9699 nucleotides(nt).Harbin had the highest identities of 98.5% with isolate GBVC 26(JQ969039)from USA and MDJ,while MDJ had the highest identity of 98.5% with isolate Harbin and GBVC 26.Harbin was a recombinant of isolates Oz from USA and N-605 from Switzerland,while MDJ was a recombinant of N-605 and isolate CW from USA.In the phylogenetic tree constructed with the complete genomic sequences,PVY was divided into nine groups,among which Harbin and MDJ belonged to group PVYNWi.All the eleven proteins of PVY were under negative selection,with NIa gene under the highest selection pressure.2.The genomic sequences of TMV isolates HEB1,HEB2 and MDJ collected from tobacco plant in Heilongjiang Province were determined.Sequence identities,recombination,phylogenetic relationship and selection pressure were analyzed.The results showed that the full-length genome of isolates HEB1,HEB2 and MDJ were 6395 nucleotides(nt)(Gen Bank accession numbers MH595919,MH595920 and MH595921).HEB1 and MDJ had thehighest identities of 98.9% and 99.6%,respectively,with isolate Beipiao(HE818412)from Liaoning province,while MDJ had the highest identity of 97.1% with isolate Chuxiong1(HE818417)from Chuxiong of Yunnan province.HEB1 was a recombinant of isoaltes MDJ and HEB2.In the phylogenetic tree constructed with the complete genomic sequence,TMV was divided into three groups,among which HEB1 and MDJ belonged to group I(U1)while HEB2 belonged to group II(Rakkyo).All four proteins of TMV were under negative selection,with CP gene under the highest selection pressure.3.Genomic fragments that can effectively mediate resistance to PVY and TMV from Heilongjiang were obtained.By aligning the genomic sequences of PVY and TMV determined in this paper and those deposited in Gen Bank,we obtained 3 fragments of PVY(PVYF1,PVYF2,PVYF3)and 3 for TMV(TMVF1,TMVF2,TMVF3)that has the potential to generate si RNA.These 6 framgents were cloned into mild mutants of TVBMV and transformed into competent cells of Agrobaterium GV3101 to produce mild vaccines PVY1,PVY2?PVY3,TMV1,TMV2 and TMV3.The plants of Nitotiana tabaccum were firstly inoculated with mild vaccines by agroinfiltration separately,and.challenged with virulent strains 15 days later.At 15 days after challenging inoculation,the protection efficiency of PVY 2 was 75%,the virus accumulation levels of PVY in protected plants reduced by89.27%;TMV3 significantly reduced the symptoms of TMV,and virus accumulation levels were decreased by 67.20%.4.Cross protection effects of mild vaccines PVY2 and TMV3 were evaluated in tobacco fields in Linyi and Weifang of Shandong province,and Harbin of Heilongjiang province.The relative control effects of PVY2 and TMV3 to PVY and TMV in Heilongjiang were 66.88%and 80.74%,respectively.The relative control effects of PVY2 on PVY in Linyi and Weifang of Shandong were 76.46% and 60.71%,respectively,while those of TMV3 on TMV in Linyi and Weifang were 87.34% and 74.40%,respectively.