Identification And Analysis Of Expression Characteristics Of Apple Necrosis Mosaic Virus Yantai Isolate

China is the world’s largest apple producer,and its apple cultivation area,total output and consumption rank first in the world.Apple mosaic disease is one of the major viral diseases affecting the apple industry in China.It occurs in various apple producing areas across the country,leading to a decline in fruit quality and yield,which has brought huge economic losses to the apple industry.Recent studies have shown that Apple Mosaic Virus(ApMV)is not the only pathogen of Apple Mosaic Disease.Prunus necrotic ring spot virus(PNRSV)and apple necrotic mosaic virus(ApNMV)are also pathogens of apple mosaic disease.To date,NCBI has registered a total of 10 ApNMV RNA3 genomic sequences,including 1 isolate from Japan and 9 isolates from China.However,research on apple necrosis mosaic virus isolates in Yantai,Shandong has not been reported.This study investigated the virus infection of apple mosaic virus plants in Yantai,cloned the full-length sequences of ApNMV RNA3 on Fuji and Red Generals,and analyzed their genome structure,taxonomic status,Genetic diversity and molecular evolution relationship,detected the differences in their spatial and temporal expression,and analyzed the sequence differences of ApNMV in apple,pear and rose.It is expected to provide a theoretical basis for the prevention and treatment of the disease,the judgment of epidemic laws and the study of the pathogenic mechanism.The main contents are as follows:1.Tested 31 apple flowers and leaf samples for poisoning in Yantai.The detection rate of ApNMV in the flower and leaf samples was 87.1%.ApMV and PNRSV specific primers did not amplify the target band,indicating that ApNMV is related to the symptoms of apple mosaic disease in Yantai.Mosaic symptoms are highly correlated.At the same time,the three latent viruses ASGV,ACLSV,and ASPV,which are widely present on apples,were tested.It was found that the infection rate of these three viruses with ApNMV was as high as 95%.2.Using reverse transcription polymerase chain reaction(RT-PCR)technology,three fragments of ApNMV of about 440 nt were obtained from the mosaic samples of Begonia(H281),Pear(L16),and Rose(Y6)in Yantai.Sequenced multiple alignments of Begonia(H281),Pear(L16),Rose(Y6)and the target fragment(F411)in Fuji Apple in DNAMAN,the results showed that the obtained three isolates of Begonia(H281),Pear(L16),and Rose(Y6)had a similarity of 93.78%,and the identity of the three fragments with the sequence on Fuji was 89.74%,90.58%,90.87 %.The results of phylogenetic analysis show that these three fragments are closely related to the Fuji apple fragment(F411)and are clustered in the same branch.It showed that ApNMV was related to the symptoms of mosaic disease in begonia,pear and rose.3.Two full length RNA3 genomes of apple ring spot mosaic virus(ApNMV)YF31and YF515 were identified in Red General and Fuji apple leaves with mosaic symptoms in Yantai,Shandong,respectively,GenBank accession numbers are MN911185 and MN911186,the two isolates are 1785 nt and 1783 nt in length.Sequence analysis results showed that the amplified YF515 and YF31 both had about 180 base deletions at the5’UTR,and the sequence variability was large;the nucleotide identity between the IR region and the registered sequence was only 58.39-91.26%.The coding region MP and CP nucleic acid sequence identity were 89.09%~97.52%,89.85%~96.21%;the amino acid sequence identity was 91.79%~98.21%,90.02%~95.43%.Sequence multiple alignment and phylogenetic analysis showed that the isolates did not show host specificity and geographical distribution.Construct phylogenetic tree based on amino acid level and nucleic acid level ApNMV isolates and other viruses of Bromoviridae family by contiguous method,the results showed that the two obtained ApNMV isolates belong to the third subgroup of the isometric unstable ring spot virus of the Brome mosaic virus family in the taxonomic status,and they have homology with APMV and PNRSV,and their nucleotide consistency is respectively 47.28~47.77%,54.7~55.61%.Construct phylogenetic tree with the MP and CP sequences of ApNMV,APMV,and PNRSV registered in GenBank to further clarify the evolutionary differences between ApNMV,APMV,and PNRSV.4.Using the Fuji apple tree carrying ApNMV as the test material,the real~timefluorescence quantitative PCR method was used to quantitatively detect ApNMV of samples collected at different parts and at different times,indicating that the spatial and temporal distribution of ring mosaic virus was different in the tree: the relative contents of ApNMV in apple plants from high to low are: bud,root,bark,leaf.From August 15 to December 15,the relative content of ApNMV was the highest on September 15,and the relative content of the virus was the highest.Thereafter,the relative content of the virus decreased.