Complete Genomic Sequence And Biological Characteristics Of A New Alfalfa Mosaic Virus Isolate

Alfalfa mosaic virus (AMV) is a main plant virus in alfalfa, clover and other legumes, causing mosaic, mottles, malformations and other symptoms. Using double-stranded RNA (dsRNA) extraction and modified single primer amplification (M-SPAT), a new AMV was isolated from Trifolium repens. Sequence analysis and biological characteristics of the new AMV isolate were carried out in this study. Fny is a typical strain of Cucumber mosaic virus(CMV)with the same family of AMV. The infection activity of pseudorecombinant between AMV and CMV-Fny was also discussed herein.Trifolium repens with yellow stripes on leaves were collected from Hangzhou Xiasha. After sap-inoculation, it showed diseased symptoms on Nicotiana glutinosa, an indicator plant. Extracted from tissue leaves of N. glutinosa, dsRNA bands similar to AMV genome in size were observed. M-SPAT was performed to amplify all dsRNA sequences, and the whole genomic sequence information was obtained after cloning and sequencing. The result of sequence analysis indicated the virus isolated from Trifolium repens was AMV, tentatively called AMV-HZ. AMV-HZ RNA1 was 3643nt in length, encoding P1 protein (1126 amino acid sequences); RNA2 was 2595nt in length, encoding P2 protein (804 amino acid sequences); RNA3 was 2041nt in length, encoding movement protein (300 amino acid sequences) and coat protein (CP) (218 amino acid sequences). The information of three genomic sequences was published in GenBank database, the accession numbers were HQ316635, HQ316636 and HQ316637 respectively. Alignment of amino acid sequences showed the CP identities were 92~99% between AMV-HZ and other 27 AMV strains reported in NCBI. Among all strains, the CP identities were more than 90%. However, strain Caa1, Dac16, Lyh 1, VRU and Lye 80 showed evident differences in site 67, 72, 84, 176, 212, 221of CP amino acid sequences by amino acid sequence comparison. Phylogenetic analysis of CP amino acid sequences divided AMV stains into two groups (I andâ…¡). Strain Caa1, Dac16, Lyh 1, VRU and Lye 80 formed groupâ…¡, AMV-HZ and other strains belonged to the major group I. There was also a restriction site difference in CP amino acid sequences between group I and groupâ…¡. Primers for amplification full sequence of AMV genome and universal probe for Northern blot hybridization of the progeny RNAs were successfully constructed. Thus, RT-PCR and Northern blot hybridization can be used to detect the infection of AMV. The study of biological characteristics indicated that symptoms induced on Nicotiana spp., Chenopodium amaranticolor and Capsicum frutescens inoculated with AMV-HZ were observed, and the symptoms were varied at different times. The typical symptoms on the leaves of N. glutinosa were systemic chlorosis, local etch and local white spots. The typical symptoms on the leaves of Nicotiana benthamian were chlorotic blotches and vein clearing. The typical symptoms on the leaves of Nicotiana tobacum cv. Samsun NN were systemic chlorosis and yellow stripes. The typical symptoms on the leaves of Chenopodium amaranticolor were yellow chlorotic stripes. The typical symptoms on the leaves of Capsicum frutescens were bright yellow blotches similar to the shape of oak leaf.We also constructed artificial pseudorecombinant F1F2A3 from AMV-HZ and CMV-Fny, in vitro transcription experiments confirmed that it was no infection activity.