Studies On Viruses Infecting Rosaceae Plants

Based on the survey of viruses infecting Rosaceae plants from in Zhejiang Province, Liaonin Province and Shandong Province in China, pathogenic viruses of this family were detected and identified with biological and molecular techniques. Sequence similarity was compared between Prunus necrotic ringspot virus (PNRSV) isolates, Apple stem grooving virus (ASGV) isolates, Apple mosaic virus (ApMV )isolates and Prune dwarf virus (PDV) isolates, which were detected from different region and different host. Phylogenetic tree and antigenic index were also analyzed. Virus infecting sweet cherry and apple tree were also detected using a newly developed slide hybridization assay (SHA).Field survey indicated that symptoms on Rosaceae plants virus infection varied intensively. ELISA and RT-PCR were used for virus detection from natural-infection of Rosaceae plants. The infection rate of PNRSV on sweet cherry was 45% when tested with ELISA, the infection rate of ACLSV was 30% by RT-PCR. It suggested that PNRSV was the main virus infecting sweet cherry and ACLSV may also one of the principal virus. ASGV and ApMV were two main viruses infecting apple trees, the detection positive rate were 26.7% and 18.3% respectively by ELISA. The infection rate of PNRSV on rose, peach and apple was low. PNRSV was not detected on Chinese cherry.For understanding the genomic variation, geological distribution and host adoption ofPNRSV, the CP sequences of different isolates were compared, after RT-PCR and cloningof the part genome. Eight Mainland isolates (PNRSV-C4, PNRSV-CTA, PNRSV-C3,PNRSV-C915, PNRSV-A1, PNRSV-A2, PNRSV-P, PNRSV-R) originally isolated fromnaturally infected apple plants, sweet cherry, peach and rose were collected and identifiedfrom Zhejiang Province, Liaonin Province and Shangdong Province respectively. The coatprotein genes of Mainland-China isolate, PNRSV-P, consisted of 675 nucleotides (nt)coding for 224 amino aids, seven other Mainland-China isolates consisted of 681nucleotides (nt) coding for 226 amino aids. The sequence data obtained in our study wasthen compared with previously reported sequences of 54 other PNRSV isolates obtainedfrom other areas of the world. Similarity of nucleic acid sequence for CP gene was notrelated with the host and geological origination. The homology of nucleotide acidsequence and amino acid sequence of all PNRSV isolates share 87.7%~100% and87.9-100% similarities respectively. The all isolates formed three subgroups, PNRSV-Pwas assigned into subgroup III, and seven other isolates were assigned onto subgroup I .There was an additional 6 nucleotide insertion at position between 118-125 nucleotideacid of CP gene in subgroup I . Therefore, this feature appears to be a generaldistinguishing feature of Group I. Analysis of antigenic index of eight Mainland isolatesindicated that PNRSV-P belonged to CH9 mild serotype, seven other isolates were similarwith CH9 cripple serotype, but there is much diversity at position of NO 5, 62 and 126 amino acid.The CP gene sequence of two ASGV isolates (ASGV-A1, ASGV-A2) and one ApMV isolate (ApMV-A) were obtained with RT-PCR from apple tree. The sequence of two ASGV isolates were compared with other ten ASGV isolates from other origin with DNAStar software, the homology analysis of CP gene sequence and amino acid sequence of 12 ASGV isolates shared 90.2%~100% homology at nucleotide acid level, while at amino acid level shared 94.1%~100%. ASGV-Al and ASGV-A2 were most identical with UV01 isolate, ASGV-Al and UV01 shared 99.2% similarity, while ASGV-A2 and UV01 share 99.6% similarity, these three isolates may have the same origin. Analysis of phylogenetic tree suggested that CP gene of ASGV isolates was not related with the host and geological origination. The sequences of ApMV collected from China and 16 other ApMV isolates were also compared, they shared 87.5%~100% similarities at nucleotide acid level and 82.3-100% similarities at amino acid level. The 17 ApMV isolates could classified into three subgroups, ApMV-A isolate belong to subgroup I , the sequence of ApMV-A was compared with isolate Fuji2, L03726, Malus domestica and Fuji1, they shared 99.9%, 99.4%, 99.1% and 99.1% similarities respectively. These five isolates may have similar origins. Analysis of phylogenetic tree suggested that CP gene of ApMV isolates was related with the host origination in certain degree, but was not related with geological origination. RNA slide hybridization assay was used for detection ASGV and ApMV from natural infected apple, the positive rate of buds tissue/ young leaves was much higher than mature leaves tissue, and we suggested that buds tissue or young leaves of apple tree should be used for detection of viruses.cDNAs containing the CP sequences of PDV isolates (PDV-DL) from sweet cherry were obtained by RT-PCR and sequenced. The sequence date was compared with other 21 PDV isolates from different origins. The sequence similarity of all PDV isolates was 88.1%~98.9% at the nucleotide acid level and was 89.4%~99.5% at the amino acid level. Phylogenetic tree analysis indicated that 22 PDV isolates could not been devidded into obvious subgroup. There were 56 amino acid mutation positions in total PDV CP amino acid sequence, 21 more mutation positions than previously reported. The N-end part of PDV CP amino acid sequence was much conserved, the hydrophilicity plot analysis of all PDV isolates at this sequence region was very similar, which supported the hypothesis that the N-terminal part of the CP is crucial for RNA-binding activity.