Study On The Molecular Characteristics Of Apple Chlorotic Leaf Spot Virus And Two Viriods From Pear

In order to understand the molecular characteristics of ACLSV (Apple chlorotic leaf spot virus, ACLSV), and two viroids ASSVd (Apple scar skin viroid, ASSVd) and PBCVd (Pear blister canker viroid, PBCVd) infecting pear plants grown in China, the genome sequences of three ACLSV isolates from three Pyrus species were sequenced. Meanwhile, the full RNA sequences of ASSVd isolates from pear and apple plants and PBCVd pear plants were analyzed. The obtained results are as followings:1. Molecular characteristics of ACLSV isolates:The genome sequences of three isolates of Apple chlorotic leaf spot virus (ACLSV) from three pear (species (Pyrus sp.) grown in China were sequenced were determined. The complete genome of ACLSV-JB consisted of7560nucleotides (nts)(excluding a poly (A) tail), and the partial genome sequences (excluding about30nt at5’ end and a poly (A) tail at3’end) of both ACLSV-KMS and ACLSV-YH consist of7528nts. They had the same genomic structure with that of previously reported ACLSV isolates. The complete genomes of those isolates consisted of three open reading frames (ORF1-3). The ORF1was5637nts and encoded a putative216kDa polyprotein, which contained replication-associated (Rep) region. ORF2and ORF3consisted of1377nts and582nts, and encoded a50kDa putative movement protein (MP) and a28kDa putative coat protein (CP), respectively. The three isolates showed an overall nucleotide identity of87.3-100%with each other, and68.4-83.1%identity with the11isolates available in GenBank. A phylogenetic tree was generated from amino acide sequences of a hyper-variable region in the Rep (at sites468-670aa) of three ACLSV pear isolates together with11completely sequenced isolates available in GenBank revealed a similar phylogenetic relationship to that obtained fron their genomic sequences, indicating the sequences in the hyper-variable region might be related to the phylogenetic evolution of the virus. The multiple aligments of CP amino acid sequences of ACLSV isolates showed the obtained three pear isolates had the the signature sites (S40-V59-Y75-T130-L184) presenting in "B6type" ACLSV isolates.2. Molecular characteristis of ASSVd: Totally,475pear samples and32apple samples were detected for ASSVd. Results showed that89pear samples and21apple samples were ASSVd positive. The full RNA of ASSVd from37pear samples and13apple samples were sequenced. The obtained sequences had90.7-100%similarity with each other, and87-99%similarity with sequences available in Genbank. Their sizes varied highly and were329-335nts long. In the phylogenetic tree, the obtained sequences grouped into two clades, of which the isolates in clades I were predominant. It was found the most of variations happened in left terminal domain(TL)、pathogenicity domain (P) and rightt terminal domain(TL) of the second structure of ASSVd.3. Molecular characteristis of PBCVd:Totally,94pear samples from four provinces of China were subjected to RT-PCR detection for PBCVd. Results showed that7samples were positive to PBCVd. The full cDNAs of PBCVd from these positive samples were312bp except that a isolate was314bp. AS they were compared to the prototype of PBCVd, the obtained PBCVd sequences had11nucleotide variations, including4deletions at sites101、119、187and249,1insertions at the site54and6nucleotide changes (G48�'A、C142�'T、T149�'C、T176�'C、C234�'G、G235�'C235).