Pear plants (Pyrus pyrifolia var. Hengshen) showing symptoms of chlorotic spots on leaves were observed in orchards in central Taiwan in 2004. The sap of diseased leaves reacted positively to Apple chlorotic leaf spot virus (ACLSV) antiserum. A purified virus isolate (LTS1) from pear was characterized by host range, electron microscopy, phylogenetic analyses, serological property, and back-inoculation experiments to pear. Fifteen of 28 species of tested plants were susceptible to this virus after mechanical inoculation. Pathogenicity of ACLSV isolate LTS1 was verified by back-inoculating to pear seedlings. Filamentous virions of ≈12 × 750 nm were observed in the preparations of purified virus. Virus particles accumulated in the cytoplasm were observed in the ultrathin sections of LTS1-infected pear leaf tissue. Sequence analyses of the coat protein (CP) gene of LTS1 and the CP gene of LTS2, which originated from a distinct symptomatic pear sample, shared 81.4% to 92.6% nucleotide and 87.6% to 98.4% amino acid identities with those of the CP of 35 ACLSV isolates available in GenBank. ACLSV isolates were grouped into four clusters, i.e., Asia I, II, III, and Europe, and isolates LTS1 and LTS2 were classified as members of cluster Asia II and Asia I, respectively, based on phylogenetic data. Moreover, the variability of amino acid sequences of the CP gene of 37 ACLSV isolates showed geographically associated clustering in the phylogenetic tree. To our knowledge, this is the first study on the characterization of ACLSV causing the leaf chlorotic spot disease of pear in Taiwan. This study also provides the phylogenetic relationships among ACLSV populations based on amino acid sequences of CPs, which are correlated with their geographic origins.
Zhong-Bin Wu, Hsin-Mei Ku, Yuh-Kun Chen, Chung-Jan Chang and Fuh-Jyh Jan
Yu-Tsung Lin, Chia-Wei Lin, Chien-Hung Chung, Mei-Hsiu Su, Hsiu-Yin Ho, Shi-Dong Yeh, Fuh-Jyh Jan and Hsin-Mei Ku
This study was undertaken to establish for the first time an efficient regeneration and transformation system for Cucumis metuliferus line PI292190, which is the source of a well-defined resistant gene, Wmv, that provides resistance against Papaya ringspot virus type P (PRSV-P) and PRSV-W (formerly known as Watermelon mosaic virus 1, WMV-1). Different combinations of growth regulators were evaluated for the regeneration of cotyledon explants. Adventitious buds or shoot primordia were obtained within 3 to 4 weeks on regeneration medium. After shoot development, adventitious buds or shoot primordia were transferred to elongation medium for 3 to 4 weeks and these shoots were subcultured onto rooting medium for another 1 to 2 weeks. Under optimal culture conditions, a total of 7 to 10 weeks was necessary to obtain C. metuliferus plantlets from cotyledons. Furthermore, transgenic plants were successfully obtained using an Agrobacterium tumefaciens-mediated transformation method as shown by polymerase chain reaction analysis and histochemical β-glucuronidase (GUS) assay. A total of nine transgenic plants were developed from 360 cotyledon explants, giving a transformation frequency of 2.5%.