Construction of an infectious clone of citrus chlorotic dwarf-associated virus and confirmation of its pathogenicity

Citrus chlorotic dwarf disease (CCDD) seriously affects the citrus industry. Citrus chlorotic dwarf-associated virus (CCDaV) is speculated to be the causal agent of CCDD. However, this speculation has not been confirmed by fulfilling Koch’s postulates. In this study, an infectious clone comprising a 1.6-fold tandem CCDaV genome in the binary vector pBinPLUS was constructed and agro-inoculated into ‘Eureka’ lemon (Citrus limon) seedlings through vacuum infiltration. At 60 days postinoculation, 25% of the ‘Eureka’ lemon seedlings developed symptoms of crinkling and curling that were the same as those associated with the wild-type virus. Western blotting and graft transmission assays confirmed that the infectious clone systemically infected ‘Eureka’ lemon seedlings. In addition, CCDaV can establish infection on three more Citrus species and one hybrid, although at different infection rates. These findings support that CCDaV is the primary causal agent of CCDD. The infectious CCDaV clone will allow further studies on the functions of viral proteins and molecular interactions of CCDaV with its hosts.