ABSTRACT
Citrus exocortis viroid (CEVd) exists as populations of heterogeneous variants in infected hosts. In vivo generated CEVd progeny variants (CEVd-PVs) populations from citrus protoplasts, seedlings and mature plants, following inoculation with transcripts of a single CEVd cDNA-clone (wild-type, WT), were studied. The CEVd-PVs population in protoplasts was heterogeneous and became progressively more homogeneous in seedlings and mature plants. The infectivity and pathogenicity of selected CEVd-PVs was evaluated in citrus and herbaceous experimental hosts. The CEVd-PVs U30C, G128A and U182C were not infectious; G50A and 108U+ were infectious but reverted back to WT and 62A+, U129A and U278A were infectious, genetically stable and more severe than WT. The 62A+ and U278A and U129A accumulated at higher levels than WT in protoplasts and seedlings respectively. The effect of specific mutations on the predicted secondary structure of the CEVd-PVs' RNA coupled with the infectivity and replication studies suggested complex structure-to-function relationships for CEVd.
Subject(s)
Citrus/virology , Plant Diseases/virology , Polymorphism, Genetic , RNA, Viral/biosynthesis , Viroids/metabolism , Viroids/pathogenicity , Virus Replication , Molecular Sequence Data , Mutation , Nucleic Acid Conformation , RNA, Viral/genetics , Sequence Analysis, DNA , Viroids/geneticsABSTRACT
A quantitative and multiplex real-time RT-PCR assay was developed to detect Citrus tristeza virus (CTV) along with plant mRNA, which serves as an internal control to ascertain RNA extraction quality. The real-time technique was validated against 39 CTV strains from around the world as well as with the aphid vector, Aphis gossypii, given a 48 h acquisition access period on a CTV source plant. The assay was effective for quantitation of the viral template in infected plants and in single aphids. CTV detection was compared from different plant tissues and for different RNA isolation methods from aphids. Less than 1 fg was consistently detected when RNA transcripts were diluted in extracts from healthy plants while RNA copies carried by single aphids were estimated to be between 12,000 and 13,000,000. The assay was more sensitive and less time consuming than ELISA or traditional RT-PCR. The real-time RT-PCR assay developed is a valuable new tool for detection and titer quantitation of CTV.
Subject(s)
Aphids/virology , Citrus/virology , Closterovirus/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction/methods , Animals , Enzyme-Linked Immunosorbent Assay , Plant Diseases , RNA/isolation & purification , RNA, Messenger/analysis , RNA, Messenger/genetics , RNA, Viral/analysis , RNA, Viral/genetics , Sensitivity and SpecificityABSTRACT
The incidence of Citrus tristeza virus (CTV) was found to increase significantly in southern Florida within 2 years after the establishment of its most efficient vector, Toxoptera citricida (Kirkaldy). Increased incidence of both mild and severe strains was documented, with the incidence of severe strains increasing more than mild strains. Molecular probes capable of differentiating mild, quick decline and various types of stem-pitting strains demonstrated that trees often were infected with more than one strain of CTV, with trees containing up to five different strains. Some CTV strains detected in the southeast urban corridor of Florida and in commercial groves in southwest Florida were found to react with probes specific for stem-pitting strains known from elsewhere in the world. The implications of the presence of these CTV strains in Florida and their possible presence in citrus budwood scion trees are discussed.
