|Yokomi, Raymond - Ray|
Submitted to: Acta Horticulturae
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/11/2011
Publication Date: 3/15/2011
Citation: Yokomi, R.K., Saponari, M. 2011. Molecular analysis among MCA13-reactive isolates reveals a rapid strategy for assessment of Citrus tristeza virus severity. Acta Horticulturae. 892:251-256. Interpretive Summary: Some severe strains of Citrus tristeza virus (CTV) cause economic damage to citrus while others are mild on CTV tolerant or resistant rootstock. Most strains of CTV in the San Joaquin Valley are mild and symptomless in commercial groves. This has put growers at odds with the CTV-eradication program which tested all trees for CTV. Any CTV-infected tree was removed immediately since the virus is spread by aphid vectors. In previous studies, a method to differentiate CTV strains by real time-polymerase chain reaction (qPCR)multiplex assay was developed. This report details a strategy to rapidly differentiate CTV strains using serology as an initial screen to select potentially severe CTV strains and then apply a multiplex qPCR assay to further differentiate these isolates. This was accomplished by testing extracts from trees with a broad spectrum CTV antiserum to determine if the tree was infected by CTV. CTV positive extracts were then tested with MCA13 antiserum. MCA13 reacts with all known virulent CTV strains along with a few mild isolates. The serological tests were performed by a user friendly method called direct tissue blot immunosorbent assay. All MCA13-reactive isolates were then subjected to a multiplex TaqMan®-based qPCR assay with strain discriminating primer/probes which differentiated CTV strains into definitive genotypes with higher correlation to disease phenology. This separated the CTV genotypes into potentially severe (VT or T3) vs. a potentially mild (T36NS genotype) phenotype. Trees infected with VT and T3 genotypes could then be targeted for removal.
Technical Abstract: Citrus tristeza virus (CTV) usually occurs as a complex of strains that vary greatly in severity and aphid transmissibility. A rapid assay, therefore, is needed to distinguish potentially mild vs. severe strains of CTV for disease mitigation. An economical and practical strategy to screen for potential severe CTV strains was developed using a two step system. Serology with a broad-spectrum and the strain-discriminating MCA13 monoclonal antibody was used to screen isolates. MCA13-reactive isolates were then subjected to qPCR assays using three strain-specific TaqMan® probes. This allowed categorizing MCA13-reactive isolates into at least three distinct CTV genotype groups which included potentially severe strains associated with VT or T3 genotypes vs. T36NS genotype associated with a mild phenotype. Trees infected with VT and T3 genotypes could then be targeted for removal.