|Yokomi, Raymond - Ray|
Submitted to: International Organization of Citrus Virologists Proceedings
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/23/2012
Publication Date: 2/3/2012
Citation: Yokomi, R.K., Saponari, M., Metheny, P., Vidalakis, G. 2012. Genetic differentiation and biology of Citrus Tristeza virus populations spreading in California. International Organization of Citrus Virologists Proceedings. Available: http://www.ivia.es/iocv/archivos/Proceedings_XVIII_Conference/Yokomi_et_al.pdf. Interpretive Summary: Molecular and biological properties were assessed of representative Citrus tristeza virus (CTV) strains collected from more than 1500 CTV-infected trees during 2008-2010 in Central and Southern California citrus groves. Serological tests were performed using the severe strain-specific CTV monoclonal antibody MCA13. MCA13-reactive strains were further differentiated by multilocus sequence-specific markers in Polymerase Chain-Reaction (PCR) assays. PCR was combined with single-strand conformation polymorphism (SSCP) to assess genetic variation among MCA13-negative and MCA13-positive strains. Coat protein (CP) gene sequences were reverse transcribed and amplified by PCR, DNA denatured with heat and electrophoresed in nondenaturing polyacylamide gels. Genetic variants (haplotypes) were identified by migration of single-stranded DNA in gels. Amplified CP and P20 gene region sequences from variants detected by PCR-SSCP assays were cloned and sequenced and phylogenetic relationships of haplotypes determined. Most Central California CTV strains were MCA13-negative, T30-genotypes, and mild or asymptomatic in greenhouse bioindex tests. Southern California CTV strains were mostly mild but ~20% were MCA13-reactive strains. A few MCA13-positive strains were found in Central California. Quantitative real-time Reverse Transcription PCR (qRT-PCR) assays differentiated MCA13-positive strains into two groups based on probe reaction: i) T36NS-positive, VT3-negative; ii) VT3-positive, 36NS-negative. Genome sequences analyzed from T36NS strains showed a close relationship with the CTV T36-decline strain from Florida and CTV resistance-breaking strains from New Zealand. VT3-positive strains had a T3 genotype and shared a phylogenetic relationship with a seedling yellows (SY) strain from Japan. T36NS strains caused mild disease in bioindexing tests; whereas the T3 strains produced severe seedling yellows (SY) disease. These data showed the VT3 probe was effective in detecting severe CTV strains and support use of genotype-specific probes to differentiate CTV strains which react with the MCA13 antibody and help identify trees infected by severe strains of CTV.
Technical Abstract: CTV strains were collected from more than 1500 CTV-infected trees in citrus groves in Tulare, Kern, Ventura, Riverside and San Diego Counties to assess molecular and biological properties of CTV strains currently in California. Tests included serology with MCA13, quantitative (q) real-time Reverse Transcription (RT) Polymerase-Chain Reaction (PCR) (qRT-PCR) assays with strain-specific markers, Single Stranded Conformation Polymorphism (SSCP) analysis of amplified products from the coat protein (CP) gene region and sequencing of PCR products from CP and P20 gene regions. Central California CTV strains typically had a T30-like genotype, were MCA13-negative and were mild or induced no symptoms on indicator plants. Southern California strains and a few from Central California were mostly T30-like strains in single or mixed infections with a non-standard (NS) and/or T3-like genotype strain. CP and P20 gene sequences from NS strains clustered on the same main clade as T36 and were named T36NS. Genetically diverse strains were selected and indexed in greenhouse tests. T36NS strains reacted with MCA13 and were mild on Mexican lime, and symptomless in other citrus indicators. T3-like strains in single or mixtures with T30-like strains also were MCA13 positive, reacted with the CPiVT3 probe and induced strong seedling yellows in indicator plants. California CTV strains, thus, grouped into three classes: i) mild strains with a T30 genotype which did not react with MCA13; ii) T36NS-like genotype strains which reacted with MCA13 but cause mild disease in bioindex tests; and iii) severe strains with a T3-like genotype which reacted with MCA13 and produced virulent seedling yellows disease in the bioindex tests. These data characterize CTV strains in California and support use of genotype-specific probes used in surveys to differentiate putative severe CTV strains.