2008 Annual Report
A quantitative PCR(qPCR) assay was developed to distinguish potentially mild vs severe strains of Citrus tristeza virus (CTV). The method is currently being tested to differentiate CTV strains in California and Florida. Characterizations of CTV from central California continued to show presence of new CTV strains in field plots having genetic diversity from those found in previous years. Some isolates caused seedling yellows and mild stem pitting in indicator plants. Rapid CTV spread was found to be occurring in fields monitored in Tulare County. The cotton aphid, Aphis gossypii, is the principal vector of CTV in California and was found overwintering on citrus suggesting development of a biotype adapted to citrus in central California. Local movement of these aphids in early spring were linked to localized rapid spread of CTV as opposed to migrating aphids from weeds and other crop hosts. A qPCR assay for Spiroplasma citri, the causal agent of citrus stubborn disease (CSD), was improved using multiplex Taqman® probes. This procedure allowed measurement of CSD incidence and documented pathogen spread in the field. Trees with severe CSD symptoms were documented to be small with less plant canopy, drop fruit prematurely and have smaller and less abundant fruit. Scaphytopius spp. leafhoppers were observed to feed, multiply, and overwinter in citrus in association with the presence of CSD in an isolated citrus grove near Coalinga, CA.
3. Development of a multiplex real time PCR assay for the simultaneous detection of CTV, S. citri, and huanglongbing (HLB)- associated disease. California has begun monitoring citrus for the presence of the Ca Liberibacter asciaticus-associated huanglongbing (HLB) in dooryards and commercial fields. A multiplex quantitative PCR assay was developed, by ARS scientists in the Crop Disease, Pests and Genetics Research Unit in Parlier, CA, capable of simultaneously detecting the HLB-associated disease as well as stubborn and tristeza in any combination of singleplex, duplex, or triplex from extracts made from citrus tissues. Application of this procedure has potential to develop new information on strains of tristeza and distribution of stubborn wherever HLB surveys are taken and can play a role in detection and delimiting surveys for these pathogens. This accomplishment contributes to National Program 303, Component 1. Problem statement 1A and 1B.
5.Significant Activities that Support Special Target Populations
Yokomi, R.K., Mello, A., Saponari, M., Fletcher, J. 2008. PCR-based detection of spiroplasma citri associated with citrus stubborn disease. Plant Disease. 92:253-260.
Saponari, M., Keremane, M.L., Yokomi, R.K. 2008. Quantitative Detection of Citrus tristeza virus (CTV) in Citrus and Aphids by Real-time Reverse Transcription-PCR (TaqMan®). Journal of Virological Methods. 147:43-53.