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United States Department of Agriculture

Agricultural Research Service

Research Project: CHARACTERIZATION & EPIDEMIOLOGY OF CITRUS TRISTEZA VIRUS & OTHER INVASIVE & EMERGING GRAFT-TRANSMISSIBLE DISEASES OF CITRUS IN CALIFORNIA
2008 Annual Report


1a.Objectives (from AD-416)
Determine genetic diversity of tristeza and stubborn disease agents in California; 2)Characterize tristeza and stubborn biologically by graft and vector passage; and 3)Examine patterns of spatial and temporal spread of tristeza and stubborn.


1b.Approach (from AD-416)
Isolate, identify, and characterize field strains of tristeza and stubborn in California with respect to phenotype, vector transmissibility, serology, molecular structure and phylogeny, and epidemiology. This will be accomplished by using a combination of the following: enzyme-linked immunosorbent assay (ELISA); reverse transcription (RT) polymerase chain reaction (PCR); conventional PCR; real time PCR; transmission electron microscopy; dark field microscopy; bacterial culturing in cell-free media; cloning; sequencing; spectrophotometry; electrophoresis; insect transmission; geostatistics; and ARC-GIS. Replaces 5302-22000-006-00D.


3.Progress Report
These research findings address NP 303, Component 1. Development of new diagnostic methods and tools and plant pathogen characterization, and Component 2. Biology, ecology, epidemiology and spread of plant pathogens and their relationships with hosts and vectors.

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.


4.Accomplishments
1. Documentation of genetic diversity and presence of potential severe CTV strains spreading in central California. No current detection method allows rapid and sensitive strain differentiation of CTV. Quantitative (q)PCR method was developed, by ARS scientists in the Crop Disease, Pests and Genetics Research Unit in Parlier, CA, and is being field tested to distinguish stem pitting CTV isolates like Dekopon and Rocky Hill from mild isolates. This tool can be used to rapidly assess presence of a potential virulent CTV isolates which can be immediately targeted for removal. This accomplishment contributes to National Program 303, Component 1. Problem Statement 1A and 1B.

2. Improvement of qPCR assay and surveys to determine incidence of citrus stubborn disease (CSD) for field epidemiology studies Bacterial culturing for diagnosis of citrus stubborn disease (CSD) is time consuming, laborious, and expensive, making large scale surveys impractical. A Multiplex Taqman® PCR assay was developed, by ARS scientists in the Crop Disease, Pests and Genetics Research Unit in Parlier, CA, which was as reliable as culturing and was used in a sub sampling protocol to estimate CSD incidence in the field. This procedure is now being used to investigate a resurgence of CSD disease in central California and to determine if rouging of infected trees is necessary. This accomplishment contributes to National Program 303, Component 1. Problem statement 1A and 1B.

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
None


6.Technology Transfer

Number of Invention Disclosures Submitted1
Number of Non-Peer Reviewed Presentations and Proceedings5

Review Publications
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.

Last Modified: 10/30/2014
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