Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 212 - Pathogens and Nematodes Affecting Plants | 910 - Grapefruit | 1160 - Pathology | 34% |
| 212 - Pathogens and Nematodes Affecting Plants | 920 - Orange | 1160 - Pathology | 34% |
| 212 - Pathogens and Nematodes Affecting Plants | 930 - Lemon | 1170 - Epidemiology | 32% |
1. Establish collections of citrus pathogens including CTV, CVV (Xylella fastidiosa), CPsV, Pospiviroids (CEVd, CVd-IV), Hostuviroids (CVd-IIa, CVd-IIb, CVd-IIc), stubborn (Spiroplasma citri), huanglongbing (HLB, ¿Candidatus¿ Liberibacter asiaticus, ¿C.¿ L. africanus), Leprosis, and Citrus leaf blotch virus (Dweet mottle) and Tatterleaf; 2. Establish reliable real-time PCR detection of these pathogens; 3. Establish rapid extraction of pathogen nucleic acid from target tissue; 4. Use results of computational design for oligonucleotides for specific identification of the panel of citrus pathogens from LANL collaborators, assemble fluorescent dye to oligo markers and employ Luminex instrument for detection by MOL-PCR; and 5. Test automated high-throughput nucleic acid extraction machines for target oligo acquisition from citrus tissue.
Results of this study are in support of Objective 1 (Develop field deployable systems that provide rapid, sensitive detection of Citrus tristeza virus and Spiroplasma citri in citrus) of the in-house project. After thorough evaluation of Multiplexed Oligonucleotide Ligation�PCR (MOL-PCR) using citrus tissue infected with various pathogens, with research partner, Los Alamos National Laboratory (developer of the MOL- PCR technology), the procedure was found to still require further improvements before it can be used reliably to detect citrus pathogens from field samples. Therefore, other lines of research were explored to accomplish multiplex pathogen detection. Annual nursery certification for pathogen-free citrus trees produced in the U.S. and many other citrus-producing countries is required. Biological indexing is still required for pathogens, including viroids and the virus causing psorosis. Bioindexing requires a temperature- controlled greenhouse and 6-months to one year to complete. Space and time constraints limit testing to 5-year intervals and is a bottleneck for production of nursery stock. A generic polymerase chain reaction (PCR) diagnostic procedure was developed using a magnetic bead-based commercial kit modified for citrus pathogen detection using a high throughput robotic instrument. Unique pathogen gene sequences were targeted, allowing simultaneous diagnosis of multiple citrus pathogens in a single test. The high throughput real time PCR assay for citrus diseases caused by viroids was certified by the California Department of Food and Agriculture. This procedure was more economical and timely than biological indexing and will allow annual testing of all ~7000 registered scion nursery trees in California. The research enables citrus nurseries to increase the number of certified varieties available for propagation through a streamlined regulatory process.