1a. Objectives (from AD-416):
We have tested thousands of psyllid and plant samples from Florida for Ca. Liberibacter asiaticus by standard qPCR assays. Our plant samples were mainly citrus relatives from wild, backyards, parks, and variety collections. Symptomatic, but qPCR negative samples were subjected to further analysis. Selected small set of samples were analyzed for other genomic regions by conventional PCR (cPCR), cloning and sequencing. This study confirmed the presence of Liberibacters not detectable by standard qPCR assays. These samples were further analyzed using a set of 384 primer pairs by qPCR confirming variability. We propose to analyze several citrus relatives for the presence of Liberibacters by various cPCR and qPCR assays. Selected isolates will be sequenced for several genome regions. Graft transmissibility and their effect on Florida commercial citrus varieties will be tested under greenhouse conditions in Florida. We also propose to develop rapid methods for detecting these population variants.
1b. Approach (from AD-416):
Since 2005, we have tested thousands of psyllid and plant samples by qPCR for Candidatus Liberibacter asiaticus (Las). From this testing, we have DNA extractions from about 750 plants of citrus and citrus relatives collected at 3-6 time points (including the GIS coordinates to relocate plants) and stored frozen. Several of these source trees have been photographed multiple times, the DNA extractions have been tested for Las as well. The data is managed in an Access database. This is excellent source materials for conducting this study. We have developed a macro array of 384 primer pairs to detect regions spanning throughout the 1.2 mb genome of Las. In addition to conventional PCR and qPCR facilities, we have recently acquired a 384 well ABI ViiA 7 qPCR machine with several new capabilities including detection of multiple targets (24-72 nos) in very small reaction volumes. Milestones: Year 1: Identify 50 extractions from HLB symptomatic plants that tested qPCR negative; characterize the bacterial isolates present in these plants by conducting cPCR of multiple genome regions, cloning and sequencing; conducting SYBR green qPCR using our 384 macro array; and analyzing the 16s rDNA region. Develop methods to identify the variant Liberibacter populations. Conduct limited graft transmission studies from citrus relatives with known Liberibacter variants on to different commercial citrus varieties (in a greenhouse at Fort Pierce). Years 2 & 3: Re-analyze existing plant DNA extractions collected over the last three years of our surveys in Southern Florida using new method/s along with standard qPCR to better understand population shift in the Liberibacter population, if any, over a period of time in citrus and citrus relatives. Conduct similar studies with psyllid DNA extractions from selected samples collected since 1998, and from different counties.
3. Progress Report:
This project is related to objective 3, "Develop more accurate and high through-put diagnostic methods for priority graft transmissible pathogens of citrus and for insect-vectored pathogens of dates, to enable the exchange of pathogen-tested accessions, and to more efficiently evaluate accessions for host-plant resistance" of the parent project. Citrus huanglongbing (HLB) is associated with three species of Candidatus Liberibacter: Ca. Liberibacter asiaticus (Las), Ca. Liberibacter americanus, and Ca. Liberibacter africanus. So far, only Las has been associated with HLB in Florida. From previous surveys conducted in South Florida, Ca. Liberibacter-like bacterium were detected by DNA testing from several plants, which would test negative by standard PCR testing. Twelve of these isolates have been recovered from the field and grafted into receptor plants at the USDA, ARS U.S. Horticultural Research Laboratory (USHRL), Ft. Pierce, Florida, and are being monitored to determine if the isolates have been established. Genome characterization is being conducted to determine the relationship with known Ca. Liberibacter species. Information developed will be useful for the development of better detection methods for HLB.