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United States Department of Agriculture

Agricultural Research Service

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Research Project: Analyzing Liberibacter Isolates Undetectable by Standard Diagnostic Methods in Florida

Location: National Clonal Germplasm Repository for Citrus

Project Number: 2036-21000-010-04-R
Project Type: Reimbursable Cooperative Agreement

Start Date: May 1, 2012
End Date: Apr 30, 2015

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.

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.

Last Modified: 09/20/2017
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