High-Throughput Inoculation of Citrus Germplasm for HLB Resistance Screening

High-Throughput Inoculation of Citrus Germplasm for HLB Resistance Screening

Report Date: 10/15/2019
Project: 18-065C   Year: 2019
Category: Horticultural & Management
Author: Ed Stover
Sponsor: Citrus Research and Development Foundation

Project rationale and focus:

The driving force for this three-year project is the need to evaluate citrus germplasm for tolerance to HLB, including germplasm transformed to express proteins that might mitigate HLB, which requires citrus be inoculated with CLas. Citrus can be bud-inoculated, but since the disease is naturally spread by the Asian citrus psyllid, the use of psyllids for inoculations more closely resembles “natural infection”, while bud-inoculations might overwhelm some defense responses. CRDF funds supported high-throughput inoculations to evaluate HLB resistance in citrus germplasm developed by Drs. Ed Stover and Kim Bowman. The funds cover the costs associated with establishing and maintaining colonies of infected psyllids; equipment such as insect cages; PCR supplies for assays on psyllid and plant samples from infected colonies; and two GS-7 USDA technicians. A career base-funded USDA technician is also assigned ~50% to the program. USDA provides greenhouses, walk-in chambers and laboratory space to accommodate rearing and inoculations.

Most recent quarter:
Stover lab: 11,560 ACP used for 1388 leaves inoculated by DLA ( detached leaf assay), 108 small trees inoculated by no choice caged assay, and 13 assays run. A total of 14 different experiments, most testing Liberibacter killing transgenics.

Bowman lab: Continued grow out for first group of budded trees that were inoculated in July. First PCR and scoring will be done in November, 2019. Rootstocks were budded with Valencia for test groups 2, 3, and 4, to be inoculated beginning in early 2020. Once these Valencia budded trees are to size, there will be a regular ACP inoculation of a new group of trees every 1-2 months.

Previous quarter:
Over 7000 infected ACP were used in the last quarter, in part to screen 450 trees, but also for other related uses. The Stover lab used 1700 ACP in no-choice inoculation of transgenic citrus. 2700 ACP were used for detached leaf assays in which leaves of putative CLas killing transgenics and related controls are exposed to CLas-infected ACP for 4 days, allowed 3 days for ACP-free metabolism and then assessed for CLas titer in leaves and the ACP. One thousand ACP were used in an assay in which CLas+ ACP are used to develop a uniform homogenate for rapid testing of putative CLas-disrupting peptides

The Bowman lab has transitioned to use of grafted trees with a commercial scion in 2.5 liter pots. The first group of test plants will be removed from ACP inoculation the second week of July to begin post-inoculation evaluation. Subsequent groups of test plants for rootstock evaluation are being prepared.

Previously achieved in this project:
The 35 day federal government shutdown, and the threat of a possible shutdown on Feb 15, directly disrupted our ability to initiate and conduct experiments using the CLas+ ACP colonies. In addition, considerable rehibilitation of colonies and supporting plants was necessary due to the minimal care that could be provided during the shutdown. Only 2400 CLas+ ACP were used for experiments in this quarter and were used for detached leaf assessments of plants expressing three different transgenic constructs. We anticipate a normal demand in the current quarter.

As of December 21, 2018, a total of 14,111 plants had passed through the inoculation process. A total of 361,255 psyllids from colonies of CLas-infected ACP had been used in inoculations. Not included in these counts of inoculated plants and psyllids used in inoculations were many used to refine inoculation procedures, which provided insight into the success of our inoculation methods and strategies for increasing success. After inoculations, plants were returned to the breeders and subsequently subjected to further inoculations when they are transplanted to the field.

In addition to inoculating germplasm, infected psyllids were supplied to other researchers for other purposes. This side of the project grew over time, and detailed records were not maintained on how many were given out until 2018. More than 10,000 infected psyllids were supplied to the research community for an array of experiments during 2018. Recipients included researchers with USDA in Fort Pierce, Ithaca and Beltsville, UF in Gainesville, Cornell in Ithaca, University of California, and University of Nevada.

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