2019-12_18-065C – High-Throughput Inoculation of Citrus Germplasm for HLB Resistance Screening

High-Throughput Inoculation of Citrus Germplasm for HLB Resistance Screening

Report Date: 02/04/2020
Project: 18-065C   Year: 2019
Category: Plant Improvement
Author: Ed Stover
Sponsor: Citrus Research and Development Foundation, Inc.

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:
· 5300 for Stover lab – for inoculations to screen antimicrobial transgenes
Stover lab:5300 ACP for inoculating 100 detahced leaves, 90 no-choice small trees and six homogenate assays of peptides.
Bowman lab:
First test group of sweet orange on rootstocks was scored in November 2019 (at 4 mai) and PCR being processed now. Grafted trees for groups 2, 3, and 4 are growing in greenhouse now. Will begin ACP inoculation of group 2 in April.Rootstock liners for groups 5, 6, and 7 are being grown in greenhouse now.
Other users:
· 500 for Dean Gabriel – UF
· 4200 for Yongping Duan – inoculations of resistant Duncan Grapefruit
· 1440 for Randy Niedz – inoculations of Ridge Pineapple and Duncan Grapefruit
Previous quarter:
Over 11,560 ACP infected ACP were used in the last quarter, to screen trees in no-choice inoculation of transgenic citrus and prescreen transgenic events using detached leaf assays
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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