1a. Objectives (from AD-416):
The purpose of this project is to determine the ability of cryotherapy to eliminate the bacterium associated with huanglongbing (HLB) from citrus and to determine effectiveness of using young indicator plants for biological indexing. Currently the breeding programs in Florida are continuing to evaluate trees under field conditions due to lack of a better alternative, even though HLB is present. While antibiotic treatment appears to successfully eliminate bacteria, application of cryotherapy (e.g. freezing small vegetative buds in liquid nitrogen and recovery of the bud by thawing and grafting onto a seedling rootstock without inducing juvenility) may provide a better alternative, and have the additional benefit of eliminating other graft transmissible pathogens. Cryotherapy has been reported to eliminate HLB in Citrus (Ding et al, Plant Cell Reports 27:241) and to eliminate numerous viruses in other crops (Wang et al, Annals of Appl. Biol. 154:351).
1b. Approach (from AD-416):
ARS scientists have developed the protocol for cryopreserve citrus (ms submitted) and had preliminary evidence that the methodology may be applied to several varieties of citrus. Scientists will develop the protocols used for cryotherapy. The citrus breeding team at USHRL, Ft. Pierce, will maintain plants under protected conditions in Florida and assist with biological indexing and extraction of nucleic acids for use for laboratory detections. ARS scientists will perform laboratory diagnostics establish biological indexing trials at the USHRL, Ft. Pierce using young indicator seedlings. Milestones distributed over the two years will be: 1) Develop cryotherapy protocols for mandarins, sweet oranges. Apply cryotherapy protocols to citrus germplasm collected as part of the germplasm recovery project and held in under protected conditions at USHRL, Ft. Pierce. Conduct biological and lab diagnostic assays to accessions before and after cryotherapy to determine effectiveness of pathogen removal. 2) Develop cryotherapy protocols for grapefruit and pummelos. Apply to germplasm held at Ft. Pierce as well as from collections made from trees in the field. Conduct biological and lab diagnostic assays to accessions before and after cryotherapy to determine effectiveness of pathogen removal. 3) Develop cryotherapy protocols for lemons, limes, papeda and citrus relatives. Apply to germplasm held at Ft. Pierce as well as from collections made from trees in the field. Conduct biological and lab diagnostic assays to accessions before and after cryotherapy to determine effectiveness of pathogen removal.
3. Progress Report:
This project is related to objective 1, Develop and apply new methods for conserving and propagating citrus and date genetic resources which improve genebank operating efficiency and effectiveness and which enable pathogen-tested materials and associated information to be distributed worldwide", of the parent project. The purpose of this project is to determine methods to effectively eliminate ‘Candidatus Liberibacter asiaticus’ (Las), the bacterium associated with huanglongbing (HLB) in Florida from citrus which are used as budwood sources. Emphasis is being placed on cryotherapy as well as other treatments which may be successful at elimation of Las, such as antibiotic treatments and heat treatments. Accessions of tangelo, grapefruit, mandarin and pummelo have been forwarded to the Plant Germplasm Preservation Research Unit, Ft. Collins, Colarado, for treatment using cryotherapy. The recovered plants are being tested for presence of Las at intervals following treatment.