2013 Annual Report
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).
Gayle Volk has developed the protocol for cryopreservation of citrus (ms in press) and has preliminary evidence that the methodology may be applied to several varieties of citrus. Dr. Volk will develop the protocols used for cryotherapy.
1b.Approach (from AD-416):
Year 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.
Year 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.
Year 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.
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. Emphasis is being placed on cryotherapy with conventional shoot tip grafting being used for comparison purposes. The project also includes determining the effectiveness of using young indicator plants for biological indexing to verify elimination of graft transmissible pathogens. During this past quarter, selections of tangelo, grapefruit, mandarin and pummelo material have been forwarded to Ft. Collins for therapy using cryotherapy and shoot tip grafting. Recovered plants are tested for presence of HLB 12-14 weeks post treatment. A total of 98 plants recovered from cryotherapy have been tested for HLB using qPCR. The COX internal control cot values ranged from 18.4 to 20.6; all but three recovered plants tested negative for HLB with cot values of 40 or higher. Three plants had cot values of 38.9, 37.6, and 38.7, respectively, and may test positive later. HLB positive controls had cot values ranging from 17.4 to 20.0. All recovered plants will be retested to verify the freedom of HLB on a continuing basis. Cryotherapy techniques appear to be highly effective in eradicating HLB from infected Citrus.