First Steps towards Rescuing Las-Infected Citrus Germplasm

Huanglongbing (HLB) disease is having a devastating impact on the citrus industry in Florida.  The disease is also having significant impact on the USDA citrus breeding program as it has shown up in a number of trees which exist only in our population of segregating hybrids or in a virtually irreplaceable  germplasm collection.  HLB is associated with infection by Candidatus Liberibacter asiaticus (Las) and it is critical that we rescue Las - budwood from elite germplasm that is Las+.  Fortunately, Las is known to be unevenly distributed within Las+ trees, and although the pathogen is graft-transmissible, the efficiency of graft transmission never is 100%.  Therefore, we reasoned that by selecting budwood that tests Las-, albeit from Las+ trees, we would produce some propagations free of Las.  We tested our hypothesis by starting with seven hybrids in our program that exist only in the field and which we felt needed to be rescued.  At least 3, and as many as 6, branches from each of these trees were tested for Las using standard qPCR methods.  Three propagations were made from each of the Las-tested braches for a total of 90 propagations.  Of the braches initially tested, 63% were Las+.  Of all the propagations, 89% survived, with no apparent difference in survival between propagations made from Las+ or Las- branches.  Of the propagations that survived, Las testing had been completed on 90% of them at the time of this writing.  Among  all of the propagations that have been tested, 29% were Las+.  Among propagations made from branches that were Las+, 55% (18/47) tested Las+ whereas among the propagations made from Las- branches, 12% (3/25) were Las+, with two of these propagations originating from the same original branch.  Average Ct value for Las+ propagations from Las+ branches was 27.9 compared to 36.0 for the Las+ propagations from Las- branches; Ct = 36 is accepted as the cutoff between Las- and Las+.  So far, our data support the notion that testing for Las prior to propagation is an important first step in the process of rescuing Las- tissue from Las+ trees.  Propagated trees will continue to be monitored for the appearance of HLB symptoms and further development of Las as determined by qPCR, to determine whether uneven Las distribution and selected propagation from Las+ trees will permit rescue of critical germplasm.