Identifying HLB-tolerance and -resistance has greatly evolved in the three years of this project. Several ambitious objectives failed, and efforts were focused on promising project areas. Progress was made and resulted in supporting data and new objectives in a new NIFA-CRDE project (Baldwin lead).� Due to this NIFA funding we did not submit a new proposal to CRDF for this project.�Evaluation of existing cultivar/rootstock combinations for HLB resistance/tolerance was completed and revealed potentially valuable tolerance.� It further indicated that early HLB symptoms and CLas titer are unrelated to growth and cropping. ‘SugarBelle’ and ‘Tango’ were the largest, healthiest in overall appearance, and had the most fruit.� Grapefruit was by far the worst performing scion in this trial.�A replicated trial is underway of 50 selections and cultivars following no-choice ACP, months in an ACP house, and 5 years in the field. Several of our best grapefruit-like hybrids looked good last year but are now declining. The one true grapefruit is among the least healthy selection in the trial. Jackson grapefruit continues to look quite good.� The best performers include hybrids containing�Poncirus, and conventional hybrids which are predominately mandarin.� It may take 2-3 more years to clearly distinguish tolerant material.� Several selections that had looked good last year suddenly dropped to very low canopy growth, and it appears this may be a relatively early indicator of HLB tolerance.��Replicated trials in multiple locations were established of our best sweet-orange-like cultivars and mandarin-types.� These trials are in the field with data collection managed by Greg McCollum of USHRL.� The easy peeling sweet-orange-like hybrid 1-76-55 continues to look better than sweet orange at most sites.� The 1/16th Poncirus hybrid we have been studying under the name Gnarlyglo (released as US SunDragon) continues to look very healthy with no evidence of HLB compromised fruit. Most of the mandarin-types in this trial continue to display marked HLB-tolerance.��A replicated field planting was established of 133 Fortune x Fairchild hybrids, 27 Ponkan-like accessions, 10 advanced ARS selections that are predominately mandarin, and trees of Fortune, Fairchild and Valencia.� Data collection is underway and this planting will be critical for the new NIFA project identifying HLB-tolerance in mandarins.�� �A replicated citrus relatives planting (85 seed source genotypes) was assessed for apparent HLB tolerance.� Within the genus�Citrus, measures of tolerance (canopy density, health, and tree size) correlated positively with % citron in pedigree, with r2 of 0.3-0.6. This has encouraged use of citron hybrids in breeding acid-citrus and introgression of citron genes into phenotypes like sweet oranges, grapefruit, and mandarin hybrids�Seedlings with a range of pedigree contributions from�Microcitrus have been grown in collaboration with M. Smith, Queensland Aus. citrus breeder, and will be planted this fall for field testing of HLB resistance.��Trees of 54 scion pairs were planted in 2000-04 on both mandarin and trifoliate hybrid rootstocks.� There was no significant effect of rootstock on CLas titer in the foliage or the roots.�� Scions which included�Poncirus in the pedigree (no pure�Poncirus), had one third the level of foliar CLas compared to other scions and less severe HLB symptoms, but scion type had no significant effect on root CLas titer. This provided further evidence that scions derived from�Poncirus can contribute to HLB-tolerance.��RNA-seq compared transcriptome responses in HLB- tolerant `Sun Chu Sha’ mandarin and susceptible `Duncan’ grapefruit, to Xcc-flg22 and CLas-flg22 (most active epitope from the pathogen flagella; project initiated with Gloria Moore at University of Florida).� Differential expression of a number of genes occurred between tolerant and susceptible citrus infected with CLas, suggesting their involvement in HLB tolerance. Genes identified may be valuable for studying HLB tolerance using CLas-flg22 as a pathogen proxy.��28 CLas effectors were predicted and transcriptional levels determined in infected citrus. Many were detected suggesting roles in pathogen virulence and host response modulation. Highly expressed effectors will be further tested for potential as biomarkers for screening breeding materials.�Small citrus seedlings or very small micrografted trees were exposed to CLas-infected ACP. CLas titer levels were detectable in most plants at 3 weeks.�� By nine weeks after exposure, susceptible genotypes can be clearly distinguished from resistant material (Carrizo and�Poncirus) by higher CLas levels in roots. There was no evidence that HLB-tolerant genotypes differed from� -susceptible material in the development of CLas titers, which is consistent with field observations.� However, this methodology should be especially useful for screening anti-Las transgenics, with recent transition to using detached leaves for a much shorter assay.�