The goal of the proposed study is to understand the mechanism of survivor trees. 1. Understanding the role of endophytic microbes from survivor trees. Three healthy and three HLB infected trees were selected for phytobiome analysis from Gapway grove based on the LasQPCR detection results. The microorganisms collected from this experiment were classified as three types: rhizosphere, rhizoplane and endosphere communities. The DNA and RNA samples were sequenced. Multiple known beneficial microorganisms, such as Bradyrhizobium, Lysobacter and Variovorax showed significantly higher relative abundance and activity in rhizoplane microbiome despite of health status. However, several beneficial taxa, including Rhodopseudomonas, Achromobacter, Methylobacterium and Chitinophaga, showed higher relative abundance and activity in healthy rhizoplane microbiome compared with rhizosphere community in healthy trees but not in HLB samples. By performing comparison between healthy and HLB samples, we found several phyla, such as Proteobacteria, Acidobacteria and Bacteroidetes were enriched in healthy root-associated microbiome. HLB altered the rhizoplane microbiome by recruiting more functional features involved in autotrophic life cycle such as carbon fixation, and abandoning the functional genes involved in microbe-host interactions identified above, collectively resulting in downward spiral in rhizoplane microbiome-host interaction. This seems to suggest the manipulation of the root microbiome is necessary. However, the challenge is how to maintain a beneficial microbiome which is under study now. Objective 2. To illustrate whether the endophytic microbes from survivor trees could efficiently manage citrus HLB. As shown in Objective 1, Bradyrhizobium and Burkholderia are the most abundant bacteria that have shown dramatic changes between survivor trees and HLB diseased trees. We determined the contribution of Burkholderia to the citrus hosts. We isolated multiple Burkholderia strains. We selected two representative strains A53 (Burkholderia metallica) and A63 (Burkholderia territori) to inoculate citrus plants using the soil drench method. The results demonstrated that the two strains could successfully colonize the root surface and maintain a relative high population even seven months after inoculation. We then conducted a greenhouse study to evaluate the effects of the selected strains on the plant fitness. One manuscript entitled: “Characterization of antimicrobial-producing beneficial bacteria isolated from Huanglongbing escape citrus trees “has been apublished by Frontiers in Microbiology. One more manuscript on the effect of induced systemic resistance against disease by rhizospheric bacteria has been accepted for publication by Phytopathology. In addition, we grafted the roots from survivor trees to healthy and HLB diseased trees in greenhouse to check the effect of endophyte changes on the grafted trees. Since endophytes appear to be enriched from the rhizosphere, we also used the soil from the survivor trees to plant both healthy and HLB diseased trees in the greenhouse. We also grafted shoots from survivor trees to further understand the putative mechanisms. Shoots from more survival trees are being grafted. We are also characterizing the potential mechanism why some branches are Las free. Multiple plants successfully grafted with leaf branches from survivor trees were subject to a test for citrus attractiveness to ACP. No significant effect on response of ACP to the grafted trees from the control. We have grafted more trees with branches from survivor trees to test their effect on Las and ACP. Consortium of bacteria of different combinations are being used to test their effect on Las and ACP. One manuscript entitled: “Huanglongbing impairs the rhizosphere-to-rhizoplane enrichment process of the citrus root-associated microbiome” has been published by Microbiome. We have requested a no cost extension for this project. We are currently finishing up the data collection and summarizing the data for publication.