The objectives of this research are 1) to develop cost effective thermotherapy protocols for field application by optimizing temperature and relative humidity conditions in the tent; 2) to develop a mathematical model derived from our data and grower�s data which will be used to determine the best treatment duration in future applications; and 3) to study gene expression of HLB-affected citrus plants that received heat treatment, and identify critical citrus genes that may be induced by heat stress for the benefit of suppressing HLB. To reach our goals in objective 1, we have exposed both HLB-affected and healthy periwinkle (40�C) and citrus (42�C) to heat stress. DNA has been extracted and amplified for Las 16S rRNA and certain phage genes. A standard curve for a normalization gene has been established and data is being analyzed using the delta Ct method. Additionally, changes in expression levels for these genes are being monitored. Reverse-transcription PCR is currently being used to confirm the results of gene expression data. As that Las cannot be cultured, a kill curve of Liberibacter crescens was determined. A dramatic decrease in viability was shown after L. crescens was exposed to 10 minutes of 46�C. For objective 2, we have conducted a comparison study between field heat-treated and non-heat-treated citrus plants. There were 31 consistent up-regulated genes and 47 down-regulated genes in the citrus trees treated with heating. Additionally, potted Las-positive and negative citrus was exposed to 4 hours for 4 days of 40�C, 85 % relative humidity (similar to heat exposure in field setting) in a controlled greenhouse. RNA-Seq data was analyzed using DESeq2 with a FRD of 0.5 and fold change above 2. Using new tender flush as a sample, there were 3,722 differently expressed genes (DE) between Las-negative and positive trees not exposed to heat. Flush that appeared after heat treatment on the positive plants had 294 DE as compared to flush on unheated positive plants and 1308 DE as compare to flush on healthy trees. Some heat shock and oxidative proteins were identified in the DE lists. Analysis and confirmation are ongoing. As for the third objective, over 3 years of prior data (tree Las Ct values, treatment procedures, and temperature logs from one location) have been summarized and are currently being used to determine an algorithm that relates environmental conditions with decreases in Las titer. Extensive analysis of temperature and humidity data using KS nonparametric test, paired T Test, and other measures have shown that each HLB-affected tree responds uniquely to heat treatment. The greatest decrease in Las titer and overall duration of this decrease varies for each tree (6-18 months) and is not solely dependent on heat but most likely affected by the biology of the tree. When comparing 7 versus 9 days of treatment, the longer treatment did not increase titer reduction. Also, six days was not more effective than a 4 day treatment. Regarding temperature, the greatest effect was present at 40, 41, and 42�C for 5 to 7 hours for 3 out of 4 test plots. A detailed correlation chart indicates other combinations of temperature and durations can also be effective at reducing Las. All statistical analysis shows that the response to heat stress is unique for each Las-infected tree. Although fruit drop did decrease for many of the treated trees, due to the large variation in data, the decrease was not statistically significant. Juice quality taste panels have just been completed. Panelists could tell the difference between juice from commercial Valencia trees that were heated and not heated with a 95-99% confidence interval. Further analysis of taste panel data is ongoing. Volatile production analysis for one group of data (out of three) has been finished. A total of 63 aromatic volatile compounds were detected by HS-SPME-GC-MS. Discriminant analysis separated the “no heat” juice from the “heat” juice. Juice made from the product of the heat-treated trees had “fruity” and “pineapple fruit” top-notes.