Thermotherapy and the Molecular Mechanism Behind the Success of Heat Treatment for the Control of Citrus Huanglongbing

Huanglongbing (HLB), a systemic and destructive disease of citrus, is associated with three species of α-proteobacteria, ‘Candidatus Liberibacter asiaticus’ (Las), ‘Ca. L. africanus’ and ‘Ca. L. americanus’.  Previous studies have found distinct variations in temperature sensitivity and tolerance among these species.  Las, the most prevalent and heat-tolerant species, can thrive at temperatures as high as 35°C.  Our earlier work has shown that Las bacteria in potted HLB-affected citrus were significantly reduced and often eliminated when exposed to continuous temperatures of 40 to 42°C for a minimum of 48 hours.  To determine the feasibility and effectiveness of thermotherapy in the field, portable greenhouses were placed over commercial and dooryard citrus to achieve therapeutic temperatures through solarization.  Within weeks after treatment, most trees responded with vigorous new growth.  Las titer was greatly reduced for more than one year after treatment and trees continued to grow well.  Unlike with potted trees, exposure to high heat was not sufficient to eradicate the Las population in field conditions. This may be attributed to reduced temperatures at night, rather than continuous high temperatures, and failure to achieve therapeutic temperatures in the root zone. Despite the presence of Las in heat-treated field citrus, many trees continue to produce large flushes and grow vigoursly two to three years after treatment. RNA-seq was used to monitor changes in gene expression of HLB-affected trees exposed to the heat treatment. Seventy eight genes were identified as differentially expressed before and after heat treatment. Thirty one genes were up-regulated after heat treatment. Among thirty one genes, there are four heat-shock related genes, one TIR-NBS-LRR gene, one peroxidase, one copper amine oxidase, two P450 genes, and one CBL-interacting protein kinase. Forty seven genes were down regulated in the heat treatment samples. Among these forty seven genes, there were one homogentisate phytyltransferase, two osmotin genes, two HXXXD-type acyl-transferase family proteins, and one WRKY transcription factor. These data indicate that heat treatment does have beneficial effects on citrus gene expression in addition to affecting Las survival.