Evaluation of Mobile Heat Treatment System for Treating In-Field HLB-Affected Trees by Analyzing Survival Rate of Surrogate Bacteria

Huanglongbing (HLB or citrus greening) is a disease caused by an insect-transmitted bacterial pathogen Candidatus Liberibacter asiaticus (CLas). Thermotherapy has been successfully used by others to reduce the population of CLas bacteria in HLB-affected citrus trees under greenhouse studies. Thermotherapy is the application of heat as a strategy to reduce the adverse economic impact of certain pests and diseases. CLas is a fastidious, non-cultivable organism. The high variance in CLas titers in canopy samples together with this lack of cultivability makes it impossible to use classical bacteriological techniques to measure the viability either before or after treatments. Therefore, we used the survival rates of a surrogate bacterium, Klebsiella oxytoca, in order to evaluate the effectiveness of a mobile thermotherapy delivery system developed for in-field treatment of HLB-affected trees. K. oxytoca is a Gram-negative, rod-shaped bacterium that was originally isolated from soil and has been used in the development of industrial applications related to ethanol fuel production. It served as a biologically-based sensor of temperature stress (biosensor) in this study. Thermocouples and biosensor packets (plastic cups with suspended small snap-top tubes) containing the K. oxytoca were attached to an HLB-affected citrus tree and their canopy locations mapped. The mobile thermotherapy treatment hood covered the canopy of the HLB-affected tree. Then, steam and hot water were injected through nozzles inside of the hood to increase the temperature of the tree canopy. A standard temperature-time combination of 54 degrees C for 90 s was chosen based on preliminary studies where heat treatment parameters caused a significant reduction in CLas populations without inflicting permanent damage to the tree. The survival ratio of the K. oxytoca in the biosensor packets was found to range from complete elimination to 5% with treatments of 250 s and a maximum temperature of 54 degrees C.