A Reductionist Approach to the Study of Citrus Huanglongbing Disease

Huanglongbing (HLB) is a citrus disease that has devastated the Florida citrus industry in the last decade.  In addition to Florida, HLB is spreading throughout citrus in Texas.  Although HLB was confirmed in California in a single residential tree (2012), there have been no additional confirmations of HLB in California; however, the threat of HLB is serious.  Huanglongbing develops in citrus trees infected with Candidatus Liberibacter asiaticus (CLas), a phloem-limited bacterium.  The CLas lives in only two places, citrus phloem and the Asian citrus psyllid (ACP), its insect vector.  Huanglongbing is an exceedingly challenging pathosystem to study, especially hindered by unique biological characteristics of host, pathogen and vector.  To overcome some of the inherent complexities of HLB research we have taken a reductionist approach.  We report here on an experiment that was conducted with the objective of determining the effect of duration of exposure to ACP on CLas infection and HLB disease development.  Three hundred CLas negative ‘Valencia’ sweet orange grafted onto US 812 rootstock were placed into an ACP inclusionary greenhouse.  At two month intervals, groups of 60 plants were removed from the inclusionary greenhouse, treated with insecticide and then placed into an ACP exclusionary greenhouse.  At the time of transfer away from ACP 2, 12,  18, 30, and 95 were CLas-positive following exposure to ACP for 2, 4, 6, 8, or 10 months, respectively.  At eleven months after initiation of the experiment, 8%, 10%, 20%, 25%, and 37% of the plants were HLB-symptomatic for plants that had been exposed to ACP for 2, 4, 6, 8, or 10 months, respectively.  CLas infection had pronounced impact on stem diameter and starch content in leaves and roots.  Mean stem diameter 1 cm below the graft union was largest (11 mm) for plants that tested CLas-negative and smallest (7 mm) for plants when CLas titer reached 10⁵ copies/100 ng nucleic acid. Starch content was low (1% to 2% dry wt) in leaves and roots of CLas-negative plants.  In CLas-infected plants, leaf starch increased and root starch decreased as CLas titer in leaves increased.  However, the CLas-induced decrease in root starch (ca. 50%) was low compared to the CLas-induced increase in leaf starch (1400%).  Our results clearly demonstrate the value of a reductionist model for the study of HLB disease development.