**3. Causal agents of citrus Huanglongbing**

The bacterium associated with citrus HLB was *Candidatus* Liberibacter species, which belongs to the alpha-proteobacteria determined by the 16 s ribosomal DNA sequences and the operon [9]. *Proteobacteria* associated with HLB disease in citrus are successively referred to as *Candidatus* Liberibacter asiaticus (Las) found in the majority of HLB-affected countries, *Candidatus* Liberibacter africanus (Laf) limited to African countries, and *Candidatus* Liberibacter americanus (Lam) limited to America [15]. How *Candidatus* Liberibacter bacterium established its association with citrus species remains unclear.

Scientific classification of *Candidatus* Liberibacter. Kingdom: Bacteria. Phylum: Proteobacteria. Class: Alphaproteobacteria. Order: Rhizobiales. Family: Rhizobiaceae. Genus: *Candidatus* Liberibacter.

#### **3.1** *In vitro* **culture of** *Candidatus* **Liberibacter species associated with HLB**

The isolation of *Candidatus* Liberibacter species, causing HLB in an artificial culture medium, was a primary target for many researchers. Davis et al. [46] attempted to isolated *Candidatus* Liberibacter asiaticus in a culture medium from young angular green shoots from HLB-affected trees. A growth film appeared on the bottom of the tube containing broth AD medium. After single-colony isolation, Las and the actinobacteria closely related to *Propionibacterium acnes* remained together. Thus, Las was not isolated in axenic culture. Moreover, actinobacteria are prevalent residents of citrus and psyllids, whether Las is present. Sechler et al. [47] successfully cultivated a single colony of all three *Candidatus* Liberibacter species from HLB-affected leaf midveins and petiole sap in a new medium designated Liber A. The isolated cells were ovoid to rod-shaped, 0.3 to 0.4 by 0.5 to 2.0 μm, often with fimbriae-like appendages. They isolated two Las and one Lam strains from non-inoculated tissues of inoculated trees and seedlings 9 and 2 months later.
