FIGURE 1.

Culture of Candidatus Liberibacter asiaticus (CLas) in vitro. (A) Metabolic pathway analysis of 23 representative species of α-Proteobacteria. Less-conserved metabolic pathways in Candidatus Liberibacter species (>50% dissimilarity to other α-Proteobacterial species) are shown. Blue, full functional unit; orange, partial functional unit; gray, no functional unit. Phylogenetic relationships were analyzed on the basis of nucleotide sequences of 16S ribosomal DNA from GenBank by the neighbor-joining method in MEGA 6 software. Stability was assessed using 1000 bootstrap replications. (B) CLas Ishi-1 was grown in newly developed culture medium supplemented with ampicillin (50 ppm). Ishi-1 was quantified by real-time PCR using DNA templates prepared from 1-cm² pieces of agar (n = 3, each value plotted individually). (C) Microscopic observation of CLas Ishi-1 by fluorescence in situ hybridization (with Las-specific probe LSS) using samples incubated for 2 weeks.