Gabriel, D. W.

‘Candidatus Liberibacter’ spp. are uncultured insect endosymbionts and phloem-limited bacterial plant pathogens associated with diseases ranging from severe to nearly asymptomatic. ‘Ca. L. asiaticus’, causal agent of Huanglongbing or citrus “greening,” and ‘Ca. L. solanacearum’, causal agent of potato zebra chip disease, respectively threaten citrus and potato production worldwide. Research on both pathogens has been stymied by the inability to culture these agents and to reinoculate into any host. Only a single isolate of a single species of Liberibacter, Liberibacter crescens, has been axenically cultured. L. crescens strain BT-1 is genetically tractable to standard molecular manipulation techniques and has been developed as a surrogate model for functional studies of genes, regulatory elements, promoters, and secreted effectors derived from the uncultured pathogenic Liberibacters. Detailed, step-by-step, and highly reproducible protocols for axenic culture, transformation, and targeted gene knockouts of L. crescens are described. In the course of developing these protocols, we found that L. crescens is also naturally competent for direct uptake and homology-guided chromosomal integration of both linear and circular plasmid DNA. The efficiency of natural transformation was about an order of magnitude higher using circular plasmid DNA compared with linearized fragments. Natural transformation using a replicative plasmid was obtained at a rate of approximately 900 transformants per microgram of plasmid, whereas electroporation using the same plasmid resulted in 6 × 104 transformants. Homology-guided marker interruptions using either natural uptake or electroporation of nonreplicative plasmids yielded 10 to 12 transformation events per microgram of DNA, whereas similar interruptions using linear fragments via natural uptake yielded up to 34 transformation events per microgram of DNA.

Citrus huanglongbing (HLB) is one of the most devastating diseases of citrus worldwide. The disease is associated with three different species of fastidious α-proteobacteria, namely ‘Candidatus Liberibacter asiaticus’, Ca. L. americanus, and Ca. L. africanus (1). ‘Ca. L. asiaticus’ was first detected in South Florida in 2005 and has spread throughout the citrus-growing areas of Florida. ‘Ca. L. asiaticus’ is transmitted naturally by the Asian citrus psyllid, Diaphorina citri, and can also be transmitted by graft propagation and via various species of dodder (Cuscuta). HLB affects most if not all citrus and citrus relatives within the family Rutaceae (2), including the ornamental shrub Murraya paniculata (4). In addition, ‘Ca. L. asiaticus’ and ‘Ca. L. americanus’ can infect tobacco (Nicotiana xanthi) and periwinkle (Catharanthus roseus) (1,4). Here we report that ‘Ca. L. asiaticus’ can infect tomato (Lycopersicon esculentum) cvs. Manapal and FL47. Manapal and FL 47 plants grown from seed were placed adjacent to ‘Ca. L. asiaticus’-infected sweet orange (Citrus sinensis) plants with dodder (Cuscuta pentagona) already well established on them. Young dodder shoots still connected to the citrus were draped over the tomato plants and subsequently also became attached to the tomato stems. After 1 month, the tomato plants were detached from the citrus and most of the dodder removed. One month later, these tomato plants started to show vein clearing and subsequently the mature leaves became thicker and leathery. Some leaves showed blotchy mottle symptoms and some fruits became lopsided in a manner similar to HLB symptom expression on citrus. PCR amplification of the ‘Ca. L. asiaticus’ 16S rDNA with primers OI1/OI2c and the β-operon with primers A2/J5 (1) revealed the presence of ‘Ca. L. asiaticus’ DNA. Sequence analysis confirmed that the sequences of the cloned amplicons were identical to those from the HLB-infected citrus source plant. Both conventional and quantitative real-time PCR (3) revealed a much lower abundance of ‘Ca. L. asiaticus’ DNA in tomato as compared with ‘Ca. L. asiaticus’-infected citrus or periwinkle, indicating that ‘Ca. L. asiaticus’ bacteria multiplied at a lower titer in these tomato cultivars. References: (1) J. M. Bové, J. Plant Pathol. 88:7, 2006. (2) S. E. Halbert et al. Fla. Entomol. 87:330, 2004. (3) W. Li et al. J. Microbiol. Methods 66:104, 2006. (4) L. Z. Zhou et al. Plant Dis. 91:227, 2007.