Evaluation of DNA Amplification Methods for Improved Detection of “Candidatus Liberibacter Species” Associated with Citrus Huanglongbing

Citrus huanglongbing (HLB), also known as citrus greening (3), is considered the most serious disease of citrus worldwide (8). The disease has long been present in Asia, Africa, the Indian subcontinent, the Mascarene Islands, and the Arabian Peninsula (2). It has only recently been found in South and North America (16,26). The pathogen is a gram-negative bacterium belonging to the alpha subdivision of the proteobacteria (12). Because the bacterium has never been cultured in vitro, the name of the organism has provisional (“Candidatus”) status in nomenclature. The disease has Asian, African, and American forms caused by “Candidatus Liberibacter asiaticus” (Las), “Ca. L. africanus” (Laf), and “Ca. L. americanus” (Lam), respectively (12,28). Besides transmission through propagation of plant materials (1,18,21), the disease can be vectored efficiently by the psyllids Trioza erytreae and Diaphorina citri (19,24). Infected citrus groves are usually destroyed or become unproductive in 5 to 8 years following infection (6). D. citri has become established in Florida since its introduction in 1998 (7) and in Texas in 2001 (4). A Las strain was first detected and confirmed in various counties of Florida in 2005 (16). The disease is now of great concern to the entire U.S. citrus industry. The nonspecific nature of HLB symptoms makes it initially difficult to distinguish this disease from nutrient deficiencies or other plant diseases. The presumed low concentration and uneven distribution of the pathogens in host plants and vector insects make the pathogens difficult to detect consistently. Many detection methods have been described, including the use of biological grafted plant assays (23), the presence of specific fluorescent substances in infected plants (25), light or electron microscopy (14,30), or enzyme-linked immunosorbent assay (ELISA) (5). Although molecular approaches have been developed for detection and/or differentiation of “Ca. Liberibacter spp.” based on DNA amplification by conventional polymerase chain reaction (PCR) (10,11,13, 27,29), real-time PCR (16,17), and loopmediated isothermal amplification (LAMP) (22), consistent detection of the pathogens in infected plants or vector insects still remains problematic (16). Suspect plants are usually identified in the field by foliar blotchy mottle symptoms and/or lopsided fruit. The diagnosis is then confirmed by conventional and real-time PCR assays. Although PCR assays are accepted as confirmatory tests for HLB surveys in locations such as the state of São Paulo, Brazil, and Florida, USA, the potential of these assays to improve detection of “Ca. Liberibacter spp.” in host plants or vector insects has never been critically evaluated. The objective of this study was to compare several conventional PCR-based assays with LAMP and with our newly developed TaqMan real-time PCR system (16) for accurate, sensitive, and specific detection of HLB pathogens in the tissues of greenhouseand field-grown plants. This is important because the various DNA amplification-based assays have been developed separately and validated with limited numbers of strains of the pathogen. A systematic comparison of the existing assays, on a uniform and extensive set of plant samples, will provide needed data to make an informed choice among the many assay options available for use in regulatory and management programs. Our results showed that the analytical sensitivity of “Ca. Liberibacter spp.” detection in plant tissues using the TaqMan real-time PCR system could be increased by 1 to 2 log units over the conventional PCR system. Thus, the TaqMan real-time protocol has the potential to become a valuable tool for early detection of “Ca. Liberibacter spp.” prior to the appearance of disease symptoms. Reliable, robust, and unambiguous identification of “Ca. Liberibacter spp.” is greatly needed for an effective regulatory response, to facilitate management of infected trees, and to contribute toward the development of “Ca. Liberibacter spp.”–free certified plant materials.