Submitted to: Journal of Microbiological Methods
Publication Type: Peer reviewed journal
Publication Acceptance Date: 10/26/2005
Publication Date: 1/18/2006
Citation: Li, W., Teixeira, D., Hartung, J.S., Levy, L. 2006. Detection and identification of candidatus liberibacter species associated with citrus huanglongbing by multiplex real-tim pcr. Journal of Microbiological Methods. 66:104-115. Interpretive Summary: Citrus Huanglongbing disease, also known as citrus greening, is the most serious disease of citrus wherever it occurs. The pathogen is spread by infected insects, or by grafting healthy trees with infected buds. Because the disease is caused by a bacterium that can not be grown in the laboratory, detection and diagnosis of the pathogen is very difficult, and the methods must be improved. During the course of this research, the pathogen was detected for the first time in the United States, so detection and diagnostic methods are of great current interest. We have developed, for the first time, a series of related assays that can be used to detect the bacterium in infected plants or insects. The assays are based on the DNA of the pathogen. The assays can be completed in hours, can be performed in large numbers, and can both detect the presence of the pathogen and count the number of pathogen cells present in the samples. Our methods will be used by the research community as well as by state and federal regulatory agencies responding to the current crisis caused by the discovery of citrus greening in Florida.
Technical Abstract: Citrus huanglongbing (HLB, ex greening) is one of the most serious diseases of citrus. Different forms of the disease are caused by different Candidatus Liberobacter species, Candidatus Liberibacter asiaticus (Las), Ca. L. africanus (Laf), and Ca. L. americanus (Lam). The pathogen is transmitted by psyllid insects and by budding with contaminated plant materials. The vector psyllid Diaphorina citri can transmit both Las and Lam. The recent establishment of this vector into Florida, and the recent reports of Lam and Las in Brazil in 2004, and the very recent confirmation of HLB in Florida in September, 2005 could be devastating for the citrus industry. Research on HLB has been hampered by the unculturable nature of the causal bacterium in artificial media. It has also been difficult to detect and identify the pathogens, possibly because of low concentration and uneven distribution in host plants and vector psyllids. In this study, we developed quantitative TaqMan PCR using 16S rDNA-based Taqman primer-probe sets specific to the different Ca. Liberobacter spp. An additional plant cytochrome oxidase (COX)-based primer-probe set was used as a positive internal control to assess the quality of the DNA extracts and reaction cocktails. The assays do not cross-react with other pathogens or endophytes commonly resident in citrus plants, and are very sensitive. HLB pathogen DNA was successfully amplified from the equivalent of 20 ng of midrib tissue from symptomatic leaves. The consistent results of the assays with DNA extracted from plants infected by various Ca. Liberibacter species grown in greenhouses and in the field demonstrated a degree of reproducibility for these TaqMan assays. Inhibitors of the PCR that are frequently present in plant extracts did not affect the assay results. The populations of the pathogens was estimated to be 2.5 x 107 and 2.0 x 106 cells per gram of fresh midribs of symptomatic sweet orange leaves infected by Las and Lam, respectively. The ratio of the pathogen DNA to host plant DNA was estimated to be 1:1,000 in DNA extracts obtained by a standard CTAB method. Our rapid, sensitive and specific TaqMan PCR assay for the detection, identification and quantification of Ca. Liberibacter spp. has been successfully used to confirm the detection and identification of Ca. Liberibacter in Florida, and will be very useful for a broad range of research programs as well as phytosanitary quarantine and management of HLB disease.