A new diagnostic system for ultra-sensitive and specific detection and quantification of Candidatus Liberibacter asiaticus, the bacterium associated with citrus Huanglongbing

Crop Diseases, Pests and Genetics Research Unit, San Joaquin Valley Agricultural Science Center, USDA ARS PWA, Parlier, CA 93648, USA.
Journal of microbiological methods (Impact Factor: 2.03). 04/2010; 81(1):17-25. DOI: 10.1016/j.mimet.2010.01.014
Source: PubMed


An ultra-sensitive and quantitative diagnostic system by combining nested PCR and TaqMan PCR in a single tube was developed for detection of "Candidatus Liberibacter asiaticus". The procedure involves two PCR steps using the species-specific outer and inner primer pairs. Different annealing temperatures allow both the first and the second rounds of PCR to be performed sequentially in the same closed tube. The first PCR with outer primers was performed at a higher annealing temperature and with limited amount of primers to prevent interference with the inner primers during the second round of PCR. The specificity of the dual primer TaqMan is high because the fluorescent signal can only be generated from the TaqMan probes that are homologous to the product amplified by the outer and inner primers. This new detection system can reliably detect as few as single copies of target DNA. The sensitivity of the dual primer system is comparable to the conventional two-tube nested PCR, but it eliminates the potential risk of cross contamination commonly associated with conventional nested PCR. This one-tube dual primer TaqMan PCR method is gel-free with reduced handling time and is cost effective. At the same time, this system provides significantly increased sensitivity, improved reliability and high through-put capability suitable for routine, large scale diagnoses of clinical plant tissue and insect samples. The technique described here is generic and can be applied to the detection of other plant pathogenic bacteria.

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    • "The line connecting contiguous data points has been added to emphasize the greater variation between the early incubation periods. (Modified from Pelz-Stelinski et al. 2010). inoculum without sufficient infectious pathogens and therefore most arthropods are never exposed to enough pathogens to be detected or propagate. "
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    ABSTRACT: Characterizing the vector competence of Diaphorina citri Kuwayama for ‘Candidatus Liberibacter asiaticus,’ the pathogen causing citrus greening, is essential for understanding the epidemiology of this disease that is threatening the U.S. citrus industry. Vector competence studies have been difficult because of the biology of D. citri, the inability to culture the pathogen, and the available diagnostic methods used to detect the bacteria in plant and insect tissues. The methods employed in many studies of D. citri vector competence may have overestimated amounts of live ‘Ca. L. asiaticus’ in both plant and insect tissues, and it is possible that the amounts of phloem ingested by psyllids may not contain sufficient detectable pathogen using current diagnostic methods. As a result of the difficulty in characterizing D. citri vector competence, the several daunting challenges for providing D. citri that are unable to inoculate ‘Ca. L. asiaticus’, as a novel method to control greening are discussed. Suggestions to overcome some of these challenges are provided.
    Journal of Economic Entomology 06/2015; 108(3). DOI:10.1093/jee/tov038 · 1.51 Impact Factor
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    • "Although the visual symptoms favor the detection of the presence of HLB (Roistacher, 1991) on citrus plantations, it is only by using more sophisticated detection methods that are based on electronic microscopy, Enzyme-Linked Immuno-Sorbent Assays with monoclonal antibodies (ELISA), HLB specific fluorescent marking substances (Schwarz, 1968). Recently, the implementation of PCR and real-time PCR methods that have been used in many countries for the detection of the three causal agents of HLB based on the 16S ribosomal DNA region and other regions of the bacterial genome (Lin et al., 2010), that the presence of the disease can be truly diagnosed. "
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    ABSTRACT: Citrus huanglongbing (HLB) is the most destructive citrus disease. Two of the three known HLB-associated Candidatus Liberibacter species were recently found to be present in the Americas. In this study, eggs, nymphs and adults of Diaphorina citri Kuwayama (Hemiptera: Liviidae) and suspect citrus plant materials were collected in 25 municipalities in the departments of Cundinamarca, Santander, Valle del Cauca, Meta and Quindio (Colombia). The detection sensitivity, specificity and assay performance of the 16S rDNA-based real-time PCR (qPCR) were validated for the field survey of the disease in Colombia. The validation confirmed the reliability and robustness of the real-time PCR method for the detection of HLB bacteria in host citrus plant tissues and the vector D. citri. The diagnosis was performed for Candidatus Liberibacter asiaticus (Ca. L. asiaticus) and for Candidatus Liberibacter americanus (Ca. L. americanus) on 168 citrus plant material samples and 239 insect samples. Neither Ca. L. asiaticus nor Ca. L. americanus were detected in the host plants or insects vector, confirming the absence of the disease in the citrus-producing areas of Colombia. © 2014, Universidad Nacional de Colombia1. All rights reserved.
    02/2015; 32(3):377-389. DOI:10.15446/agron.colomb.v32n3.44069
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    • "The titer of phytoplasma cells in the phloem of infected plants varies by season and plant species, and it is often very low in woody hosts, presenting a major obstacle to the diagnosis of these phytopathogens (Marzachi, 2004). The diagnosis of Huanglongbing disease is based on symptoms such as foliar blotchy mottle (Bové, 2006), as well as the use of DNA hybridization (Villechanoux et al., 1992) and the polymerase chain reaction (PCR) (Jagoueix et al., 1996; Hocquellet et al., 1999; Teixeira et al., 2005; Li et al., 2006; Lin et al., 2010). These molecular techniques make it possible to accurately and rapidly diagnose Ca. "
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    ABSTRACT: Huanglongbing (HLB), one of the most devastating citrus diseases in the world, was detected in Mexico in 2009. Currently, HLB is associated with the bacteria Candidatus Liberibacter spp., although several phytoplasmas have been found from trees showing HLB-like symptoms in Brazil and China. The aim of this study was thus to determine if, in addition to ‘Ca. L. asiaticus’ (CLas), phytoplasma species are also associated with HLB-like symptoms in citrus groves of Mexico. Citrus plants exhibiting symptoms such as diffuse chlorosis, blotchy mottle and vein yellowing were collected in the Mexican States of Nayarit, Colima and Sinaloa between August 2011 and September 2012. Samples were then evaluated for phytoplasmas and CLas by PCR, using primers that respectively target the genes for the 16S ribosomal RNA and 50S ribosomal protein of the β operon (rplA-rplJ). Out of 86 HLB-symptomatic citrus plants, 54 were positive for CLas, 20 were positive for phytoplasmas, 7 were found in mixed infections with both pathogens and 19 samples were negative for CLas and phytoplasmas. Actual and virtual RFLP analyses of the 16S rDNA sequences enabled us to classify two HLB phytoplasma strains as members of the aster yellows group (16SrI) ‘Candidatus Phytoplasma asteris’, which was confirmed by phylogenetic analysis. The HLB phytoplasma strain identified from Nayarit (HLBpc-Nay-IB) belongs to subgroup B (16SrI-B), and the strains identified from Colima (HLBpc-Col-IS) and Sinaloa (HLBpc-Sin-IS) belong to subgroup S (16SrI-S). The partial ‘Ca. L. asiaticus’ rplA-rplJ gene sequences were 100% identical to the ‘Ca. L. asiaticus’ strains isolated from several countries affected by HLB. These results confirm the association of ‘Candidatus Phytoplasma asteris’ with HLB-like symptoms in citrus groves in Mexico. Nonetheless, further studies are required to fully describe the ‘Ca. L. asiaticus’ and ‘Ca. P. asteris’ interactions in citrus, which will greatly assist the design of efficient management strategies.
    Crop Protection 08/2014; 62:144–151. DOI:10.1016/j.cropro.2014.04.020 · 1.49 Impact Factor
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