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Title: EVALUATION OF DNA AMPLIFICATION METHODS FOR IMPROVED DETECTION OF CANDIDATUS LIBERIBACTER SPECIES ASSOCIATED WITH CITRUS HUANGLONGBING

Author
item LI, WENBIN - USDA APHIS CPHST
item Hartung, John
item LEVY, LAURENE - USDA APHIS CPHST

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/16/2006
Publication Date: 12/20/2006
Citation: Li, W., Hartung, J.S., Levy, L. 2006. Evaluation of dna amplification methods for improved detection of candidatus liberibacter species associated with citrus huanglongbing. Plant Disease. 91:51-58.

Interpretive Summary: Citrus Huanglongbing, or greening disease, kills infected trees and makes entire groves unproductive within a short period following infection. The pathogen has recently been introduced into both São Paulo, Brazil and Florida, USA, the homes of the two largest citrus industries in the world. Diagnosis is difficult based on symptoms alone, and so DNA based detection methods are needed to confirm the diagnosis of the disease. The diagnosis must be accurate, because all disease control programs require the removal of infected trees to limit further spread of the pathogen. We have recently developed a rapid and specific test for the pathogens that cause this disease. In this study we compared our new test to four previously described tests for the pathogen. We found that our new test was just as specific as the previously accepted tests, but was 100 times more sensitive and could be completed in much less time. Thus the speed and sensitivity of identification has been improved without compromising specificity. Our new test can also detect the pathogen before symptoms appear in the plant, which no other test has been able to do. USDA APHIS as well as regulatory officials in Florida, California and São Paulo will be interested in this work, as will other researchers.

Technical Abstract: Citrus huanglongbing (HLB), also known as citrus greening or citrus yellow shoot, is considered the most serious disease of citrus worldwide. The disease has Asian, African and American forms caused by Candidatus Liberibacter asiaticus, Ca. L. africanus and Ca. L. americanus, respectively, which can be spread by efficient vector psyllids Diaphorina citri and Trioza erytreae and through contaminated plant materials. Infected citrus orchards are usually destroyed or become unproductive in 5 to 8 years. The presumed low concentration and uneven distribution of the pathogens in citrus plants and vector insects make the phloem-limited bacterium difficult to consistently detect. Although molecular diagnoses based on conventional polymerase chain reaction (PCR) and real-time PCR are usually used as confirmatory tests for symptomatic samples, the potential of PCR assays to improve detection of Ca. Liberibacter spp. has never been evaluated. In this study, we compared and validated four conventional PCR-based protocols, one loop-mediated isothermal amplification (LAMP) protocol and three TaqMan® real-time PCR protocols. The detection sensitivity of the validated conventional PCR assays was improved compared to the original protocols. All of the validated conventional and the newly developed real-time methods were reliable for confirmatory tests for the presence of Ca. Liberibacter spp. in symptomatic samples. There were no differences in assay specificity among the standard format PCR-based methods evaluated. The TaqMan® real-time PCR was 10 to 100 fold more sensitive than conventional PCR and LAMP, showing the potential to become a valuable tool for early detection and identification of Ca. Liberibacter spp. prior to the appearance of disease symptoms. The methods validated in this study will be very useful for regulatory response, effective management of infected trees, and development of a Ca. Liberibacter spp.-free nursery system.