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Title: COMPARISON OF METHODS FOR THE DETECTION OF CANDIDATUS LIBERIBACTER ASIATICUS IN PLANT SAMPLES

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

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 10/10/2005
Publication Date: 11/7/2005
Citation: Hartung, J.S., Li, W., Levy, L. 2005. Comparison of methods for the detection of candidatus liberibacter asiaticus in plant samples. Meeting Abstract.

Interpretive Summary:

Technical Abstract: The Exotic Pathogens of Citrus Collection (EPCC) at Beltsville maintains an inventory of several hundred potted citrus trees infected with exotic, graft transmissible pathogens, including Ca. Liberibacter spp. Plants from the EPCC were selected and used to compare published PCR-based detection methods for Ca. Liberibacter spp. with Loop Mediated Isothermal Amplification (LAMP) and a new Taqman based assay for quantitative, real time PCR. The 76 trees selected for the study included trees entered into the collection with Ca. Liberibacter spp. as the pathogen of record, as well as trees entered with other pathogens of record. The tree samples also included sub-propagations of the original accessions. DNA was extracted using the Qiagen Plant DNeasy kit from ~200 mg of the proximal portion of midribs pooled from three mature leaves from each tree. Symptomatic tissue was chosen when present. PCR amplification with PCR primers A2/J5 (Hocquellet et al., 1999) and OI1/OI2C; GB1/GB3 (Teixeira et al., 2005) was carried out first, followed by LAMP (Okuda et al., 2005) and RT-PCR (Li et al., submitted). Primer pairs A2/J5 and OI1/OI2C each detected Ca. Liberibacter asiaticus in the same 15 trees (20%). LAMP assays also detected the pathogen in the same 15 trees. The Taqman assay also detected the pathogens these fifteen trees, as well as one additional tree. Ca. Liberibacter asiaticus could be detected in a 10-5 dilution of extracts, using the Taqman assay. Primer pair GB1/GB3 did not detect Ca. Liberibacter americanus in any of the trees, as expected. Ca. Liberibacter africanus was also not detected in any sample, although trees thought to be infected with this species were tested. We also noted two instances where the original accession tested negative for Ca. Liberibacter asiaticus, but sub-propagants were positive. It is likely that these trees were cured of Ca. Liberibacter spp. by high greenhouse temperatures. We further noted several instances where different sub-propagants from a source tree gave different reactions, suggesting that transmission by grafting is not 100 % efficient. In our hands, the LAMP assay was prone to contamination, yielding obvious false positives. The fact that the four assay systems gave congruent results suggests that each assay system is reliable.