|Deng, X - SO. CHINA AGR UNIV. CHINA|
|Feng, Z - SO. CHINA AGR UNIV. CHINA|
|Li, H - SO. CHINA AGR UNIV. CHINA|
Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 22, 2007
Publication Date: July 15, 2007
Citation: Deng, X., Chen, J., Feng, Z., Li, H., Civerolo, E.L. 2007. Nested-PCR Detection and Sequence Confirmation of Candidatus Liberibacter asiaticus from Murraya paniculata in Guangdong, China. Plant Disease 91:1051. Interpretive Summary: Murraya paniculata (jasmine orange) is an ornamental plant that is commonly planted in southern states from California to Florida, and in tropical and subtropical regions of Asia, Africa, and South America. It is utilized in hedges and as bonsai. Jasmine orange is a preferred host for the Asian citrus psyllid, Diaphorina citri (Kuwayana), a primary vector of Candidatus Liberibacter spp. causing citrus Huanglongbing (HLB). However, it remains unclear if M. paniculata is a host of Ca. L. species due to the difficulty of pathogen detection and identification from this host. We developed a nested-PCR technique that can amplify DNA associated with Ca. L. asiaticus from HLB symptomatic jasmine orange leaves. Specificity of the diagnostic technique was confirmed by sequence analysis. Our data strongly suggest that M. paniculata is a host of Ca. L. asiaticus.
Technical Abstract: Murraya paniculata (jasmine orange) is a popular ornamental plant and is a preferred host for Asian citrus psyllids, Diaphorina citri (Kuwayana), the primary vector of Candidatus Liberibacter spp. that causes citrus Huanglongbing (HLB). However, the presence of Ca. L. species in M. paniculata remains uncertain. In 2006, we identified five M. paniculata trees in Guangdong Province, People's Republic of China. Psyllids were found on all trees and some leaves showed yellowing and mottling symptoms. DNA was extracted from M. paniculata leaves and assayed by nested-PCR. The general bacterial 16S rDNA primer set fDl/rD1 (AGA GTT TGA TCC TGG CTC AG / AAG GAG GTG ATC CAG CC) was used for the outer round of amplification. The primary amplicon (2 µl) was then used for an inner round of amplification with primer set OI1/OI2c (5' GCG CGT ATG CAA TAC GAG CGG CA 3' / 5' GCC TCG CGA CTT CGC AAC CCA T 3'). A 1.1 kb DNA band was unambiguously associated with symptomatic but not asymptomatic leaf samples. Non-nested PCR using primer set OI1/OI2c alone did not yield a target DNA band, or yielded trace amount of product unsuitable for further analysis. XbaI digestion of the nested PCR DNA product yielded two fragments of 520 bp and 640 bp, characteristic of Ca. L. asiaticus. PCR amplicons were sequenced and found to be 1,095 bp. This sequence shared >98% similarity to sequences of Ca. L. asiaticus in the GenBank database. We note that nested-PCR is necessary for consistent amplification of DNA of Ca. L. asiaticus from M. paniculata. Sequence comparisons validated specificity of the assay and excluded the possibility non-specific amplification associated with nested-PCR.